A QUANTITATIVE PROTEOMICS STUDY OF THE
ADDITIVE EFFECT OF INFLAMMATORY CYTOKINES AND
INJURIOUS COMPRESSION ON CARTILAGE DAMAGE
JMASSACHUSniTS INSTITUTE
OF TEcr-,
Krishnakumar Swaminathan
JUL 142011
B.Tech Biotechnology
Indian Institute of Technology Madras, 2006
ARCHIVES
Submitted to the Department of Biological Engineering in Partial Fulfillment
of the Requirements for the Degree of
Masters of Science in Biological Engineering
at the
Massachusetts Institute of Technology
February 2011
© 2011 Massachusetts Institute of Technology
All rights reserved
V(
Signature of Author
Krishnakumar Swaminathan
Department of Biological Engineering
-
Certified by
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Alan J. qrdznsky, th7TAdvs r
Professor of Biological, Electrical and Mechanical Engi
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Accepted by
Forest M. White, Graduate Program Committee
Department of Biological Engineering
ABSTRACT
Objectives: 1) To perform a quantitative comparison of proteins released to media on
combination with cytokine (IL-1p or TNF-a) and Injury as compared to either treatment
alone, and to thus identify proteins which may be responsible for the synergism seen
between cytokine and injury in causing catabolism of cartilage in vitro. 2) To identify
proteins which contribute most to some commonly observed phenotypes on treatment of
cartilage with cytokine or injury or both.
Methods : Cartilage explants from calves were treated with (i)IL-1 (10 ng/ml), (ii)TNFa (100 ng/ml), (iii)Injurious compression (50% strain at 100%/sec) and IL-1p (10 ng/ml)
or (iv)Injurious compression (50% strain at 100%/sec) and TNF-a(10 ng/ml), cultured for
5 days post treatment, and the pooled media collected, labeled with one of four iTRAQ
labels and subjected to nano-2D-LC/MS/MS on a quadrupole time of flight instrument.
Peptides were identified and quantified using Protein PilotTM, and MATLAB scripts used
to obtain protein ratios. These results were analyzed using different statistical techniques.
Data from two iTRAQ experiments were combined to generate data for all possible
injury and cytokine treatment conditions, and proteins on which injury and cytokines
acted synergistically identified. PLSR analysis was performed using Unscrambler@X
software with the combined data set to determine which proteins are most relevant to
some observed phenotypes. The phenotypes chosen were sGAG released to media in 5
days post treatment, proline and sulfate incorporation rates on day 6 post treatment, and
nitrite accumulation in media in 5 days post treatment
Results and Discussion: TNF-a+injury and IL-1p +injury treatment conditions show a
very high correlation with each other. Most cytosolic, ER lumen and nuclear protein
levels are highly elevated with both cytokine+injury conditions, while ECM proteins are
either highly down regulated or marginally elevated. Many collagen telopeptides are
down regulated, possibly indicating reduced anabolism. However, attempts at repair
exist, as shown by increased levels of TGF- and activin A, and reduced levels of
LTBP1. Also, biglycan and lumican, SLRPs known to be involved in early development
are significantly increased, possibly indicating repair attempts. Other SLRPs such as
PRELP and chondroadherin are also highly elevated, with one or both injury+cytokine
treatments. While MMPs are mildly down regulated or remain the same, ADAMTS1
increases with TNF-a+injury, indicating increased catabolism. Among ECM structural
proteins, COMP shows high down regulation with TNF-a+injury, possibly due to
reduced synthesis. Proenkephalin, a signaling molecule possibly involved in tissue/repair
and apoptosis, AIMPI, a multifunctional proapoptotic, inflammatory and pro-repair
cytokine and Annexin A5, a protein indicating mineralization and apoptosis are all
highly elevated with cytokine+injury indicating heightened apoptosis and/or repair.
When results of two 4-plex iTRAQ experiments are combined to obtain data for all
possible combinations of injury and cytokine, we again find a very high correlation
between TNF-a+injury and IL-1 +injury (-95%), slightly higher than the correlation
between TNF-c alone and IL-1$ alone (-90%), and much higher than the correlation of
either cytokine+injury condition with cytokine alone (-70%) or injury alone (-75%).
This shows that IL-1P and TNF-a in combination with injury act through very similar
pathways in chondrocytes to produce their effect on cartilage tissue. TNF-a and injury
were seen to act synergistically in a positive fashion on aggrecan, CILP-2, COL6A3 and
histone H4, and in a negative fashion on SPARC and IGFBP7, suggesting that these
proteins may be involved in causing synergism between injury and cytokine in releasing
sGAG to the media. A PLSR analysis shows that SPARC and IGFBP7 project close to
proline and sulfate incorporation, and far away from sGAG, indicating that SPARC and
IGFBP7 may be proteins involved in anabolism. The highest phenotype-protein positive
correlations obtained using PLSR are sGAG with Perlecan, SAA3, Complement factor B,
CILP-2 and pleiotropin, indicating that all these 5 proteins are associated strongly with
catabolism and can serve as markers of catabolism. The correlation of inflammatory
proteins SAA3 and complement factor B with sGAG indicates the role of inflammation
with catabolism.
Conclusion: The combination of injury and cytokine affects tissue differently at a
molecular level as compared to either chemical or mechanical stresses alone. Increased
catabolism and increased attempts at tissue repair are observed due to a combination of
injury and cytokine, and a combination of injury and cytokine may thus serve as a useful
model to study OA in vitro.
ACKNOWLEDGEMENTS
I am grateful to the Biological Engineering Department and MIT for giving me the best
of education, and teaching me what true scientific research and thinking is all about. My
sincere thanks to Linda Bragman and Han-Hwa Hung for help in many small ways and to
Dalia Fares for taking care of administrative issues. Dr. Anna Stevens performed the
cartilage explantation experiments, and Dr.Ramesh Indrakanti ran the Mass Spec, my
grateful thanks to them. My heartfelt thanks to Anna, who guided me into this work, and
was always there for any questions I had (and there were many!). And most important of
all, I am indebted to Prof. Alan Grodzinsky who stood by me through thick and thin, and
was a constant source of motivation and inspiration.
TABLE OF CONTENTS
A bstract...........................................................................
.. 2
Acknowledgements................................................................4
L ist of Figures.........................................................................7
L ist of T ables........................................................................
8
9
Chapter 1: Introduction ..............................................................
1.1 Arthritis - An Overview......................................9
1.2 Osteoarthritis : Pathology, Symptoms and Treatment........9
1.3 A rticular Cartilage...............................................10
1.4 Cartilage Components........................................10
1.5 Post-traumatic GA.............................................18
20
1.6 Cytokines in GA .................................................
1.7 In vitro models combining injury and cytokine..............23
1.8 Thesis Objectives.............................................23
25
Chapter 2: M ethods..............................................................
25
2.1 O verview .......................................................
2.2 Cartilage explantation, Mechanical Injury and Cytokine
T reatm ent..............................................................25
2.3 sGAG removal, trypsin digestion and iTRAQ labeling.....26
2.4 SCX chromatography and LC/MS/MS.....................28
2.5 Data Analysis..................................................29
2.6 Comparison of biological duplicates and final protein list
31
com pilation.........................................................
2.7 Global proteomic analysis and clustering..................31
2.8 Merging data between two iTRAQ experiments............32
2.9 Finding proteins on which injury and cytokines act
synergistically.........................................................33
2.10 Partial Least Square Regression (PLSR).................33
. .. 35
Chapter 3: R esults.............................................................
3.1 Reproducibility between biological replicates...............35
3.2 Global proteomic analysis...................................36
3.3 Effect of combination of cytokine and injury on specific
p rotein s................................................................37
3.4 Effect of combination of injury and cytokine as opposed to
either treatment alone.............................................40
3.5 Partial Least Square Regression (PLSR) between protein
ratios and phenotypes................................................43
Chapter 4: Discussion
4.1 Methodology for correcting for multiple comparisons - The
Benjamini Hochberg Procedure.................................63
4.2 Variation between biological replicates....................63
4.3 A global proteomic analysis of the effect of
cytokine+injury.......................................................64
4.4 Effect of cytokine+injury on specific protein classes.......66
4.5 Effect of combination of cytokine and injury as opposed to
either treatment alone.............................................86
4.6 Partial Least Square Regression (PLSR) Analysis..........92
4.7 Limitations of this study.....................................97
Chapter 5: Conclusion...........................................................99
References...........................................................................103
Appendix ............................................................................
124
LIST OF FIGURES
Figure 1: Histograms of biological replicate comparisons.....................45
Figure 2: Projection plot of log transformed protein ratios with the axes as
(IL-iI+injury/IL- 1P) and (TNF-a+injury/IL- I1P) as the axes..................47
Figure 3: 3D plot and 2D projections of log transformed protein ratios,
clustered by K-means clustering with (TNF-a/IL-11), (IL-1 +injury/LL-lp)
and (TNF-a+injury/IL- 11P) as the axes.........................................48
Figure 4: Profile of means of log transformed protein ratios sorted by
clusters identified by K-means clustering.....................................49
Figure 5: Profile of means of log transformed protein ratios sorted by
50
cellular location...................................................................
Figure 6: Behavior of different protein classes on cytokine+injury
1
treatm ent..............................................................................5
Figure 7: Histograms of comparison of log transformed protein ratios
obtained by two iTRAQ experiments...........................................54
Figure 8: PLSR plots : Explained variance in protein ratios and phenotypes
by the latent vectors, projection plots of treatment conditions, log
transformed protein ratios and log transformed phenotype ratios on the first
57
tw o latent vectors.................................................................
LIST OF TABLES
Table 1: Matrix of correlation coefficients between TNF-ac alone, IL-1p
+injury and TNF-x+injury treatment conditions.............................46
Table 2: Matrix of correlation coefficients between IL-lIP alone, TNF-a
alone, injury alone, IL- I+injury and TNF-a+injury conditions.............55
Table 3: Proteins on which TNF-a and injury act in a positive or negative
synergistic fashion...................................................................56
Table 4: List of protein-phenotype pairs showing the top 5 positive and
negative correlations................................................................60
Table 5: Effect of injury or cytokine or both on different phenotypes.......60
INTRODUCTION
1.1 Arthritis: An Overview
The word "Arthritis" literally means inflammation of joint, and encompasses
more than 100 different rheumatic diseases and conditions. It is the leading cause of
disability in the US 1. Arthritis affects around 21.6% of the US population, and limits the
activity of -40% of people having the disease 2 . It is projected to affect nearly 67 million
people by 2030 3. Osteoarthritis (OA) is the most common form of arthritis and is
estimated to affect nearly 27 million people currently
4. It
poses an immense societal and
economic burden, with OA alone estimated to cost $185.5 billion annually 5.
1.2 Osteoarthritis: Pathology, Symptoms and Treatment
Osteoarthritis is a condition representing an imbalance between degradation and
repair involving the whole joint and its component parts, with secondary inflammatory
changes in the synovium 6. Pathological features of OA include degradation of hyaline
cartilage leading to loss of joint integrity, elevation in bone density at areas of cartilage
loss (bony sclerosis), subchondral cyst formation and endochondral ossification
(osteophyte formation). The synovium, ligaments and neuromuscular apparatus may also
show pathological changes. Symptoms of OA include joint pain, swelling and stiffness of
joint and loss of mobility. Several factors increase the risk of OA, including body weight,
age, gender, physical activity, bone mineral density, injury and genetic factors 6
Pharmacological treatment of OA is usually symptomatic, with analgesics, NSAIDs and
neutraceuticals like glucosamine and chondroitin sulfate used to combat inflammation
and pain. Non-pharmacological interventions including rest, physical therapy and
lifestyle changes seem to help, however, surgery is usually the only option in end stage
disease. Common surgical procedures are osteotomy (joint realignment), arthroscopy
(removal of loose bodies and inflamed synovium, and smoothing irregular joint surfaces),
arthrodesis (joint fusion) and arthroplasty (joint replacement). Total joint replacement is
the recommended procedure for older patients experiencing significant pain and highly
impaired mobility, however, the life time of the joint may not be more than 10-15 years.
No permanent cure currently exists for OA.
1.3 Articular Cartilage
Articular cartilage is a thin, smooth and stiff layer covering bone surfaces in
synovial joints. It consists of an extensive hydrated extracellular matrix (ECM),
maintained by a population of specialized cells, the chondrocytes, which can occupy as
little as 2% of the total cartilage volume. It serves as a low-friction surface between the
bones of the diarthrodial joint, and is responsible for load support, load distribution and
joint motion. Cartilage is avascular, alymphatic and aneural, and the major source of
nutrition for chondrocytes in adult articular cartilage is through diffusion from the
synovial fluid
6
1.4 Cartilage Components
1.4.1 The Collagens
The major components of the ECM in cartilage are collagens and the large
aggrecating proteoglycan aggrecan. Collagen accounts for two-third of the dry weight of
cartilage, while collagen type II fibrils are responsible for the tensile strength of cartilage,
extensive cross linking of the collagen fibrils is responsible for the mechanical strength of
cartilage (for review
7).
Collagen fibers also help counteract the swelling caused due to
hydration of proteoglycans. Several different collagen types have been identified. In
articular cartilage, Collagen types II, IX and XI form a fibrillar network which constitutes
the structural framework of cartilage, in the form of an anisotropic and inhomogeneous
mesh of fibers. While collagen type II is the predominant fibrillar collagen, collagen IX
molecules have several cross-linking sites through which they interact with collagen type
II and other collagen type IX molecules. Collagen Type XI on the other hand is believed
to form a template which constrains the lateral growth of type II collagen heterofibrils 8
Two predominant classes of proteinases are known to degrade collagens, the matrix
metalloproteinases (MMPs) and the cathepsins, and cleavage of collagen by MMPs is an
important feature of OA 9. Collagen type II synthesis is much lower in adult tissue as
compared to immature cartilage; however, synthesis is seen to significantly increase after
injury 10
Cartilage can be divided into four different depth dependent zones, superficial or
tangential, intermediate, deep or radial and calcified, all of which have different
orientations of their collagen fibrils. The inherent inhomogeneous nature of collagen
fibrils along with depth dependent variation in their orientation is responsible for the
anisotropic and non linear properties of cartilage in compression and tension
1.4.2 Aggrecan
Proteoglycans are a family of molecules with a central core protein, to which one
or more glycosaminoglycan (GAG) chains are added post-translationally. GAG chains
are large extended structures with highly charged sulfate and carboxylate groups, which
dominate the properties of the proteins to which they are attached. Common GAG chains
include Keratan sulfate (KS) and Chondroitin sulfate (CS). (for review 12). Aggrecan is
the major proteoglycan in articular cartilage, where it associates with hyaluran and link
protein to form large aggregates. The large fixed negative charge of these aggregates (due
to the large number of GAG chains on aggrecan) causes an osmotic imbalance, and draws
water from the surrounding areas, thus keeping cartilage hydrated. This hydration of
cartilage by aggrecan influences nutrient and solute transport in cartilage. The high
negative charge of aggrecan aggregates is the main reason for the compressive stiffness
of cartilage (for review
13)
1.4.3 Other ECM Molecules
Besides the collagens and aggrecan, several other molecules are present in the
ECM, including thrombospondins, matrilins, cartilage intermediate layer proteins (CILP),
fibulins, laminins, SPARC, fibronectin and members of the Small leucine rich
proteins/proteoglycans (SLRP) family. Some of these proteins such as matrilins and
laminins have structural roles, while others such as some thrombospondins and SPARC
are involved in modulating cell function, tissue development and remodeling. The latter
set of proteins are termed as matricellular proteins
1
Thrombospondins (TSPs) are multidomain proteins containing either three (TSP
1,2) or five (TSP 3,4 and 5) identical subunits. The thrombospondins identified in
cartilage are TSP-1,3 and 5, of which TSP-5 (COMP) is dominant (for review
15).
TSP-1,
TSP-2 and TSP-5 (COMP) function at the interface of the ECM and cell surface, thus
regulating cellular behavior and hence can be said to be members of the class of
16,17
matricellular proteins 1,.
To accomplish their roles in ECM remodeling as matricellular
proteins, TSPs 1,2 and 5 interact with a wide variety of proteins and proteoglycans. TSP1 is the only thrombospondin which can activate TGF-, which leads to synthesis of
collagens and other molecules. It is known to bind to heparin, perlecan, syndecans-1,3
and 4, versican, Collagen type V, fibrinogen and decorin. It also inhibits several proteases
including MMPs and Cathepsin G
17.
Cutaneous wounds in TSP-1 null mice showed
delayed wound healing, reduced levels of active TGF-3, decreased collagen and reduced
inflammatory response 18.
TSP-3 is expressed in early proliferative zone and hypertrophic zone in cartilage.
Its function has not yet been elucidated, while single knockouts of TSPs does not affect
stature, mice that lack TSP-3,TSP-5 and Type IX collagen have 20% lesser limb length,
suggesting a role for TSP-3 in ECM function 119
COMP, made of five identical domains, is one of the major non collagenous
proteins composing cartilage ECM and comprises up to 1% cartilage wet weight.
Mutations in the COMP gene can cause inherited osteochondrodysplasias such as
seudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED)
been shown to bind to Collagen type I and II
interact with Type IX collagen
23
, fibronectin
2
24
and aggrecan
and matrilins
22.
25
20.
COMP has
It is also known to
. Its interactions with
several ECM structural molecules may serve to organize cartilage matrix and regulate the
load bearing function of cartilage. COMP interacts with BMP-2 to regulate mesenchymal
chondrogenesis in vitro
integrins
assembly.
27.
26.
It aids chondrocyte attachment through interaction with
Thus, it has roles in chondrocyte attachment, differentiation and ECM
SPARC is a matricellular protein expressed highly in developing tissue compared
to mature, and increased SPARC production is associated with adult tissue remodeling. It
can bind to several different collagens, including collagens I, III, IV and V, and is
possibly involved in ECM assembly and turnover
28
Perlecan is another molecule which plays an important role in the organization of
the ECM through its interaction with many ECM molecules including fibronectin,
nidogen-1 and nidogen-2, PRELP, collagen types XIII and XVIII (for review
29).
It also
plays a crucial role in cartilage development, possibly by interacting with and altering
distributions of several growth factors
30
A family of non collagenous ECM proteins which may be involved in structural
roles are the matrilins. There are four matrilins known currently, of which Matrilin-1 and
3 are almost exclusively present in cartilage (for review
31).
In the adult knee, all
matrilins are present in the growth plate and peripheral areas of articular cartilage.
Matrilin-1 binds to aggrecan and associates with collagen type II containing fibrils.
Matrilins -1, -3 and -4 form complexes with SLRPs biglycan and decorin
COMP
25
.
32
and bind to
Matrilin-3 additionally interacts with collagen type IX 33. Matrilin-3 mutations
have been associated with skeletal disorders such as Multiple epiphseal dysplasia (MED)
34
and bilateral hereditary micro-epiphseal dysplasia (BHMED)
35
Cartilage intermediate layer proteins 1 and 2 (CILP-1 and CILP-2) are two related
structural proteins found in articular cartilage. CILP-1 is a monomeric glycoprotein
predominantly expressed in the intermediate zone of cartilage
36.
CILP-1 forms a
functional feedback loop with TGF-, with TGF-p inducing CILP-1 mRNA expression,
and CILP-1 in turn binding TGF-
37.
CILP-2 is a protein which exhibits -50%
homology with CILP-1, and its function is not yet clear 38.
Another set of proteins involved in collagen fibril formation are members of the
Small leucine rich proteoglycan (SLRP) family (for review
39).
Studies by Vogel et al 40
were among the earliest to show that small dermatan sulfate proteoglycans inhibited
fibrillogenesis in both Type I and Type II collagen from tendon and cartilage respectively
in vitro. Since then, various SLRPs have been identified, including biglycan, decorin,
PRELP, chondroadherin, osteoadherin, fibromodulin and lumican, and probably have
unique functions in collagen assembly, as knockout of any SLRP cannot be compensated
by other SLRPs. SLRPs can also be categorized as matricellular proteins as different
members of this family interact with different molecules in the ECM, and thus help in
remodeling the ECM 41. Recent studies have identified that MMPs can cleave some
SLRPs at precise sites, and it has been postulated that the metabolites thus released may
serve as bio-markers to monitor the progression of OA
42
1.4.4 Matrix proteases
Degradation of the ECM by various proteolytic enzymes is a primary feature of
OA. These proteinases can be classified based on the chemical group which participates
in hydrolysis of the peptide bond. The primary extracellular proteinases are the
metalloproteinases and the serine proteinases, both of which act at neutral pH. The
metalloproteinases are distinguished by a highly conserved motif containing three
histidines that bind zinc at the catalytic site, and a conserved methionine turn beneath the
active site zinc. Metalloproteinases can further be divided into different groups based on
structure, of which MMPs and the ADAMTSs are the most active in the cartilage ECM
43
The Matrix metalloproteinases (MMPs) are a family of enzymes that digest
components of the ECM. Most MMPs have three domains, a prodomain which must be
removed to activate the enzyme, a Zn** containing active domain where the substrate is
catalyzed, and a hemopexin-like C-terminal domain which determines the substrate
specificity of the enzyme
44.
Members of the MMP family can be broadly divided into
four groups based on their substrates and cellular location: (i) The collagenases (MMP-1,
-8, -13) cleave major fibrillar triple helical collagens into characteristic %/ and
1/4
length
fragments. (ii) The gelatinases (MMP-2, -9) have high activity against gelatins (denatured
component chains of collagen) and Type IV collagen, the predominant constituent of
basement membrane. However, they have broad substrate specificities and can also
cleave fibrillar collagens and other ECM molecules such as fibronectin and decorin (iii)
The stromelysins (MMP-3,10,11) cleave a wide variety of ECM and non ECM substrates
including aggrecan, laminin, fibronectin, etc and (iv) The membrane type MMPs (for
review 9). It is highly likely that MMPs are responsible for digestion of collagens in vivo
in OA, as MMPs digest collagens in vitro, are expressed in OA tissue at the correct time
necessary to cause degradation, and activities altering expression of MMPs alter the
progression of OA
44.
However, MMPs may have more complex roles than pure
catabolism in normal and OA cartilage, as they are known to regulate matrix biology by
means of varied functions including release of growth factors, enhancing/inhibiting
angiogenesis and modification of the cell-ECM interface
45
A second important group of proteinases involved in cartilage remodeling are the
ADAMTSs (A disintegrin and metalloproteinase with thrombospondin repeats) (for
review
46).
ADAMTS proteins can be categorized into different subgroups based on
sequence similarities and substrates, if known. ADAMTS-1,4,5,8 and 15 form a subgroup
called "aggrecanases" as they all cleave aggrecan, though with different efficiencies.
Some aggrecanases can cleave other proteoglycans such as versican47 and brevican48 as
well. ADAMTS-4 and 5 are the predominant aggrecanases in human OA4 9 , these cleave
aggrecan at the Glu 373 -Ala 374 bond in the IGD region, and also at four other distinct sites
between the G2 and G3 domainssosi. Aggrecanases may also have roles besides aggrecan
degradation, with ADAMTS-1 being implicated in growth and development, organ
morphology and fertility
52
. While aggrecanase mediated degradation is probably more
destructive to cartilage than MMP mediated degradation
53
, cartilage may have the ability
to recover better from aggrecanase degradation than from MMP degradation 54
Another major group of ADAMTS proteins involved in cartilage remodeling are
the procollagen N-proteinases (ADAMTS-2,3,14) . As the name suggests, these are
involved in the removal of N-terminal propeptides of procollagen 55-57, and hence are
anabolic in nature.
Regulation of metalloproteinases can happen at different levels. MMP activity in
vivo can be controlled by regulation of gene expression, activation of the pro-enzyme
form and
inhibition of active enzymes by inhibitors 58. Tissue
inhibitor of
metalloproteinases (TIMP) refer to the family of proteins which inhibit active MMPs by
binding to them (for review
59).
Four TIMPs (TIMP 1-4) have currently been identified in
vertebrates. Mammalian TIMPs have two domains, a larger N-terminal domain and a
smaller C-terminal domain, each having three disulfide bonds. All active MMPs are
inhibited by TIMP, however, the four TIMPs differ in affinity for different MMPs.
TIMPs may also form non-inhibitory complexes with proMMPs, with TIMP-2 known to
form a tight complex with proMMP-2 to activate it
60
. ADAMTS proteins are also
inhibited by TIMPs. TIMP-3 seems to be the strongest inhibitor of the ADAMTS family,
inhibiting ADAMTS-4 and 5 with Ki values in the sub nanomolar range
shows some inhibitory activity against ADAMTS-4
ADAMTS-1
63.
62
61.
TIMP-1
and TIMP-2 and TIMP-3 against
TIMPs have also been shown to be involved in regulation of cell growth,
migration and apoptosis, either through inhibiting metalloproteinases, or by directly
binding to cell surface receptors
64
1.4.5 Other molecules
Several signaling molecules, such as annexins A2 and A5, proenkephalin,
pleiotropin and many others play important roles in tissue morphogenesis and
differentiation, and their expression levels are significantly altered in diseased state
65-67
1.5 Post-traumatic OA
OA is broadly classified into two forms, primary and secondary. Primary, or
idiopathic OA, is the common age-related onset of the disease, with no obvious cause. In
contrast, secondary OA has clear identifiable causes, such as trauma or a congenital or
developmental abnormality
6. Post-traumatic
OA, a type of secondary OA, refers to joint
degeneration, dysfunction and pain which develop after joint injuries. Unlike primary OA
which affects aged people, post traumatic OA affects younger individuals primarily, as
they are more prone to joint injury
68.
A study by Gelber et al 69, for example, found that
cumulative incidences of knee OA by 65 years was 13.9% for participants who had an
injury during adolescence and young adulthood, and 6% for those who did not. Joint
impact loading; tears in the meniscus, ligament and joint capsule, intraarticular fractures
and joint dislocations all increase the risk of posttraumatic OA
70.
In vivo joint injury
causes immediate mechanical damage to cartilage, followed by an acute inflammatory
phase which can last up to two months, and a chronic phase which eventually leads to
OA like symptoms 7 1
1.5.1 In Vitro Cartilage Injury Models
The stress distribution of articular cartilage in vivo depends on several factors,
including contact force, surface geometry, tissue properties, and cartilage thickness
72,
thus making it extremely difficult to quantify and study the effect of mechanical injury on
cartilage biochemical properties. In vitro models enable precise application of load, and
accurate measurements of the stress and strain profiles thus generated, which can then be
correlated with cellular and biochemical changes in cartilage. Usually, high stress/stress
rate mechanical compression is applied, and the resulting maximal strain measured or
vice versa. Also, mechanical compression injury can either be confined or unconfined.
The extent of damage to cartilage by blunt trauma in vitro depends on the magnitude of
stress applied 73 , the orientation of load relative to the articular surface 74 and the rate of
loading
7.
Injury immediately affects the collagen network, which in turn reduces the
tensile load carrying capacity of cartilage
76,
affects compressive and shear stiffness
77
and causes increase in matrix water content 73 and cartilage swelling because of reduced
ability of damaged collagen to withstand swelling pressure of the GAGs. Rapid GAG loss
to the media post injury occurs, possibly due to mechanical disruption of the ECM78
Kurz et al showed that injury lowers biosynthetic rates of chondrocytes immediately, and
biosythesis remains impaired even after 3 days post injury
'7.
Within 24 hours of a single
injurious compression, gene expression of MMP -1,-3,-9,-13; TIMP-1 and ADAMTS 4,5 increase significantly 79. Pooled media for 5 days post injury show that MMP -1,-3,13 are highly up regulated, and MMP-9,TIMP-1 and TIMP-2 mildly up regulated at the
protein level 80 . Injurious compression also causes chondrocyte cell death by a
combination of necrosis and apoptosis, with necrosis occurring earlier at the site of injury
and apoptosis responsible for subsequent spread of chondrocyte death beyond the initial
impact area 81,71 The extent of apoptosis is directly related to the load applied, and
apoptosis seems to occur at stresses lower than that required for matrix degradation
.
it
is however unclear if apoptosis drives matrix degradation or whether matrix degradation
causes apoptosis in vivo. Apoptosis of chondrocytes subsequently reduces the ability of
cartilage to repair itself and contributes to the evolution of an OA like phenotype.
1.6 Cytokines in OA
Cytokines such as IL-1$ and TNF-a are key mediators in the post traumatic
inflammatory phase. These cytokines are produced by the synovial membrane, diffuse
through the synovial fluid into cartilage, and activate chondrocytes, which in turn
produce pro-inflammatory cytokines and proteases
83.
Therefore, these cytokines act in an
autocrine and a paracrine fashion. IL-1p and TNF-a are present in OA cartilage, but not
in normal
84.
Also, both IL-1$ and TNF-a have been isolated from the synovial fluid of
patients undergoing ACL rupture, with the concentrations of IL-1
proportional to the severity of chondral damage
85,86
and TNF-a
1.6.1 IL-1p in cartilage damage
IL-16 is synthesized as a 31 kDa precursor, which is then cleaved to form the
active 17.5 kDa protein
87.
IL-1$ acts upon cells through specific cell surface receptors,
type I and type II, of which type I IL-1 receptor is elevated in OA cells, and is mainly
responsible for signal transduction by IL-16 in OA
87.
Direct injections or gene
transfection of IL-IRa, a soluble receptor antagonist of IL-1, reduce the progression of
experimental OA in animal models
88.
IL-1 promotes collagen degradation
89
by stimulating metalloproteases
90.
IL-iP
causes strong up regulation of MMP-1, MMP-3 and MMP-13 at both the mRNA 91 and
the protein levels 80 in vitro. It causes aggrecan degradation by stimulating aggrecanases
in vitro, but it is unclear whether ADAMTS-4 or ADAMTS-5 is the predominant
aggrecanase responsible for this. Species specific differences may be possible, with
ADAMTS-4 the predominant aggrecanase in cultures of human OA chondrocytes
stimulated by IL-la
92,
and ADAMTS-5 in murine cartilage explants stimulated by IL-la
. IL-1 also decreases proteoglycan and collagen synthesis in cartilage in vitro
4
Exposure of cartilage to IL-1f also affects physical properties of cartilage, causing an
increase in hydraulic permeability and decrease in streaming potential and equilibrium
modulus, possibly due to the degradative activities of MMPs and aggrecanases
96
1.6.2 TNF-a in cartilage damage
TNF-a is synthesized as a type II transmembrane protein arranged
as
homotrimers, which is then released by proteolytic cleavage to form the homotrimeric
soluble form. It activates cells through two kinds of receptors, TNF-R1 and TNF-R2, of
which TNF-R1 is the type present in several kinds of tissues including cartilage. TNF-R1
is responsive to both the soluble and membrane trimeric forms of TNF- 97 , and is over
expressed in OA cartilage on the surface of chondrocytes located near sites of high GAG
loss
98.
99,100,
Like IL-1P, TNF-a also causes aggrecan degradation by stimulating aggrecanases
and suppresses proteoglycan synthesis, though less efficiently than IL-1 01. TNF-a
also highly up regulates several MMPs including MMP-1, MMP-3, MMP-13 and MMP-9
80.
Additionally, TNF-a stimulates IL-6 production 101, which in combination with its
soluble receptor can contribute to further proteoglycan catabolism
102
1.6.3 Comparison between IL-1 and TNF-a in cartilage damage
TNF-a exerts effects similar to IL-1, and can also act synergistically to IL-1
103
however, animal models of RA indicate that TNF-a and IL-1p may have distinct
functions, with TNF-a important in initiating joint swelling, and IL-1
playing a role in
subsequent inflammation and cartilage erosion 104. Chondrocytes stimulated by IL-1
alone or IL-1 in combination with TNF-a produce inflammatory molecules such as
inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) and phospholipase
A2 (sPLA2). Molecules such as nitric oxide and E-series prostaglandins produced by the
activity of these enzymes have further anabolic/catabolic roles downstream 105
1.6.4 Anti-Cytokine Therapies for OA treatment
In spite of the fact that IL-1 and TNF-a seem to be key molecules involved in the
pathogenesis of OA, the inhibition of IL-1 and TNF-a using IL-iRa and anti-TNF alpha
therapies in OA patients is not very successful
106
, generating the need for further
investigation of the roles of these cytokines and better therapies.
1.7 In Vitro Models combining injury and cytokine
Therefore, to get a more realistic in vitro model of post traumatic OA, we need to
capture the effect of cytokines as well. Patwari et al used an in vitro model where
cartilage explants from newborn bovine and human knee tissue were subjected to a single
injurious compression followed by incubation with exogenous cytokines IL-la and TNFa for three days 107. They found that incubation of injured bovine cartilage with cytokines
increased sGAG loss to the media greatly, from a mean of -7% sGAG loss from injured
cartilage without exogenous cytokine, to a mean of 60% and 54% sGAG loss for injured
cartilage incubated with 10 ng/ml IL-la, and 100 ng/ml TNF-a respectively. Interaction
between injury and cytokine in both cases was highly significant, with P<0.001 by two
way anova. IL-la and TNF-a showed synergism with injury for human knee tissue
explants too, though the magnitude of synergism was far lower. Sui et al found that antiIL-6 Fab reduced the amount of sGAG loss caused by synergism between TNF-a and
injury in newborn bovine cartilage explants, but did not completely eliminate it,
suggesting that the synergism between TNF-a and injury is at least partly mediated by
IL-6 produced by chondrocytes 108. Further, addition of exogenous IL-6 along with its
soluble receptor (sIL6R) increased the synergism between exogenous TNF-U and
mechanical injury in newborn bovine and in human knee and ankle cartilage explants.
1.8 Thesis Objectives
Injury and cytokines both play a significant role in the progression of post
traumatic OA, and interplay between these two factors possibly drives OA progression in
vivo. Therefore, an in vitro model system in which injured cartilage explants are
incubated in IL-16 or TNF-a is used to try to elucidate the mechanism by which injury
interacts with cytokines to drive OA. A proteomic profile of media released from
cartilage under different treatment conditions (injury, cytokine and combination of injury
and cytokine) is generated using a quantitative iTRAQ 2D-LC-MS-MS approach. The
levels of several different proteins under different treatment conditions are compared, and
proteins on which injury and cytokine act synergistically, in a positive or a negative
fashion are identified. This approach thus helps understand the mechanism by which
injury and cytokine interact with each other in driving the pathogenesis of OA.
The next part of our study uses a partial least square regression (PLSR) approach
to relate protein ratios across different treatment conditions with measured phenotypes
across the treatment conditions. This approach removes redundant data, and determines
which proteins contribute most to specific observable phenotypes. Using this approach,
we are able to see hidden associations between proteins, between phenotypes and
between proteins and phenotypes.
Thus, using a simplistic in vitro model, the overall goal of this project is to
determine molecules which may be important to OA disease progression, and may serve
as biomarkers or possible therapeutic targets for OA.
METHODS
2.1 Overview
Equal volumes of cartilage were treated in equal medium volumes with one of the
following treatments (i) 10 ng/ml IL-iI3 (ii) 100 ng/ml TNF-a (iii) unconfined mechanical
compression to 50% strain at 100%/sec followed by incubation with 10 ng/ml IL-l $3(iv)
unconfined mechanical compression to 50% strain at 100%/sec followed by incubation
with 100 ng/ml TNF-a. Medium was pooled, deglycosylated by chondroitinase ABC and
digested with trypsin. A 4-plex iTRAQ labeling strategy was used, with sample from
each treatment condition labeled with one of the four iTRAQ labels as follows: IL-1
iTRAQ 114; TNF-a : iTRAQ 115; IL-1f
:
+injury : iTRAQ 116 and TNF-a +injury :
iTRAQ 117. The samples were then combined, fractioned by SCX chromatography,
subjected to RP-LC/MS/MS and analyzed using ProteinPilot TM (ABI). Matlab scripts
were used to generate protein ratios from the peptide summary reports of protein pilot.
These data were subjected to subsequent analysis. To study the effect of biological
variance, all steps are performed on biological replicates, replicate 1 consisting of
cartilage from a set of three animals, and replicate 2 from a different set of three animals.
2.2 Cartilage Explantation, Mechanical Injury and Cytokine Treatment
Cartilage explants were obtained from the femoropatellar groves of 2-3 week old
calves as described previously 109. Briefly, cartilage was cored and punched to obtain
disks (1 mm thick X 3 mm diameter). Between 8-12 disks (explants) from each of 3
animals were used for each treatment condition for biological replicate 1, and the same
procedure followed for biological replicate 2 with 3 different animals. These disks were
equilibrated for 5 days prior to start of treatment in 1% ITS (high glucose (25 mM))
DMEM medium supplemented with 10 mM HEPES, 0.1 mM non essential amino acids,
115 uM ascorbic acid, 400 uM L-proline, 100 U/ml penicillin, 100 ug/ml streptomycin,
0.25 ug/ml amphotericin B powder (PSA for tissue culture) and 1 mM sodium pyruvate.
On treatment day, location matched explants were placed in four different groups and
cultured in 2 ml medium without ITS supplementation.
Cartilage explants meant to be treated with cytokines alone were placed in
medium without ITS containing 10 ng/ml IL-1p or 100 ng/ml TNF-a. The explants were
then cultured for 120 hours with 10% medium removal and supplementation every 24
hours. Cartilage explants meant to be treated with cytokine+injury were individually
injured first by mechanical compression in a custom designed, incubator housed
apparatus as described previously
110,79,108
. Explants were subjected to a single
unconfined compression at a strain of 50% and strain rate of 100%.second. After injury,
they were placed in medium without ITS containing 10 ng/ml IL-1IP or 100 ng/ml TNF-a,
and cultured for 120 hours, with medium removal and supplementation as before. After
treatment, explants and cartilage were stored at -80 C
2.3 sGAG Removal, Trypsin digestion and iTRAQ labeling
3 ml of the medium from each condition (for biological replicates 1 and 2 each)
was supplemented with 5 mM EDTA, 100 ug/ml PMSF and 5 mg/ml iodoacetamide. The
samples were dialyzed for 3 hours against a buffer with 10 mM Tris acetate, 40 mM
NaCl and 5 mM EDTA, mixed with 70 milliunits of Chondroitinase ABC (the
deglycosylation agent and the internal standard) and left to dialyze and deglycosylate
overnight at 37 C. The samples were further dialyzed against 2 mM TEAB and 0.5 mM
EDTA for 8 hours, and then against 500 mM TEAB. The samples were then frozen,
concentrated 10 fold, and then further concentrated to a final volume of 150 ul. Samples
were then reduced by 2 mM TCEP (in 100 mM TEAB) for 2 hours, and alkylated by 5
mM iodoacetamide (in 100 mM TEAB) for 2 hours. The samples are then precipitated by
6 volumes of ice cold acetone and then centrifuged at 15000 x g for 30 min at 4C. The
pellets were re-suspended in 25 ul of 50 mM TEAB containing 0.1% SDS. Trypsin (0.1
mg/ml in 50 mM TEAB) was added to each sample in the ratio 1:37.5. Sample volume
was increased by addition of water and acetonitrile to 40 ul, following which samples
were incubated at 37 C overnight, speedvae'd to dryness, and resuspended in 500 mM
TEAB.
The peptides were then iTRAQ labeled according to manufacturer's instructions.
Briefly, iTRAQ labels were equilibrated at room temperature, diluted with ethanol,
vortexed and centrifuged at 16000 x g for 1 minute. The iTRAQ reagent was then added
to 100 ug peptides from each sample. The samples were then vortexed for 1 minute,
sonicated for 5 minutes, centrifuged at 16000 x g for 1 min, and equilibrated at room
temperature for 55 minutes. Following this, samples were diluted to 200 pl total volume
with water and combined in equal proportion according to volume equivalents. The
combined sample volume was reduced to 50 ptl, further combined with 1% phosphoric
acid and 1 ml of strong cation exchange buffer A (10 mM Potassium phosphate pH 2.8,
25% acetonitrile), made to pH 3.5 and diluted with 3 ml SCX buffer A.
2.4 SCX Chromatography and LC/MS/MS
The combined labeled sample was injected onto a 2.1 mm X 100 mm strong
cation exchange column on an Agilent 1100 HPLC at a flow-rate of 250 ul/min; with
100% Buffer A for 20 minutes, 0 to 40% gradient SCX buffer B (10 mM phosphate
buffer pH 2.8, 400 mM KCl, 25% acetonitrile) for the next 40 minutes, 40 to 90%
gradient SCX buffer B for the next 5 minutes and finally 95% buffer B for the next 5
minutes before returning to starting conditions (Total run time ~ 80 minutes). Peptide
elution was monitored by UV absorption at 214 nm. Fractions were collected every 0.5
minutes and stored at -80 C. Fractions were then concentrated by speedvac and desalted
by zip tip. Desalted factions were loaded onto a 75 um x 160 mm column with 10 um tip
packed in house with Vydac protein/peptide C18 packing material. Peptides were loaded
at flow-rate 250 nl/min at 2% buffer B (1.2% acetic acid in 90% acetonitrile) for 12.5
minutes, eluted with 2 to 40% buffer B gradient for 147.5 minutes and washed out with
40 to 60% buffer B gradient for 20 minutes. This was followed by holding at 60% B for 5
minutes, returning to 2% B over 15 minutes and equilibrating at 2% B for 60 minutes.
The Buffer A is 1.2% acetic acid in water. Following LC, mass spectrometry was
performed using QStar, a Quadrupole time of flight mass spectrometer (ABI) with a
nanospray source. Precursor ions between m/z 400 and 1600 with a charge state between
two and four were selected with Analyst 1.1. Each cycle (10 sec) consisted of an MS scan
followed by 3 data dependent MS/MS scans (m/z 100-1600), with ions excluded for 100
seconds after a single spectra. All fractions from the first replicate were run first, before
running samples from the second replicate.
2.5 Data Analysis
Data were analyzed using the Paragon
and Pro Group algorithms of
ProteinPilot TM Software 2.0.1 (ABI). The variable modifications allowed were NH 2
terminal iTRAQ labeling, iTRAQ labeled lysine, iTRAQ labeled tyrosine and oxidized
methionine. The number of missed trypsin cleavages was restricted to 1. The search was
conducted against the NCBInr bovine database assembled in June 2008.
The peptide summary report generated is then exported into excel, and scripts are
written using Matlab (Mathworks) to calculate mean protein ratios, standard deviation
and standard error, and p values for the null hypothesis that the protein ratios are different
from 1. The Matlab script also makes sure that only peptides which are identified by
Protein Pilot with confidence greater than 90%, which are quantified well for both labels
in the ratio under consideration (i.e. the ratios generated from protein pilot are not 0 or
9999), and whose spectra are manually verified are chosen for calculating protein
information. All proteins identified by less than two peptides satisfying all the above
criteria are discarded. All mathematical operations for calculating mean, standard
deviation, standard error, p value, etc are done using log transformed ratios to make sure
that ratios lesser than and greater than one are treated identically. The means and standard
deviation are calculated using (
1
%Error
) listed by protein pilot as weights. The weighted
mean, the weighted standard deviation and the weighted standard error in the log space
are calculated using the following formulas
N
__
_Wii___
X=
SE =
Sd
Sd_
NN
(N -1)w
Wi
N
Where
X
w is the weighted mean of the log transformed peptide ratios Xi , Wg is the
weight of the peptide i (calculated as
1
%Error
), N refers to the number of well quantified
manually verified peptides identified with confidence > 90%,
standard deviation, and
SEW is the weighted standard
Sdw
is the weighted
error.
Normalization of the protein ratios thus obtained was done using the weighted
mean of chondroitinase ABC, the enzyme used for glycosylation. This protein is a non
mammalian protein and can thus serve as an internal standard
80.
Also, the ratio of this
protein is almost 1 in all cases, thus illustrating that the levels of this protein do not get
affected by treatment, as expected. To obtain the p values for the null hypothesis that log
transformed protein ratios were different from zero, the normality of log transformed
peptide ratios was first checked using Lilliefors test for normality in MATLAB, if the log
transformed ratios were normal, a paired t test was used, if not, the Wilcoxon sign rank
test was used. To correct for multiple comparisons, the Benjamini Hochberg procedure
m was used and false discovery rate (FDR) controlled at 0.1. The Benjamini Hochberg
procedure was performed for each ratio (i.e. 115:114, 116:114 and 117:114) separately.
2.6
Comparison of biological
duplicates
and final protein list
compilation
To test whether there exists significant variation between biological replicates, for
proteins identified in common between biological replicate 1 and 2, histograms of the
difference between log transformed ratios from biological replicates 1 and 2 are plotted,
and the mean and standard deviation of this difference computed, for each ratio.
A final protein list is then compiled for use in further analysis. To do so, a list of
proteins which satisfy Benjamini Hochberg procedure across all three ratios from the first
biological replicate is selected. This is supplemented with proteins identified from the
second biological replicate alone which satisfy Benjamini Hochberg procedure across all
three ratios, after correcting for any major inter experimental bias. This correction is done
using the difference between the means of log transformed ratios from replicates 1 and 2
obtained by comparing the biological replicates in the previous step. A total of 199
proteins are thus chosen for the final compiled list, for each of which we have ratios for
three conditions, TNF-ax:IL-1p, IL-1f +injury:IL-1
and TNF-a +injury:IL-1p.
2.7 Global Proteomic Analysis and Clustering
A correlation matrix is generated using MATLAB between different ratios (TNFa:IL-1p, [IL-1f+inj]:IL-1p and [TNF-u+inj]:IL-1p) using the compiled protein list. Kmeans clustering analysis was done using the statistics toolbox in MATLAB on the
compiled protein data set. This methodology partitions the 199 proteins into k clusters, by
minimizing the sum of distances between each protein and the centroid of the cluster the
protein is partitioned into. The number of clusters is chosen such that the mean of the
silhouette values is the maximum. The Mann Whitney U test or the Unpaired t test is
used to check for the uniqueness of each cluster.
2.8 Merging data between two iTRAQ experiments
We seek to combine data from two iTRAQ experiments, an earlier experiment
80
and this experiment. The earlier experiment has ratios (IL-1IP:untreated), (TNFa:untreated) and (injury:untreated), while the current experiment has ratios (TNF-a:IL1p), (IL-1p +injury:IL-1p) and (TNF-a +injury:IL-1p). By merging data from both
experiments, we hope to obtain data for all possible ratio combinations with respect to
untreated
control
(i.e.
IL- 13 :untreated,
TNF-a:untreated,
Injury:untreated,
IL-l$
+injury:untreated and TNF-a +injury:untreated). Before doing so, we need to compare
data for proteins found in common between these two experiments to ensure that there is
no significant inter experimental error. The comparison is done only between proteins
which satisfy Benjamini Hochberg procedure across all ratios at FDR 0.1 in both
experiments. 74 proteins are thus found in common between both these experiments, a
histogram of the difference between the log transformed (TNF-a /IL- 10) ratios from both
these experiments is plotted, and the mean and standard deviation of this difference
computed.
If there is no significant inter experimental error, we merge data from both these
experiments to obtain data for all five treatment conditions for 74 proteins. A correlation
matrix is generated using Matlab to ascertain how these five conditions (IL-1p, TNF-a,
injury, IL- 11 +injury and TNF-a +injury) correlate to each other.
2.9 Finding proteins on which injury and cytokines act synergistically
We want to identify proteins on which injury and cytokine act synergistically in
either a positive or a negative fashion. To do so, if the ratio of (cytokine:untreated) were
RI, the ratio of (injury:untreated) R2 and (cytokine+injury:untreated) R3 for a given
protein, if all the three conditions R3>R1, R3>R2 and R3>R1*R2 are satisfied for any
protein, injury and cytokine are determined to exert positive feedback on that protein. On
the other hand, if the three conditions R3<R1, R3<R2 and R3<R1*R2 are satisfied for
any protein, injury and cytokine are determined to exert negative feedback on that
protein.
2.10 Partial Least Square Regression (PLSR)
We want to find out which protein ratios contribute most to observed phenotypes.
We take the compiled protein list consisting of log transformed protein ratios for 74
proteins across 5 conditions, and eliminate two proteins because they act as outliers and
add noise to the PLSR model. We thus have an independent 72*5 data matrix (X)
consisting of log transformed protein ratios across five treatment conditions. For the
dependent Y matrix, we choose phenotypes for which data are already available for all
the five treatment conditions. The phenotypes thus chosen are amount of sGAG loss to
the medium during the 5 day treatment regime, amount of nitrite accumulation in medium
during the 5 day treatment regime, proline incorporation rate on the
treatment and sulfate incorporation rate on the
6 th
6 th
day post start of
day post start of treatment 11
We thus create a (4*5) matrix Y, with each element of the matrix
= log -Ilj , where
P is the value of the phenotype i on receiving treatment
j,
and Pe is the value of the
phenotype i on receiving no treatment. (For example, Pj may be sGAG released to the
medium on treatment with TNF-a, with PI, the sGAG released to the medium on absence
of treatment)
PLSR
114
is used to find a relation between Y and X, by choosing dimensions
(latent vectors) such that superfluous information in X and Y is eliminated, and the
maximum possible co-variation between X and Y is captured in the first few latent
vectors. Both the X and Y matrices are mean centered and set to unit variance along the
columns (i.e. proteins and phenotypes respectively) before performing PLSR.
PLSR is performed using the NIPALS algorithm
115
with The Unscrambler @ X Version
10.0.1 (Camo) software. The PLSR model is trained and validated by cross-validation,
where portions are omitted from the input matrix in turn, omitted data predicted from the
remaining data in the matrix, and predicted data compared to actual omitted data. Once
the latent vectors are determined, the proteins, phenotypes and treatments are projected
on the first few latent vectors, this enables one to reduce the dimensionality of data and
see relationships between proteins, phenotypes and treatments which were previously
hidden.
RESULTS
3.1 Reproducibility between biological replicates
We want to check whether the methodology and our data are sufficiently robust as
to show reproducibility between biological duplicates (Unlike technical replicates which
are designed to show variability in the process, biological replicates capture variability in
both the target population and the process) . For this, only protein ratios whose p values
satisfy Benjamini Hochberg procedure at FDR 0.1 are chosen, for each ratio separately.
Proteins identified in common between the two biological replicates are compared
by dividing the geometric mean of the peptide ratios for any protein in the first biological
replicate (ratiol) by the geometric mean of the peptide ratios for the same protein in the
second biological replicate (ratio2). These ratios of ratios (ratiol/ratio2) are log
transformed and plotted as histograms for each treatment condition (Figure 1) We expect
to see a mean of zero and negligible standard deviation for these histograms if biological
replicates were perfectly reproducible. We note that the mean +/- standard deviation is
0.1010 +/- 0.3015 for log 2 (ratiol 115:114/ratio2 115:114), 0.0807 +/- 0.3134 for
log 2(ratiol 116:114/ratio2 116:114), and 0.4317 +/- 0.4292 for log2(ratio1
117:114/ratio2 117:114). The huge error (43.17%) between 117:114 ratios is probably
due to incomplete protein digestion in the sample from biological duplicate 2 containing
the 117.1 m/z label (a white precipitate was seen in this particular sample). To correct for
this error, 0.4317 was added to log 2(ratio2 117:114) for all protein readings obtained
from the second biological replicate. The % variation observed in the other two cases
(10.1% and 8.07%) is well within the 25% variation found between biological replicates
and the 23% variation between experimental replicates (variation due to different iTRAQ
experiments) in the study by Gan et al 116
3.2 Global proteomic analysis
A total of 199 proteins identified by either iTRAQI or iTRAQ2 or both and which
satisfies Benjamini Hochberg procedure at FDR 0.1 across all ratios were used to perform
a systems level analysis of the effect of the combination of injury and cytokine on the
cartilage sample. A matrix of correlation coefficients between the log transformed protein
ratios is shown in Table 1. (All ratios are expressed with respect to IL-1P because a
4-plex iTRAQ analysis (using 4 labels) were chosen). A high correlation of ~ 91.4% was
seen between the two (Cytokine+Injury) combination conditions indicating either that the
proteins were affected similarly by the two cytokines in combination with injury or that
the effect of injury was much higher than the effect of cytokines , thus dominating the
overall (Cytokine+Injury) response. This high correlation between the two
(Injury+Cytokine) conditions is verified by a plot between log2(IL-1p +inj/IL-1p) and
log 2 (TNF-a+inj/IL- 1p ) (Figure 2) , which shows a linear relation with R2 equaling
0.8353.
3.2.1 K-means clustering analysis
K-means clustering was used to cluster the proteins into groups which would
enable better understanding of how groups of proteins varied with treatment. Silhouette
plots were used to determine that 3 clusters yielded optimum inter-cluster variation as
compared to intra-cluster variation. Figures 3a-d represent the proteins in 3D space and
their projections on 2D planes. The proteins are determined to be well separated into
three clusters by means of unpaired t test (All p values are << 0.001). Figure 4 shows
how the centroid of each cluster varies with treatment. Broadly, cluster 2 proteins are
depressed by all conditions wrt IL- 13, and additionally depressed in the (TNF-a +injury)
condition. Cluster 1 proteins are almost unchanged with all treatments, showing a mild
increase on treatment with injury and cytokine. Cluster 3 proteins are in general elevated,
and show increased elevation on treatment with injury and cytokine.
3.2.2 Effect of Cytokine+injury on proteins based on cell location
Proteins are sorted by location and average ratios are found out for all proteins
belonging to a particular cellular location. We observe that proteins inside the cell
(Including the cytosol, ER lumen and nucleus) all show highly elevated levels in response
to both (TNF-a +injury) and (IL-1P +injury) (p<0.05) (Figure 5). ECM proteins and
membrane protein levels remain almost the same on treatment with (cytokine+injury).
Secreted proteins are down regulated to a greater extent with TNF-a alone than with
(cytokine+injury).
3.3 Effect of combination of cytokine and injury on specific proteins
In cartilage, a few collagens are synthesized in a procollagen form and require
processing by removal of N- and C-terminal propeptides to form mature collagen. The
release of C-terminal propeptides is hence an indicator of collagen synthesis. From
Figure 6a, we see that C-terminal propeptide levels of many collagen chains are mildly
to moderately down regulated with IL-1 +injury, and moderately to highly down
regulated with TNF-a +injury, indicating reduced synthesis. When we examine other
major structural proteins found in the ECM, we find that fibronectin is highly down
regulated with TNF-a treatment alone or (TNF-ct +injury) and COMP is highly down
regulated with (TNF-a + injury) (Figure 6b). All the Matrilins are down regulated with
TNF-a or (IL-1
p + injury), but elevated with (TNF-a
+ injury), with Matrilin-3 showing
the most significant down regulation (Figure 6b). Aggrecan shows marginal down
regulation with all three treatments with respect to IL- 1P. Members of the Small leucine
rich proteoglycan (SLRP) family respond significantly to the combination of treatment
and injury, with chondroadherin, biglycan, lumican and PRELP all showing significant
up regulation with (TNF-a + injury) (Figure 6c). Biglycan also shows up regulation with
(IL-IP + injury).
We next look at proteases which are known to be important in ECM degradation
(Figure 6d). Both MMP 2 and MMP3 are highly suppressed with TNF-a compared to
IL- 1p, but only very mildly so on treatment with a combination of cytokine and injury
MMP 13 is down regulated with (IL-1P + injury) treatment. ADAMTS-1 is the only
ADAMTS protein found in our study, it is up regulated with (IL- 13 + injury) and more
significantly with (TNF-a + injury). The metalloproteinase inhibitor TIMP-2 is
marginally up regulated on treatment with IL-1I and injury, but not so with TNF-a and
injury.
The next figure (Figure 6e) shows the effect of the combination of cytokine and
injury on members of the TGF- super family and their inhibitors. TGF- family
members are known to be anabolic and to stimulate proteoglycan synthesis by
chondrocytes. While TGF-p2 itself is up regulated significantly with (Cytokine + injury)
for either cytokine and especially for (TNF-a + injury), inhibin PA shows extremely high
elevation with (Cytokine + injury) treatment for both IL-1
and TNF-a. Among the
LTBP's , proteins known to help in TGF-p secretion, LTBP1 and LTBP2 are both down
regulated with all three treatments, LTBP1 down regulated more with (TNF-a + injury)
and (IL-1I3 +injury) than with TNF-a; and LTBP2 more with TNF-a. Connective Tissue
Growth Factor (CTGF) is significantly down regulated with (TNF-a +injury) , while (IL1f +injury) does not seem to have much effect. TGF-p inhibitor vasorin is almost
unchanged with all treatments, while BMP inhibitor chordin-like 2 is highly down
regulated with cytokine+injury.
IGF is also an important anabolic agent. IGF binding proteins (IGFBP) show
varied response to (Cytokine+Injury) (Figure 6f). While IGFBP7 is down regulated on
treatment with both (IL-1 B3
+injury) and (TNF-a +injury), IGFBP5 is up regulated with
(TNF-a +injury) but not with (IL-1 +injury). IGFBP3 is elevated with (IL-1 P +injury)
and (TNF-a +injury), and IGFBP6 is marginally down regulated with (IL-I P +injury) and
(TNF-a +injury)
Figure 6g looks at proteins involved in the innate immune response. Complement
factors C3, Cis, Clq and C1r all show mild down regulation in the presence of (IL- 1
+injury). C3 and Cir show significant down regulation in the presence of (TNF-a
+injury), while Clq is mildly up regulated. C1 inhibitor and PGLYRP2 show significant
down regulation with (IL-1p +injury), but remain almost the same with (TNF-a +injury).
Lactotransferrin shows mild down regulation with all treatments, while
Lipopolysaccharide binding protein (LBP) is highly down regulated with TNF-a alone,
and moderately with (IL-1p +injury) and (TNF-a +injury). CD14 shows significant down
regulation with (IL- 1P +injury), and mild down regulation with (TNF-a +injury)
We next look at some proteins involved in signaling including cytokines (Figure
6h). Proenkephalin shows the highest response to the combination of injury and cytokine,
with eight and thirteen fold elevation to (IL-1f +inj) and (TNF-a +inj) respectively.
Calcium binding proteins annexin A2 and annexin A5 are also elevated significantly by
injury and cytokine. Angiopoietin-like 7, on the other hand, is depressed by both the
injury+cytokine conditions. Pleiotropin shows mild elevation with (IL-I
P +inj)
and
(TNF-a +inj) treatments. Granulin shows a more than two fold depression with (TNF-a
+inj) alone. The membrane protein CD44 is significantly depressed by (IL-1p +inj), and
mildly by (TNF-a +inj).
3.4 Effect of combination of injury and cytokine as opposed to either
treatment alone
Data from a previous iTRAQ experiment in our lab 80 are used in combination
with data from this experiment to generate all the information necessary to perform this
study. To combine data from both the experiments, we first verify that there is no
significant experimental error/bias between both these experiments.
3.4.1 Comparison between two iTRAQ experiments
We want to check whether there are no major sources of variation between the
results of both these iTRAQ experiments. As earlier, this is done by first identifying
proteins found in common between experiment A (the current experiment) and
experiment B
8.
(The final data set used for comparison from experiment A is the set of
199 proteins identified by merging proteins identified by either or both of the biological
duplicates and which satisfy Benjamini Hochberg procedure at FDR 0.1; similarly, the
final data set used from experiment B consists of 136 proteins found in either or both
technical duplicates combined which satisfy Benjamini Hochberg procedure across all
ratios at FDR 0.1). 74 proteins are thus found in common between Experiments A and B.
We compare reproducibility between exp A and B by taking the ratio of (TNF-c /IL-1 )
ratios for proteins in both experiments ((TNF-a /IL- 1P) is the only ratio in common
between experiment A and experiment B). This ratio of (TNF-a /IL- 1P) ratios is log
transformed and a histogram is plotted (Figure 8). The mean+/- standard deviation is
seen to be 0.1067 +/- 0.2191. There thus seems to be good reproducibility between
experiments A and B with variation of around 10.7%.
3.4.2 Correlation coefficient matrix between different conditions
Since variance between experiment A and B is around 10.7% only, the data for
the 74 proteins identified in common between both these experiments are fused.
Experiment A data which are ratios wrt IL-iI are converted to ratios wrt untreated
control by multiplying with (IL- 1P:untreated) data from experiment B. Thus, all possible
ratios i.e. (IL- I :untreated), (TNF-a:untreated), (injury:untreated),(IL- 13
+injury):untreated and (TNF-a +injury):untreated are obtained for these 74 proteins. A
matrix of correlation coefficients is generated from log transformed ratios (Table 2).
Around 90.3% correlation between TNF-a and IL-1$, and around 95.3% correlation
between (TNF-a +injury) and (IL- 1P +injury). There is very little correlation between
injury and cytokine treatment, and both injury and cytokine show almost equal
correlations with (cytokine+injury). Curiously, TNF-a shows greater correlation with (IL1P +injury) than with (TNF-a +injury).
3.4.3 Proteins on which injury and cytokine act independently
If the effect of injury and cytokine were almost independent on a protein, we
would expect that the effect of the combination of injury and cytokine would be a product
of the effect of injury and the effect of cytokine. Therefore, if the ratio of
(cytokine:untreated) were R1 and the ratio of (injury:untreated) were R2 for a given
protein, if cytokine and injury acted independently, the ratio of
(cytokine+injury:untreated) (R3) would be R1*R2. We therefore check for all proteins
for which
log 2(R1*R2) -0.1 <= log 2(R3) <= log 2(R1*R2) + 0.1
The addition and subtraction of 0.1 is to specify error range.
Under this criteria, we observe that IL-13 and injury act almost independently on
aggrecan, CILP-2 and SPARC. TNF-ax and injury act almost independently on Collagen
VI alpha II, MMP-2 and C-type lectin domain family 3 member A.
3.4.4 Positive and negative feedback by cytokine and injury
If the effect of combination of cytokine and injury is greater than that of cytokine
alone, injury alone and the effect obtained if cytokine and injury acted independently,
cytokine and injury positively reinforce each other, in other words, exert a positive
feedback on the protein. Similarly, if effect of combination of cytokine and injury is
lesser than that of cytokine alone, injury alone and the effect obtained if cytokine and
injury acted independently, cytokine and injury exert a negative feedback.
To formulate the above statements as equations, if the ratio of
(cytokine:untreated) were R1, the ratio of (injury:untreated) R2 and
(cytokine+injury:untreated) R3 for a given protein,
If R3 > R1, R3 > R2 and R3 > R1*R2, a positive feedback is seen.
If R3 < R1, R3 < R2 and R3 < R1*R2, a negative feedback is seen.
Under these criteria, TNF-a and injury are seen to show positive feedback for aggrecan,
collagen VI alpha III, histone H4 and CILP-2. TNF-a and injury show negative feedback
for SPARC and IGFBP-7.We do not see any protein for which IL-l $3and injury show
positive or negative feedback.
3.5 Partial least square regression (PLSR) between protein ratios and
phenotypes
We want to find out how protein ratio under different treatment conditions (the
independent variables) causes changes in various observed phenotypes under the same
treatment conditions (the dependent variables). The phenotypes available to us for the
treatment conditions IL-iI3, TNF-a, injury, IL-1P +injury and TNF-a +injury are: amount
of sGAG released into medium at the end of five days post treatment, amount of nitrite
accumulation at the end of five days, and rate of proline and sulfate incorporation
measured on the sixth day (Table 5). We thus want to find out a simple relation between
the protein ratio matrix (a matrix consisting of log transformed protein ratios as rows and
treatment conditions as columns) and the phenotype matrix (a matrix consisting of log
transformed phenotype ratios wrt control as rows and treatment conditions as columns).
Since there exists a lot of redundancy in the protein ratio matrix, we perform PLSR in
order to find a set of components (called latent vectors) which explain as much as
possible of the co-variation between the protein ratio matrix and the phenotype matrix.
First, from Figures 8a and 8b, we see that two latent vectors (factors) seem to
explain most of the variation in both X (the protein matrix) and Y (the phenotype matrix).
Therefore, a projection of the treatment conditions on the first two latent vectors is shown
in Figure 8c. From this, we see that both the cytokine only conditions are relatively
close, both the (injury+cytokine) combination are also close to each other, whereas the
injury alone condition lies away from the other conditions. This tallies with the
correlation matrix in Table 2. Figure 8d shows the projection of the phenotypes and
proteins on the first two latent vectors. Proline and sulfate are seen to project nearby,
corresponding to the fact that protein and proteoglycan synthesis are related.
Complements C3,B and C1r cluster together in the second quadrant. Cytoskeletal
proteins such as beta actin, vimentin, actinin alpha 4, alpha actinin and fascin are all
found close together in the fourth quadrant. Both the histones H2B and H4 are in the third
quadrant. A matrix containing the correlation shown by each protein with each phenotype
is generated.
In Table 4, the protein-phenotype pair showing the strongest positive and
negative correlations are listed. Perlecan-sGAG, pleotropin-sGAG, serum amyloid A3sGAG, CILP2-sGAG and complement B-sGAG are the pairs showing the highest
positive correlation. The pairs which show the highest negative correlation are Perlecansulfate, perlecan-proline, pleiotropin-sulphate, pleiotropin-proline and cathepsin BsGAG.
Figure 1: Comparison of the first and second biological replicates. Proteins are identified
in common between the first and second biological replicates and their mean ratios after
internal standard correction compared. Only ratios whose respective p values satisfy
Benjamini Hochberg procedure at FDR 0.1 are chosen for the respective plot. Histograms
of log 2 transformed ratio of ratios (i.e. log2(ratiol/ratio2) ) are plotted for each of the
ratios 115:114 (A), 116:114 (B) and 117:114 (C). The Mean +/- standard deviation is
0.1010 +/- 0.3015, 0.0807 +/- 0.3134 and 0.4317 +/- 0.4292 for A, B and C respectively.
403530
252015
10-
2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
01
-2
-1.5
Lom(ratio1 115:114 /ratio2 115:114)
-2
-15
-u.5
U
U.0
I
1.b
1
Log(ratio1 117:114 / ratio2 117:114)
-1
-0.5
0
0.5
1
1.5
Lo&(ratiol 116:114 /ratio2 116:114)
2
2.5
2
2.5
Table 1: Matrix of correlation coefficients. Correlation coefficients are found between
TNF-a only, combination of IL- 1s and injury and combination of TNF-a and injury
treatments. Log transformed protein ratios obtained from iTRAQ experiments for the
corresponding treatments are used as vectors to calculate these coefficients. Only
proteins, the p values of all whose ratios satisfy Benjamini Hochberg procedure at FDR
0.1 are chosen.
log 2(TNF-a/L-1
log 2 (TNF-a/IL-1 P)
log 2 (IL-1 p+inj/IL-1 p)
1og 2 (TNF- a+iinj/IL-1p)
log 2(TNF-a+inj/L-1 p)
Iog 2(IL-1 P+inj/L-1 p)
0.6957
0.6786
1
0.9139
1
0.6786
1
0.9139
0.6957
p)
.........
Figure 2: Expresses the relation between effect of cytokines IL-1$ and TNF-a in
combination with injury on proteins. Log 2(IL- 1p +Injury/IL- 1p) (x axis) is plotted against
Log 2 (TNF-a +injury/IL-1 [) (y axis). Figure 2 has been fitted to a line with a slope of
1.0729, y-intercept of 0.1353 and an R2 value of 0.8353. This indicates a highly
correlated relation between the two (Injury+Cytokine) conditions in their effect on
protein expression.
4
-2-
LL
z
..
I-
-2
0
log2( IL- 1 A4i nj/IL- 10)
.. . .....
....................
..
..
....
.....
....
....
Figure 3: Figure 3A is a plot of proteins in three dimensions with log transformed ratios
as axes, the x, y and z axis representing log 2(TNF-a /IL- 1$), log2(IL- 1p +Injury/IL- 1p)
and log2 (TNF-a +injury/IL- 1p) respectively. Colors represent protein clusters as
computed by k-means clustering. Figures 3B-D represent projections of the 3D plot on
different axes pairs.
K-i
O
K-2
LK=3
2
M
**
10
now
0
dP
EU
U.
*
1
2K=
8..
-4
-1
log2(L1
+inj:L1)
-4
Iog2(INF:M~)
2
-14
log2(rNF-uAL-1p)
2.
4:..
* K=I
*P~*4~
2
A
2**
KL=J
-2-2
7-
an
-2
U
-
-3
og L-1pSi
L1)
log2(TNF-AL-10)
IK=3
Figure 4: Profiles of the means of the three clusters identified by K-means clustering.
Error bars represent standard error of the mean. The clusters are well separated (p<0.001,
significance measured by Mann Whitney U test). Clusters 1, 2 and 3 contain 59, 55 and
85 proteins respectively.
2.5
2
1.5
H1
00.-+-
K=12
K=3
0.
TNF-a/J*4P
UIP4k*H
/i*4-1
-0.5-
-15-
--
nij
Figure 5: Profiles of means of log 2 transformed ratios of proteins sorted by location.
Error bars represent standard error of the mean. Asterisks indicate p<0.05, the null
hypothesis being that means of log transformed ratios are 0. Significance is measured by
Mann Whitney U test or Two Sample T test, depending on whether distribution is normal
or not.
2.5
2
OTNF-a:IL-lb
* L-1b-inj:L-1b
0.5 .
0>
-9
0 TNF-a+inj-IL-1 b
T
T1
Figure 6: Figure 6 shows the behavior of different sets of proteins to the combination of
Injury and Cytokine. All data represent mean log transformed ratios of the respective
treatments wrt IL-1p +/- standard error. The proteins whose effects are measured are (A)
C-terminal propeptides of Collagens. (B) Key structural proteins belonging to the ECM.
(C) Members of the SLRP family, key structural molecules in the ECM. (D) Proteases
including members of the MMP family and ADAMTS 1, and MMP inhibitors. (E)
Members of the TGFp super-family and their inhibitors. (F) IGF binding proteins. (G)
Proteins involved in the innate immune response. (H) Key signaling molecules. Asterisks
represent ratios whose p values are significant. Significance is measured by Wilcoxon
sign rank test or paired t-test under the null hypothesis that protein expression does not
change under treatment. Multiple comparisons are corrected for using Benjamini
Hochberg procedure at FDR 0.1
A
0.5
T
0.
-0.5 .TNF-a.IL-lb
.L-1b+inj
:L-1b
-- TNF-e+inj:IL-1b
-1.5 --
- -
-
-2
-2.5 -
B
1.5
*TNF-a: IL-lb
14 0
0.
*L-ib+!rn:
L-lb
o TNF-a+inj
:L-1b
MTNF-a: IL-lb
ML-lb+inj: IL-lb
E3TW-a-Hnj: IL-lb
0.5
0
2-0.5
mTW-a: IL-lb
mL-lb+inj: IL-lb
c3TW-a4jnj: IL-lb
el -
STNF-a: IL-lb
mIL-lb+ini: IL-lb
c3TNF-a+ini: IL-lb
1.5
1
0.5
ci
0
-0.5
STM--a: IL-1b
* L-1b+inj: IL-1b
E TM--a+inj: IL-lb
-1.5
-2
2
C-
1
0
mTNF-a: IL-lb
m IL-1b+inj: L-1b
o TiF-a+inj:
.- 2
-3
-4
*TNF-a:IL-lb
* L-lb+inj:IL-lb
0 TNF-a+inj:IL-lb
IL-lb
Figure 7: Comparison of iTRAQ data generated from this experiment (experiment A)
with iTRAQ data generated from previous experiment performed in our lab (experiment
B) 80. Proteins are identified in common experiments A and B and mean (TNF-a /IL-1p)
ratios from experiment A compared with mean (TNF-a /IL-1p) ratios from experiment B.
Only ratios whose respective p values satisfy Benjamini Hochberg procedure at FDR 0.1
are chosen for the respective plot. Histogram of log 2 transformed ratio of ratios (i.e.
log2(exp B (TNF-a IL- 1p) / exp A (TNF-a /IL-i ) is plotted.The Mean +/- standard
deviation is 0.1067 +/- 0.2191
-3
-2
2
0
1
-1
log2( expB(TNFIL1) / expA(TNFAL1))
3
Table 2: Matrix of correlation coefficients. Correlation coefficients are found between
IL-1p only, TNF-a only, injury only, combination of (IL-l p and injury) combination of
(TNF-a and injury) conditions. Log transformed protein ratios obtained from combining
data from experiments A and B and converting to untreated basis are used as vectors to
calculate correlation coefficients. Only ratios whose p values satisfy Benjamini Hochberg
procedure at FDR 0.1 are used in this calculation
log 2(IL-1 P/untreated) log2(TNF-a/untreated) log2(inj/untreated)
log2(IL-1p/untreated)
log 2(TNF-a/untreated)
1
0.9028
0.9028
1
0.1416
0.3473
log2(inyuntreated)
log 2(IL-1 P+inyuntreated)
Iog2(TNF-a+inj/untreated)
0.1416
0.6839
0.5944
0.3473
0.7707
0.7046
1
0.7581
0.7486
log 2(IL-1 P+inyuntreated)
0.6839
0.7707
0.7581
1
0.9574
Iog2(TNF-a+inyuntreated)
0.5944
0.7046
0.7486
0.9574
1
Table 3. List of proteins showing positive and negative synergism. The columns are
protein name, log 2(TNF-a /untreated), log2(injury/untreated), expected log 2 (TNF-a
+injury/untreated) if injury and cytokine acted independently, actual log 2 (TNF-a
+injury/untreated), and percentage synergism (A). Lists proteins showing positive
synergism. If the maximum ratio among log 2(TNF-a/untreated), log 2(injury/untreated)
and expected log 2(TNF-a+injury/untreated) were called R1, the percentage positive
synergism is calculated as
(( actual log2(TNF-a+injury/untreated) - R1)/R1 )*100 (B). Lists proteins showing
negative synergism. If the minimum ratio among log2 (TNF-a/untreated),
log 2 (injury/untreated) and expected log2(TNF-a+injury/untreated) were called R2, the
percentage negative synergism is calculated as
(( actual log 2 (TNF-a+injury/untreated) - R2)/R2 )*100
A
log2(1NF-a/untreated)
log2(injury/untreated)
Expected
Actual log2(TNF-
% hi gher than
log2(TNF-+
a+injury/untreated)
maximum of
injury/untreated) if
independent
[TNF-a, injury,
TNF-m+injury if
indep endent]
Aggrecan
1.014
-0.111
0.903
1.059
4.377%
Col6A3
0.468
0.684
1.152
1.389
20.588%
Histone H4
replacement-like
0.931
0.941
1.873
2.379
27.032/o
CLP-2
0.406
-0.147
0.259
0.735
81.115%
log2(TNF-aluntreated)
log2(injury/untreated)
Expected
log2(TNF-m+
injury/untreated) if
independent
Actual 1og2(TNF-
%lower than
minimum of
[TNF-a, injury,
TNF-a+irnjury
if independent]
SPARC
-0.765
-1.084
-1.849
-3.599
94.631%
IGFBP7
-0.644
-1.110
-1.754
-2.141
22.025%
B
a+injury/untreated)
Figure 8: Figure 8 is obtained by performing Partial Lease Square Regression (PLSR)
between protein ratios and phenotypes (sGAG loss into medium in five days post
treatment, nitrite accumulation in five days post treatment, rate of proline incorporation
and rate of sulfate incorporation on the 6 h day post treatment). X represents matrix of log
transformed protein ratios under five different treatment conditions, and Y matrix of log
transformed phenotype ratios (wrt control) under the same conditions. The matrices X
and Y are mean centered and set to unit variance before PLSR (A) Percent variation in
the protein ratio matrix (X) explained by 1,2 and 3 Principal components (factors). Each
vertical line represents a single protein (B) Percent variation in phenotypes explained by
1,2 and 3 principal components. The blue line is the training data set, and the red line the
validation data set. In both (A) and (B), two factors explain the variation very well. (C)
Projection plot of the five treatment conditions on the first two principal components. (D)
Projection plot of proteins (blue) and the phenotypes (red) on the first two principal
components. Points close to each other are positively correlated, and in opposite
quadrants are negatively correlated. The Unscrambler @ X Version 10.0.1 used for
generating plots A-D
A
Explained Vaniance Each line represents one protein
100
90
aO
70
60
50
(D
40
130
-
20
10
0
-10.
-20
-30
Factor-0
Factor-i
Factor-2
Factors
Factor-3
Factor-4
Explained Variance
100
90
80,
70
60
so
40
30
20
10
0
-10
-20 Factor-0
Factor-4
Factor-2
Factors
76
4
3.1
2 -
15
injury
-2
-3
L1 +inj
-5
-6
-7 TNF+inj
-8
-4
-3
-2
-1
0
3
2
1
Factor-i (31%. 74%)
4
5
6
7
8
0.2MMP2
IGFBP7
SPARC
Iaminin A4.
lactorannn~f?~ 4
0.1
EDIL3
proine
cathepsin B
CILP2
COL6Al
.COLGA3
FK8P 10
*ieaCnnexin A8
sema
3
Factor-i (31%, 74%)
u
Table 4: List of protein-phenotype pairs showing (A) The top five positive correlations
and (B) The top five (in magnitude) negative correlations, as generated by PLSR.
Top 5 positive correlations
Protein
phenotype
Perlecan
sGAG release
pleiotropin
sGAG release
serum amyloid A3
sGAG release
CILP-2
sGAG release
compl B
sGAG release
Top 5 negative correlations
Protein
phenotype
pleiotropin
proline incorporation
cathepsin B
sGAG release
perlecan
proline incorporation
pleiotropin
sulfate incorporation
perlecan
sulfate incorporation
Table 5: Effect of injury or cytokine treatment or both on different phenotypes. Rows
represent treatment conditions and columns represent phenotypes. This data matrix forms
the Y matrix in the PLSR analysis.
Experimental Condition
nmol proline
microg sGAG per set nmol sulphate incorporation lincorporation / nmol Nitrite
day (Measured accumulation (in
of 4 explants (in 120 day (Measured at the 6th
at the 6th day) 120 hrs)
day)
hrs)
Control
194.7035484
139.4045
68.32229
9.397892
injury
199.7683616
85.85213
50.74262
10.31592
L-1p
1045.690457
58.85496
37.03414
71.23021
L-1p+ injury
1164.124135
36.51318
28.51041
34.39866
TNF-a
829.7448476
66.59978
41.7247
104.7924
2080.996078
34.77035
32.56093
152.8823
TNF-a
+injury
DISCUSSION
The aim of this study is to understand the molecular basis behind the synergism
seen between cytokine and injury in causing sGAG loss to medium from cartilage
explants as observed by Patwari et al' . With this aim in mind, we generate a
quantitative profile of proteins released to medium on treatment of cartilage explants with
cytokine (IL-1p or TNF-a), injury or a combination of cytokine and injury. By doing so,
we hope to identify those proteins whose expression values differ most on combination of
injury and cytokine, as compared to either treatment alone. Since we used a 4-plex
iTRAQ labeling strategy, we could analyze only four treatment conditions at a time.
Therefore, in the first part of this study, we divided cartilage explants into four groups,
and subjected each group to one of the treatments: IL-1f alone, TNF-a alone, IL-1p and
injury and TNF-a and injury. Medium from each group was collected, iTRAQ labeled
and a 2D-LC/MS/MS analysis performed. iTRAQ ratios from this experiment were
specified with respect to IL-1p alone values and proteins whose levels differed
significantly on treatment with cytokine and injury, as compared to IL- 13 alone screened.
Clustering and correlation analyses were performed to observe which proteins and which
treatment conditions behaved similarly.
The next part of this study combined data from this experiment with earlier
iTRAQ experiments in our lab 80 which contained treatment conditions not present in the
first experiment (ie injury alone, and a control without any treatment). Data from both
these experiments were combined after suitably correcting for inter experimental bias.
This helped generate all possible ratios for several proteins with respect to control (ie IL1p:control,
TNF-a:control,
Injury:control,
IL-1f
+injury:control
and
TNF-a
+injury:control). These ratios were used to determine the proteins on which injury and
cytokine acted synergistically, in either a positive or a negative fashion. These proteins
thus identified are probably the proteins involved in causing synergistic sGAG loss to the
medium at a macro level.
Since we have protein levels for several proteins across 5 treatment conditions
with
respect
to
control
(IL-1:control,
TNF-a:control,
Injury:control,
IL-1
+injury:control and TNF-a+injury:control), we have a huge data matrix in which we
expect to see a lot of redundant information. To get the core non redundant information
from this highly "collinear" data matrix, we next use a PLSR (partial least square
regression) analysis, to determine those combinations of protein ratios (latent vectors)
which contribute most to some observed phenotypes. The phenotypes used in our PLSR
model are sGAG loss to the media in 5 days post treatment, rate of proline incorporation
on day 6 post treatment, rate of sulfate incorporation on day 6 post treatment, and total
nitrite accumulation in 5 days post treatment. Since redundant information is eliminated
by this analysis, PLSR helps unearth non-obvious groupings of proteins which behave
similarly on treatment, and proteins which are most responsible for an observed
phenotype. For example, because sGAG loss to media is directly indicative of
catabolism, and proline and sulfate incorporation rates of anabolism, the choice of these
phenotypes in the PLSR model helps to identify proteins which closely correlate with
catabolism and anabolism.
4.1 Methodology
for correcting for multiple comparisons - The
Benjamini Hochberg procedure
Instead of correcting for multiple comparisons by controlling the Family wise
error rate (FWER) using Bonferroni correction, we opt to adopt the procedure adopted by
Benjamini and Hochberg
112
to control the False Discovery Rate (FDR). The advantage of
this methodology over the Bonferroni correction is that Bonferroni correction is
exceedingly stringent and hence a lot of protein ratios which represent real differences
may not be counted. Using the Benjamini - Hochberg procedure thus ensures fewer false
negatives and more "hits" than the Bonferroni correction
117
. A FDR of 0.1 is chosen for
our analysis.
4.2 Variation between biological replicates
The LC/MS/MS analysis was done on biological replicates. The source of
variation between any two experiments can be broadly classified as technical,
experimental and biological
116.
Briefly, technical variation measures the variation
observed by repeated tests on the same test subjects under identical conditions. In the
iTRAQ experiment, this includes all steps from protein extraction till quantification.
Experimental variation in the context of iTRAQ experiments refers to variation caused
due to repetition of an identical iTRAQ experiment.
Biological variation measures
biological variability in the test subject. Since the biological replicates in our experiments
are also subjected to separate iTRAQ experiments using a different set of labels, the
source of variation between these replicates is biological as well as experimental. We
observe that log(ratiol/ratio2) for proteins identified in common between experiments 1
and 2 (only proteins which satisfy Benjamini Hochberg procedure are used in this
comparison) has a mean of 10.1 % for the 115 label and 8.07 % for the 116 label (Figure
1). Both these are lesser than the 23 % average variation between experimental replicates
and 25 % average variation between biological replicates as reported in an earlier iTRAQ
16
study with a different model system 116.
The log(ratiol/ratio2) for the 117 label however
shows a difference of 43.17 % . This huge difference is possibly due to incomplete
digestion in the iTRAQ2 117 label sample, where a white precipitate was observed after
trypsin digestion. This implies that only a part of all proteins released to medium is
subjected to LC/MS/MS analysis, and hence quantitation of this sample is naturally
inaccurate. To correct for this difference, for all proteins identified in biological duplicate
2 alone and not in biological duplicate 1, 0.4317 is added to log transformed 117:114
ratios alone. For proteins identified by biological duplicate 1 alone or in common by both
experiments, experiment 1 values are used for further analysis.
4.3 A global proteomic analysis of the effect of Cytokine+injury
4.3.1 Correlation between IL-1p +injury and TNF-a+injury treatment conditions
It is highly interesting that a global proteomic analysis between different
treatments yields a very high correlation between the two cytokine+injury treatments,
with a correlation coefficient of 0.914 (Table 1). (Only proteins whose p values suggest
good quantification across all ratios are chosen for all global analysis). This implies one
of two things, either that TNF-a and IL-1
interact similarly with injury and activate
similar pathways in the cell, or that the effect of injury dominates over the effect of
cytokine. To confirm which of these two hypothesis is right, we require data representing
the effect of injury alone on cartilage, if the injury alone condition were to show a high
correlation with both the (cytokine+injury) conditions, it would imply that injury
dominates over cytokine; if not, it would imply that both the cytokines act on the cell
through very similar pathways, thus causing very similar change in protein levels. This
study is performed later in this discussion when we combine results from two iTRAQ
experiments.
4.3.2 K-means clustering of proteins
K-means clustering is performed on the data matrix, comprising 199 proteins with
3 conditions, and the optimum number of clusters was chosen as three. Figure 3a shows
a 3D plot of log transformed protein ratios, with the three treatment conditions as three
axes, and clusters differentiated by color. The centroids of these three clusters are plotted
versus treatment in Figure 4, to better illustrate the differences between the clusters.
Broadly, cluster 1 proteins show a mild elevation with (cytokine+injury), cluster 2
proteins are significantly depressed and cluster 3 proteins are highly elevated. A closer
examination of the proteins present in each cluster reveals that about 73% of the proteins
present in cluster 3 are cytoplasmic proteins. Therefore, most of the cytoplasmic proteins
are highly elevated on treatment with (cytokine+injury). This is presumably because of
lysis of the cell membrane caused by mechanical injury, and subsequent release of
proteins in the cytosol to the medium .This is consistent with the study by Stevens et al
118
which also finds a high proportion of cytosolic proteins to be released to the medium
on treatment with injury alone. We also find that all ECM proteins identified are present
in either cluster 1 or 2 (i.e. slightly increased or highly depressed with (cytokine+injury)).
The
structural
proteins aggrecan,
matrilin-1,
matrilin-2, matrilin-3;
the SLRPs
chondroadherin, fibromodulin, epiphycan and biglycan all cluster together in cluster 1.
While collagens III, VI, XI and XII cluster together in cluster 1, collagens I and II cluster
together in cluster 2. MMPs 2, 3 and 13 also cluster together in cluster 2, and are
depressed by cytokine+injury.
4.4 Effect of Cytokine + Injury on specific protein classes
We next look at specific classes of proteins in greater detail, to find out how these
key proteins vary with (cytokine+injury), to find out how (cytokine+injury) affects
catabolism and synthesis. From this, we try to see if this in vitro model is representative
of OA.
4.4.1 The Collagen Family
Collagen accounts for two-thirds of the dry weight of adult articular cartilage, and
collagen fibers provide cartilage with the ability to withstand tensile and shear forces.
Fibrillar collagen chains are generally synthesized as procollagens, and formation of
mature collagen fibrils requires cleavage of the N and C terminal propeptides of
procollagens (for review 119). The cleavage and release of Collage C-terminal propeptides
to the medium is thus indicative of the ability of cartilage to synthesize new collagen, and
is a useful marker of the anabolic state of cartilage. In Figure 6a, we see that both
(cytokine+injury) treatments inhibit collagen synthesis of several collagen chains, with
TNF-a+injury inhibiting collagen synthesis to a greater extent than IL-1
+injury. This
suggests that the combination of injury and cytokine reduces anabolic activity in the cell,
and impairs the ability of cartilage to recover from damage.
4.4.2 Aggrecan
Aggrecan is the major proteoglycan present in hyaline cartilage, and is
responsible for compressive stiffness of cartilage (for review 13). The core protein of
aggrecan has a modular structure, with three disulfide-bonded globular domains G1, G2
and G3 and an extended central portion between G2 and G3 for GAG attachment
(chondroitin sulfate (CS) and keratan sulfate (KS)). G1 and G2 are separated by an interglobular domain (IGD)
120.
Depletion of aggrecan due to its degradation is an important
characteristic of OA. This occurs due to increased proteolytic cleavage of the core protein
by MMPs' 2 1 123 and primarily aggrecanases 2. In our study (Figure 6b), we find that
aggrecan levels remain almost constant on treatment with (injury + cytokine) with very
mild down regulation (<0.5 difference on log 2 scale)
4.4.3 Other structural and matricellular molecules
Besides collagen and aggrecan, several other molecules are present in the ECM.
These either aid in structural roles, such as collagen fibril formation and collagen crosslinking, or in remodeling of ECM. Thrombospondins are multi-domain, calcium binding
ECM proteins which have a high affinity to collagen (for review' 5 ). There are five
thrombospondins currently discovered. We identified all five in our study (Appendix),
but only Thrombospondins 1 and 5 were quantified well across ratios (Figure 6b). We
see that while thrombospondin-1 is only slightly elevated with (TNF-a + injury), COMP
(Thrombospondin-5) is highly down-regulated with TNF-a + injury. COMP is cleaved
by ADAMTS-4 and MMP-19 and 20
1'1
and elevated levels of COMP/COMP
fragments in the serum and the synovial fluid have been correlated with OA severity and
joint injury
12,127 .
However, a decreased amount of COMP remains in OA cartilage and
the distribution also changes, with higher COMP in the fibrillated superficial zones, and
almost no COMP in the middle and deeper zones
128.
Since we are measuring proteins
released to medium from cartilage explants, we would expect elevated levels of
COMP/COMP fragments on treatment with injury + cytokine, due to diffusion of newly
synthesized COMP unanchored to the matrix due to proteoglycan loss; and cleavage with
proteases respectively. Hence, it is surprising to see that (TNF-a + injury) decreases
COMP released to the medium significantly compared to IL-1
alone. One possible
reason is that synthesis of new COMP is reduced. Since we did not observe ADAMTS-4
among our proteases (Figure 6d) , it is possible that the COMP we observe released to
the medium is not due to degradation of existing COMP, but due to synthesis of new
COMP. Hence, if synthesis were reduced, we would observe reduction in COMP levels
released to medium. Further studies have to be undertaken to find out if the COMP
released is due to synthesis (i.e. whether the COMP released to medium represent intact
COMP) , and if so, reason out why COMP synthesis is reduced on treatment with (TNFa+injury).
Among the matrilins, Matrilin-1 and 2 are almost unchanged with (injury +
cytokine) (< 0.5 on the log scale). Matrilin-3 is moderately down regulated with (IL-1 +
injury) alone. Matrilins are proteins thought to play a role in the formation of filamentous
networks in the ECM through interacting with collagen and other molecules 31.
A
mutation in Matn3 gene was found to be linked to hand OA in a genome wide linkage
analysis
129.
While Ko et al
130
found Matrilin-3 deficient mice to have no skeletal
malformations and normal life spans , Van der Weyden et al
131found
an expanded
hypertrophic zone of the growth plate in the embryonic stage due to premature
chondrocyte differentiation and a higher predisposition for severe OA in one year old
animals. An examination of the collagen architecture of Matn3- knock-out mice with
electron-microscope showed mild abnormalities with slightly increased collagen fibril
diameter and collagen volume densities
132.
In view of these studies, the decrease in
Matrilin-3 we observe with (IL-1p +injury) may indicate premature differentiation of
chondrocytes, with impaired collagen fibril network structure resulting in altered
mechanical properties.
4.4.4 Small leucine rich proteins (SLRP)
Since SLRPs have been postulated to serve as biomarkers of OA
42,
we next look at how
these ECM proteins respond to treatment in our in vitro model. SLRPs are currently
classified into four classes. Our study (Figure 6c) identifies decorin and biglycan (class
I); fibromodulin, lumican, PRELP and osteoadherin (class II); epiphycan and osteoglycin
(class III); and chondroadherin (class IV).
Decorin shows mild up regulation with IL-1
+injury (not attaining statistical
significance) and moderate down regulation with TNF-a+injury. Disruption of the
decorin gene in mice leads to fragile skin with markedly reduced tensile strength, with
collagen fibrils thicker than wild type mouse, indicating that decorin may have a role in
inhibiting fibrillogenesis. 133 . Decorin has a role in connecting fibrillar collagens such as
collagens I or II with minor collagens such as collagens VI, XII and XIV
32,134,135
As opposed to decorin, Biglycan is highly over expressed with IL-1P +injury and
moderately with TNF-a+injury. This is similar to the result obtained by Robbins et al
136
who found that mechanical injury up regulated biglycan in tendons. The precise
interaction of biglycan with collagen is unclear; with some studies claiming that biglycan
binds collagen 1137 and others that biglycan does not
138.
Besides connecting collagen VI
to collagen II, biglycan also appears to organize collagen VI microfibrils into a hexagonal
network in vitro 139,32. Biglycan is known to be cleaved by MMP-13 at a specific location
140,
but this is not the reason for increased biglycan levels with (cytokine + injury) in our
study, as MMP-13 levels are seen to decrease with (cytokine + injury) (Figure 6d)
Biglycan and decorin differ in their pattern of temporal expression in corneal stroma
development. While decorin was expressed at high levels in all stages of development,
biglycan was expressed highly in early stages of development, decreasing until expressed
at very low levels in mature cornea
141
Looking at Class II SLRP's, fibromodulin and osteoadherin does not change
much with treatments. Lumican is slightly over expressed with IL-1p + injury, and highly
with TNF-at + injury. PRELP is moderately over expressed with IL-1
+ injury, and
highly with TNF-ca + injury. Fibromodulin has two sites of different affinities at which it
binds to collagen, and lumican has one site. The lumican site and the low affinity
fibromodulin sites are homologous and compete in binding collagen I, but fibromodulin
has a higher affinity to collagen I because of the presence of an additional high affinity
site
12.
During tendon development, lumican is expressed earlier and fibromodulin later,
with lumican
deficient mice
showing distorted fibril morphology
earlier, and
fibromodulin deficient mice later. This could possibly be because lumican aids in
forming early thinner fibrils, and once fibromodulin levels rise later in development, it
displaces lumican, and helps join the thin fibrils to form thicker fibrils 39.
It is highly interesting that both lumican and biglycan, both of which are
expressed earlier in development show high expressions with cytokine and injury. This
possibly indicates an attempt to repair tissue damage by development of new collagen
fibrils.
Also, our results for decorin, biglycan, fibromodulin and lumican are identical to
the results obtained by Young et al in a sheep model of OA
(143.
They found increased
mRNA levels of lumican and biglycan, decreased decorin levels and unchanged
fibromodulin levels, identical to what we observe at a protein level in our in vitro model
of injury. This indicates that the changes in protein levels we observe are probably the
result of new protein synthesis of SLRPs which again suggests an attempt to repair
cartilage. This also shows that the combination of injury and cytokine shows a
remarkably similar SLRP profile as compared to OA tissue, and hence this in vitro
treatment condition approximates OA reasonably well.
PRELP is highly up regulated with cytokine+injury in our study. While most
SLRPs have amino terminal domains with acidic properties, PRELP has a basic amino
terminal region. It also has no GAG chains. PRELP is capable of binding heparin and
heparin sulfate containing proteoglycans such as perlecan through its amino terminal
region, and collagen type I and type II through its Leucine rich repeat region. Hence, it
possibly connects the basement membrane (rich in perlecan) to the underlying connective
tissue
144
145
. PRELP is expressed highly in cartilage 4. Over expression of PRELP in skin,
where it is normally present at low levels, results in a structural change, with decrease in
collagen fiber bundle content and size, and decreased thickness of hyperdermal fat layer
146
. However, the collagen fibrils obtained are of normal structure. It is not clear what the
high up regulation of PRELP with (cytokine+injury) means, and further knowledge about
PRELP and its functions is necessary to interpret this result.
Among class III SLRPs, epiphycan shows moderate up regulation with (IL-1p +
injury), and is not changed much with (TNF-a + injury). Epiphycan-deficient mice
generated by disrupting the epiphycan gene in mouse embryonic stem cells developed
OA, and male epiphycan-deficient mice had shorter femurs than wild type mice. Also,
epiphycan-biglycan double deficient mice developed OA earliest suggesting possible
interaction between both in vivo
47
Osteoglycin does not show statistically significant
change with (cytokine + injury).
The class IV proteoglycan chondroadherin shows high up regulation with TNFa+injury, and is not changed much with IL-1p + injury. Chondroadherin is particularly
enriched in cartilage, but also found in bone. It binds to collagen type II
and C terminal globular domain of collagen type VI
32.
148
, and also to N
It binds to integrins on the surface
of cells, and causes intracellular signaling through tyrosine phosphorylation of factors
like ERK 149. Like PRELP, chondroadherin too has a heparin binding domain via which it
can bind to perlecan and cell surface receptors carrying heparan sulfate proteoglycans 15.
The elevation of chondroadherin with (TNF-a+injury) may thus indicate an attempt to
repair the collagen type II network. Chondroadherin elevation may also cause changes in
synthesis of other proteins through intracellular signaling via cell surface receptors.
4.4.5 Matrix proteases and their inhibitors
A healthy cartilage lies in equilibrium between synthesis and degradation. Matrix
degrading proteases such as collagenases and aggrecanases play a crucial role in
maintaining this equilibrium 151; however, aberrant regulation accompanied by over
expression of these proteases result in a shift of the cartilage away from equilibrium
towards degradation resulting in degenerative diseases such as OA 9 . It is believed that
aggrecanases act in early stage OA, and collagenases in late stage OA. A study by
Karsdal et al 54 shows that aggrecanase mediated aggrecan degradation in cartilage can be
reversed by anabolic stimuli, provided catabolism has not been too severe. However,
MMP mediated collagen and aggrecan degradation impaired the repair capacity of
cartilage, and was irreversible.
MMP-13 is thought to be the major collagenase in OA
152
and is highly over expressed in
OA cartilage. Mice knockout experiments showed significant protection against OA in
ADAMTS-5 single knockout and ADAMTS-4/ADAMTS-5
double knockout mice, but
lack of protection in ADAMTS-4 single knockout mice
A study by Kevorkian et al
155
153,154.
profiling several MMP, ADAMTS and TIMP (Inhibitors of MMP and ADAMTS) in
end stage hip OA cartilage found increased genetic expression of the gelatinases MMP2
and MMP9, MMP13, ADAMTS2 and TIMP3 and decreased expression of MMP1, MMP3,
ADAMTS1, TIMP1, TIMP4 and ADAMTS5 at various levels of statistical significance.
However, MMP1 was increased in knee cartilage indicating differences in regulation of
MMP1 between hip and knee. Another global study by Swingler et al
156
which profiled
expression levels of several genes in hip OA and compared them to normal cartilage
found an increase in expression of MMP13, MMP3, MMP9, ADAMTS2, MMP2 and a
decrease in expression of ADAMTS9, ADAMTS1, MMP1 among others at P<0.01
Figure 6d shows the response of members of the MMP, ADAMTS and TIMP
families to the combination of cytokine and injury. Several MMPs identified in our study
(MMP's-1,2,3,9) show very mild down regulation with (IL-1
+injury) as compared to
IL-13 alone, with MMP-2 alone showing moderate down regulation with (TNFa+injury). The gelatinases MMP-2 and MMP-9 seem to be almost unchanged .MMP-13
alone shows high down regulation with (IL-1
+injury), and a mild down regulation with
(TNF-a+injury), both showing statistical significance. This is contradictory to studies
which show that MMP-13, whose main substrate is Collagen 1I, is the major collagenase
involved in OA 1.57 It is surprising that the collagenases (MMP-1, MMP-13), the
gelatinases (MMP-2, MMP-9) and stromelyin-1 (MMP-3) all mostly mildly down
regulated with (cytokine+injury) with respect to IL-1.
With the mild down regulation
possibly due to apoptosis of some cells due to injury, this means that addition of injury to
cytokine does not change the expression of MMP as compared to cytokine in general.
Therefore, addition of injury to cytokine does not increase catabolism due to MMP.
However, ADAMTS-1, the only ADAMTS identified in our study, shows significant up
regulation with (cytokine+inury) for both IL-1p and TNF-a. ADAMTS-1 does not cleave
any member of the collagen family, but cleaves aggrecan and versican
63
Among the TIMPs, both TIMP-1 and TIMP-2 were identified. TIMP-1 shows
decrease with TNF-a+injury, without reaching statistical significance. TIMP-2 on the
other hand is moderately up regulated with IL-1
and injury, and not changed with TNF-
a+injury. TIMP-1 has previously been shown to be significantly down regulated and
TIMP-2 up regulated in OA hip cartilage
155.
The up regulation of TIMP-2 might be an
attempt to inhibit ADAMTS-1, which is seen to be up regulated, and against which
TIMP-2 has inhibitory activity.
4.4.6 Members of the TGF-p superfamily and their inhibitors
The TGF-P superfamily refers to a large family of growth factors which are
predominantly anabolic in nature and includes the TGF-3 subfamily, the activin/inhibin
subfamily, bone morphogenetic proteins (BMP) and some other members
158
.
TGF-p
causes net synthesis of the ECM by inhibiting collagenases and up regulating TIMP 159.
Treatment of chondrocytes with TGF-p1 is known to down regulate mRNA levels of
MMP-1 and MMP-13 and also reduce mRNA levels of IL-1 and TNF receptors
160,
thus
moving the chondrocyte towards an anabolic state. TGF-p also increases net proteoglycan
synthesis as measured by sulfate incorporation in vivo
161.
TGF-1 and TNF-a are known
to increase their own mRNA levels and reduce each others', resulting in an autocrine
feedback loop shifting between anabolism and catabolism.
Connective tissue growth factor (CTGF) is a growth factor, known to be inducible
by TGF-, which synthesizes collagen and fibronectin in fibroblasts
162.
It is involved in
fibrotic diseases, characterized by excessive collagen deposition. CTGF null mutant mice
develop cartilage which has impaired mechanical properties due to reduced aggrecan and
link protein production 163. CTGF has previously been identified in human osteoarthritic
cartilage
164 ,
but at very low levels. CTGF synthesis as measured by mRNA levels has
been found to reduce in a sheep model of OA
143
Cartilage derived morphogenetic protein - 2 (CDMP-2) is a bone morphogenetic
protein belonging to the TGF-3 super-family. Along with CDMP-1 and CDMP-3, they
form a distinct subgroup within the BMP family
165
Expression patterns of CDMP-2 in
bovine cartilage indicate that it is involved in terminal differentiation of chondrocytes and
at the earliest stages of endochondral bone formation, including angiogenesis and
osteoblast differentiation. CDMP-2 implantation subcutaneously or intramuscularly is
known to induce bone or cartilage; or tendon, depending on the degree of mechanical
166
stimulus, higher mechanical stimulus leads to bone formation, and lower, tendon'
Activin and inhibin are two closely related members of the TGF- superfamily which
have opposing functions. While activin enhances FSH biosynthesis and has roles in cell
proliferation, differentiation, apoptosis, inflammation, fibrosis and wound repair
167,168.
P chains
alone,
inhibin down regulates FSH synthesis
169.
Activin consists of inhibin
while inhibin contains both inhibin a and inhibin
p 1.
Proinhibin PA and mature inhibin
PA were detected in all OA samples but only in half the control samples in a study by
Hermannson et al 164 indicating presence of activin A in OA cartilage. Hermannson et al
also found that activin A was induced in chondrocyte monolayers by IL-13, TGF-31,
FGF-2, PDGF,EGF and activin A itself. Also, it is seen to induce TIMP-1 production in
culture medium, and thus acts as an anabolic factor.
Members of the TGF-$ superfamily such as TGF-,
regulated at several different levels (for review
1.
BMPs and activin are
LTBP (Latent TGF-p binding
proteins) are proteins, which as the name suggests, bind to TGF-p and another protein,
called Latency associated proteins (LAP), thus maintaining TGF-3 in an inactive form.
to be active. LTBPs are also involved in
Cleavage of LTBP is necessary for TGF-
assembly, secretion and targeting of TGF-p to its site of action. (for review
172).
MMP-2,
MMP-3 and MMP-9 have been shown to cleave LTBP, thus releasing active TGF-p
LTBP-3
null
osteoarthritis.
mice develop craniofacial
abnormalities
and eventually
41
develop
73
Looking at other inhibitors of the TGF- superfamily, the ECM proteins decorin
and biglycan have been seen to inhibit TGF- activity 174175. Chordin and gremlin are
soluble extracellular inhibitors of BMPs
176.
Chordin-like 2 is a protein structurally
similar to chordin which inhibits BMP in vitro and in vivo. It is specifically up regulated
in the middle zone of OA cartilage and may delay chondrocyte hypertrophy, thereby
ameliorating cartilage degeneration7
Vasorin is a type 1 membrane protein which is
observed to directly bind to TGF- and inhibit its action 178. Follistatin is an autocrine
glycoprotein whose primary function is the inhibition of activin 79. Follistatin-like protein
1 is a secreted glycoprotein sharing a characteristic structural molecule (the FS domain)
with follistatin. It has a role in inflammation and has been shown to both be highly over
expressed in early arthritis, leading to up regulation of pro-inflammatory cytokines like
IL-1
and TNF-a
180;
and contradictorily to ameliorate arthritis severity in a mouse
model' 81 .
In our results, looking at members of the TGF-P superfamily (Figure 6e), we see
that TGF-2 itself is highly elevated with TNF-a + injury, and moderately with IL-1f +
injury. LTBP-1 is highly down regulated for both the (cytokine + injury) conditions.
Down regulation of LTBP implies that TGF-p is activated from its latent form, thus
increasing net synthesis and moving towards anabolism. Inhibin
BA
is extremely elevated
with the (cytokine + injury) treatments. Only inhibin PA is found and not inhibin a,
which implies that activin A which is a homodimer of inhibin PA is up regulated with
cytokine+injury. This again indicates a shift towards anabolism, and tallies with the fact
that activin A is found to be upregulated in OA samples
164.
Follistatin-like 1 is also
moderately down regulated with (TNF-a + injury), which could mean reduced
inflammation, this tallies with the high levels of activin A observed.
CTGF is extremely down regulated with (TNF-a + injury), indicating cartilage with
impaired mechanical properties. However, its levels don't change much with (IL-1p +
injury). One possible reason why CTGF is inhibited is the sequestering of TGF- by
SLRPs like biglycan which show increased levels with (cytokine+injury) (Figure 6c).
However, it is unclear why CTGF levels are unchanged with (IL-1p + injury) if this be
the case, as biglycan levels are increased both with (IL-1P + injury) and (TNF-a +
injury). Other inhibitors of the TGF-6 superfamily such as chordin and gremlin remain
almost the same with (cytokine + injury) treatment.
4.4.7 Insulin-like Growth Factor Binding Proteins
IGF (Insulin-like growth factors) are ubiquitously expressed mitogens showing
high sequence similarity to insulin and which affect cell growth and metabolism. They
are predominantly anabolic in nature and known to stimulate DNA synthesis, protein
synthesis, proteoglycan synthesis and glycosaminoglycan synthesis 1 82 . There are two
forms of IGF, IGF-1 and IGF-2.
In cartilage, IGF-1 acts as an enhancer of matrix
synthesis and a growth promoter, and IGF-2 acts as a growth stimulator in a nondifferentiated state 183. Their actions are mediated by IGF receptors. IGFBP (IGF-binding
proteins) are soluble proteins which bind to IGF in biological fluids. They have higher
affinity for IGF than IGF receptors, and thus act as carriers of IGF and also modulators of
IGF availability and activity (for review182,184). IGFBPs also have actions independent of
IGF. For example, IGFBP-5 administration increased bone formation parameters both in
vitro and in vivo with a magnitude comparable to IGF-1185 and IGFBP-7 plays a potential
186
tumor suppressor role in colorectal carcinogenesis186
In our study (Figure 6f), four IGFBP's were identified, IGFBP-3,5,6 and 7.
IGFBP-3 is moderately up regulated with (IL-13 +injury), and up regulated with (TNF-a
+ injury) without approaching statistical significance. This represents a move towards
catabolism, as IGFBP-3 will sequester IGF and prevent IGF mediated anabolism. IGFBP-
3 has previously been found to be higher in severe OA cartilage as compared to mild OA
in a statistically significant manner, but difference between mild and intermediate or
intermediate and severe does not approach statistical significance
187.
A regression done
in the same study between OA score and IGFBP-3 levels show a very weak but
statistically significant dependency. The up regulation seen in IGFBP-3 may be because
of increase in IGFBP-3 in either matrix or in cytoplasm or nucleus of chondrocytes,
where it is seen to localize 188. This might be because of a role of IGFBP-3 independent
of IGF.
IGFBP-5 is highly up regulated with (TNF-ct + injury) in our study, but the level
remains the same with (IL-1
+ injury). This possibly represents an attempt to repair
cartilage as increase in IGFBP-5 has previously been seen to increase IGF-1 and thus
improve joint architecture during OA development
89 .
IGFBP-5 is also increased in
chondrocytes and bone matrix during arthritic joint deterioration
90 .
IGFBP-5 is degraded
by complement component Cis. Figure 6g shows that complement component Cis is
mildly down regulated with both the (cytokine + injury) conditions. This mild down
regulation might partly explain the elevation of IGFBP-5 in the (TNF-a + injury)
condition, but it isn't known why the same down regulation of Complement Cis in the
(IL-13 + injury) condition does not cause an increase in IGFBP-5.
IGFBP-6 is one of the predominant IGFBP's found in bovine cartilage, and not
found much in human 191,192. We find IGFBP-6 to be mildly down regulated in both the
(injury + cytokine) conditions.
IGFBP-7 is moderately down regulated with (IL-1
+injury), and highly with
(TNF-a + injury). This protein is discussed in greater detail in a later section. It is
difficult to say with certainty how change in levels of IGFBPs are affected/affect any
roles of IGFBP independent of IGF, as these roles are yet unclear.
4.4.8 Proteins associated with the innate immune response
The complement system has traditionally been associated with the innate immune
response, functioning in host defense against invading pathogens (for review 19. It does
this through the release of inflammatory mediators and has been implicated in the
pathogenesis
' . The
of several autoimmune, ischemic and vascular diseases 194 195
complement system also serves as a vital link between innate and acquired immunity196
Complement component C1 triggers the classical component pathway. It is a complex
formed by association of the recognition protein Clq with two copies of two proteases
Cir and Cis. Clq binds the target, thus triggering self-activation of C1r, which converts
pro-enzyme Cis into a protease which cleaves C4 and C2, thus activating the classical
component pathway197 . The component Clq is also known to bind to, and induce
clearance of apoptotic cells198 .
The complement system is also involved in functions very different from its
traditional role in the immune response. It may be involved in the replacement of
cartilaginous matrix by endochondral bone in early fetal development'
199
.
The
complement component C3 may be involved in osteoclast development by potentiating
M-CSF dependent proliferation of bone marrow cells and induction of osteoclasts
200
Complement proteins C3, B, C5, C9 and properdin are localized to distinct areas in the
developing endochondral bone, suggesting that complements proteins are involved in the
cartilage cell death, vascularization and hence the transformation of cartilage to bone
201
A significant role for the complement system exists in rheumatoid arthritis (for
review 202 ). Complement component C3 and C4 are drastically reduced relative to total
protein in the synovial fluid of patients with RA. Genetic deletion of complement
component C5, C3 or factor B in arthritis susceptible DBA/1 mice resulted in each case
in mice resistant to collagen induced arthritis (CIA) 203,204 . Wu et al 205 found complement
components such as complement factor B, complement component 4 binding protein,
complement 9 and complement factor H-related protein 5 to be over expressed in OA
cartilage compared to normal cartilage.
We identify complement factors B, I, components C3, C4-A, and subcomponents
C1r, Cis, and the A, B and C subunits of subcomponent Clq in our study (Figure 6d).
Cl-inhibitor is also present. Factor B is moderately down regulated with (IL-1P + injury)
and almost unchanged with (TNF-a + injury). Factor C3 is highly suppressed with both
(cytokine + injury) conditions. Injurious compression of cartilage is known to induce
apoptosis82. Since both factor C3 and B are known to be increased with inflammatory
cytokines such as TNF-a 20 6
,
it is likely that the combination of injury with these
cytokines results apoptosis of some cells, thus causing reduced levels of complement B
and C3 production. Subcomponents Cis, Cir and Clq are mildly downregulated with
(IL-13 + injury), and CIr moderately down regulated with (TNF-a + injury). C1 inhibitor
is highly down regulated with IL-1 + injury, but almost remains the same with (TNF-a +
injury). Deficiency of C1 inhibitor is associated with autoimmune diseases such as
systemic lupus erythematosus2 0 7
Peptidoglycan recognition proteins (PGLYRP) are innate immunity proteins
which recognize bacterial peptidoglycan and function in antibacterial immunity and
inflammation (for review 208). Four mammalian PGFLYRP's (PGLYRP 1-4) have been
identified. While human PGLYRPs have the highest affinity for peptidoglycans, bovine
PGLYRP1 has the highest affinity for LPS (Lipopolysaccharide) and LTA (Lipoteichoic
acid). PGLYRP2 plays a pro inflammatory role in a model of peptidoglycan-induced
arthritis in mice, with PGLYRP2- mice resistant to peptidoglycan induced arthritis and
inflammation209
PGLYRP1 on the other hand has an anti inflammatory effect.
In our
study, we identify both PGLYRP1 and PGLYRP2. PGLYRP1 is down regulated mildly
with IL-13 + injury and moderately with TNF-a + injury compared to IL-1
alone.
PGLYRP2 on the other hand remains almost the same with (TNF-a + injury) (without
attaining statistical significance) and is highly down regulated with (IL-1B + injury). It is
curious that both PGLYRP1 and PGLYRP2 are down regulated with (IL-1P + injury),
since they have anti and pro inflammatory activities respectively. Since PGLYRP2 is
down regulated to a greater extent than PGLYRP1, this might imply an attempt to reduce
inflammation and repair tissue. Since IL-1
and TNF-a are pro-inflammatory cytokines,
we would expect that IL-1 alone/TNF-a alone would increase PGLYRP2 and decrease
PGLYRP1 compared to [IL-13 + injury]/[TNF-ax + injury], unless mechanical injury
increases chondrocyte access to the cytokines in the combination treatment. Some as yet
undiscovered function of the PGLYRPs may account for the way their levels change with
(cytokine + injury).
CD14, a membrane protein commonly found on monocytes/macrophages, is
critical for activation of the innate immune
system by lipopolysaccharides2".
Lipopolysaccharide binding protein (LBP) binds to LPS on bacteria and transfers it to
CD14 on macrophages. CD14 and LBP are necessary and sufficient for the activation of
the Toll-like receptor system211. CD14 is moderately down regulated with IL-1P + injury
and very mildly with TNF-a + injury. LBP is moderately down regulated with both the
(cytokine + injury) conditions. The down regulation of CD14 and LBP with both the
(cytokine + injury) conditions as compared to IL-1 alone signifies that the inflammation
and macrophage activation caused by cytokine alone conditions is reduced on addition of
injury.
4.4.9 Signaling molecules
Proenkephalin is one of the proteins whose expression changes the most with
(cytokine + injury) in our study, showing -8 fold increase and -13 fold increase with IL1p + injury and TNF-a + injury respectively as compared to IL-1P alone (Figure 6h).
Proenkephalin
is
the
precursor
of
enkephalin,
opioid
peptides
which
are
neurotransmitters, neurohormones and neuromodulators. Initially associated with the
nervous and neuroendocrine systems, proenkephalin was found to be highly elevated in
non-differentiated cells of diverse mesodermal lineages in the process of development
into adult tissues, including cartilage
mm. It was, however, almost undetectable in
differentiated tissue, suggesting a role in cell proliferation/differentiation. Proenkephalin
expression was closely associated with cell proliferation and stimulated by TGF-
in
chondrocytes. While very few primary chondrocytes express proenkephalin mRNA, a
high proportion of subcultured cells do
. Rosen et al hypothesize that a small proportion
of undifferentiated cells in differentiated tissue retain the ability to synthesize
proenkephalin, and do so in situations demanding repair 66 . Recent studies by McTavish et
a12 15 show that proenkephalin assists stress activated apoptosis by transcriptional
repression of p53 and NF-KB gene targets. Therefore, the high elevation of proenkephalin
with cytokine+injury could indicate an attempt to repair damaged tissue. It could also
help assist in apoptosis of cells, which has been observed with mechanical injury8 2
AIMP1 (p43) is another signaling protein highly elevated with (cytokine+injury).
AIMPI was first identified as a factor associated with a macromolecular tRNA synthetase
complex in mammalian systems 216. It was later found to be secreted and acts on diverse
target
cells such as endothelial cells, macrophages
and fibroblasts to control
angiogenesis, inflammation and dermal regeneration respectively. It is seen to activate
MAPK and NF-KB,
and activates cytokine
and chemokine genes involved
in
inflammation such as TNF-a, IL-1p, IL-8, macrophage inflammatory proteins MIP-la
(CCL3), MIP-2a, MIP- 13 (CCL4) and RANTES (CCL5)m. It promotes endothelial cell
death by apoptosis at high dose 218 , but also promotes fibroblast proliferation and wound
repair2.
AIMPI has not been identified in cartilage in any study so far. The up
regulation of AIMPI with cytokine+injury might indicate inflammatory response, but it
is doubtful that cytokine+injury might have such highly elevated levels of inflammation
compared to cytokine alone (as the cytokines IL-1
and TNF-ax are highly pro
inflammatory themselves, and addition of injury would not increase inflammation). It
could represent an attempt to repair tissue, in line with its role in dermal wound repair, or
it could be involved in apoptosis caused by injury. Since AIMP1 seems to be a highly
multi-functional protein with different domains contributing to different functions220 , it
could play any of these roles here, and further studies are needed to clarify the role of
AIMPI in stressed cartilage.
Annexins A2 (II) and A5 (V) show high elevation with TNF-a + injury. Annexin
A5 is also highly elevated with IL-1 + injury, and Annexin A2 moderately. Annexins
are a family of proteins with the ability to bind to acidic phospholipids in the presence of
Ca*. Three annexins, 1I, V and VI are known to be highly expressed in calcifying
cartilage and bone
2.
Annexins II, V and VI are major components of matrix vesicles
(MV), particles released from plasma membrane of hypertrophic chondrocytes or
osteoblasts, which initiate mineralization of cartilage. These annexins form calcium
channels in MV's and mediate rapid influx of Ca** into the vesicles which serves as the
start of the mineralization process. Annexin mediated Ca"* influx also leads to terminal
differentiation and apoptosis of growth plate chondrocytess,222 . The up regulation of
Annexins II and V in our study with (cytokine+injury) therefore indicates the onset of
mineralization, terminal differentiation and apoptosis in cartilage, which are features of
OA cartilage as well. SPARC (osteonectin) shows high down regulation with (TNF-a +
injury), and is discussed in greater detail later in this discussion.
4.5 Comparing the effect of combination of cytokine and injury as
opposed to either treatment alone
4.5.1 Combining data from two iTRAQ experiments
Since we used a four-plex iTRAQ experimental design, we chose iTRAQ
114,115,116 and 117 labels to represent IL-1
alone, TNF-a alone, IL-1
+ injury and
TNF-a + injury treatments respectively. Since we had only four labels available to us, we
could not introduce an untreated control, and a sample for injury alone treatment.
However, for a true study of whether there exists synergy between injury and either
cytokine in their effect on protein levels, we require data for six treatment conditions,
namely untreated control, IL-1
alone, injury alone, IL-1
+ injury, TNF-a alone and
TNF-a + injury. Therefore, we combine data from this experiment with a previous
experiment done in our lab (which had untreated control, IL-13 alone, TNF-a alone and
Injury alone treatment sample data8 0) to generate all possible ratios with respect to
control.
4.5.2 Variation between the two iTRAQ experiments
We first check for reproducibility between the two iTRAQ experiment data, by
looking at the only common ratio between the two experiments, TNF-a/IL-1. To do this,
we first identify proteins which are found in common between experiment A (this
experiment) and experiment B
80,
and which are quantified well across all ratios. We
identify 74 such proteins. The log of ratio of (TNF-a/IL-1P) ratios from experiment A
and B for these proteins has a mean of 0.1067. This variation of 10.67% is again lesser
than the 23% and 25% variation seen between experimental and biological replicates in
an earlier iTRAQ study1 1 6 . Therefore, for these 74 proteins, data from both experiments
are merged and converted to ratios with respect to untreated controls.
4.5.3 Correlation between cytokine alone, injury alone and cytokine+injury
treatment conditions
A matrix of correlation coefficients is thus generated for these 74 proteins for the
five ratios (IL-1I:control), (TNF-a:control), (injury:control), (IL-1IP+injury:control) and
(TNF-a+injury:control) (Table 2) . We see that while IL-1 alone and TNF-a alone have
a correlation of -90.2%, (IL-1
+ injury) and (TNF-a + injury) have a 95.7% correlation.
It is highly interesting that in the presence of injury, the two cytokines show an even
higher similarity in their effect on proteins than in the absence of injury. This shows that
while IL-1
and TNF-a themselves have very similar effects on cells globally, the
addition of injury further augments this similarity. This high similarity between the two
(cytokine+injury) conditions is not due to the effect of injury masking the effect of
cytokine, as the correlation between injury alone and (cytokine+injury) (-75%) is much
less than the 95% correlation between the two (cytokine+injury) conditions. One possible
hypothesis to explain higher correlation the (cytokine+injury) treatments (as compared to
correlation between cytokine alone treatments) is that the combination of injury with IL1p and TNF-a may activate some new pathways in the chondrocyte, in addition to
pathways activated by cytokine alone and injury alone, and that these new pathways
activated are common for both (IL-1 + injury) and (TNF-at + injury).
4.5.4 Proteins on which injury and cytokine act in a synergistic fashion
To identify proteins on which injury and cytokine act synergistically, we look at
proteins for which the effect of (injury + cytokine) is greater than that caused by injury
alone or cytokine alone. If the ratio of (injury + cytokine) for a protein is greater than the
injury alone ratio, the cytokine alone ratio, and the product of injury alone and cytokine
alone ratios (the ratio expected if injury and cytokine act independently), injury and
cytokine are identified as exerting a positive feedback on each other's action (positive
synergism) for that protein. On the other hand, if the ratio if (injury + cytokine) is lesser
than the injury alone ratio, the cytokine alone ratio, and the product of these ratios, injury
and cytokine are said to exert a negative feedback on each other's action for that protein.
By having such a stringent criterion, we make sure that we pick only proteins on which
injury and cytokine truly act synergistically, and avoid artifacts due to quantitation error,
and any error in combining data from the two experiments.
When we thus look at proteins which satisfy this criterion, we find that among our
list of 74 proteins, for no protein does IL-1P and injury exhibit positive or negative
feedback. However, we identify a few proteins for which TNF-a and injury act
synergistically. TNF-a and injury exhibit positive feedback for Aggrecan, Histone H4,
CILP-2 and COL6A3 ; and negative feedback for SPARC and IGFBP7 (Table 3).
Aggrecan is almost doubled with (TNF-a) alone, and slightly down regulated with
injury alone, but when injury and TNF-aL are combined, the level rises to more than that
by TNF-ax alone. The percentage synergism is 4.3%. This small synergism may be due to
enhanced transport in the ECM caused by injury, which allows better access of aggrecan
to ADAMTS and MMP, thus causing higher cleavage and greater release of cleaved
fragments to medium, resulting in increased levels.
CILP-2 shows the highest percent positive synergism (81%) among these four
proteins. Cartilage intermediate layer protein 2 (CILP-2) is an isoform of the ECM
protein CILP-1. CILP-1 and CILP-2 are 50.6% identical and around -66% of their amino
acids are conserved
38.
CILP-1 has been found to bind to TGF- and act as a negative
regulator of TGF-p in chondrocytes, thus preventing TGF-p induction of aggrecan and
Collagen type I
223.
It is also seen to block the ability of IGF-1 to decrease extracellular
inorganic pyrophosphate; however, CILP-2 has no such effect 38. A protein profile of
human OA cartilage yielded CILP-2 fragments, suggesting possible CILP-2 cleavage in
OA
224.
The increase in CILP-2 levels we observe due to synergism between injury and
TNF-a may thus be due to cleavage of CILP-2. Further studies on the function of CILP-2
may help in interpreting these results better.
The alpha III chain of Type VI collagen (COL6A3) shows 20.6% positive
synergism between TNF-a and injury. Collagen VI is structurally composed of three
different peptide chains, al(VI), a2(VI) and a3(VI); a3(VI) (260-300 kDa) is much
larger than al(VI) and a2(VI) (both around 140 kDa). It is an ECM protein which forms
a microfibrillar network around or in between collagen type II fibers in cartilage; it is
concentrated in areas surrounding chondrocytes22 5 2 2 6 . It interacts with several matrix
constituents including collagens type Im and type II228 ; SLRP's biglycan, decorin,
chondroadherin 32 , fibronectin 229 and basement membrane Collagen type IV2
.
It also
binds to alp1 and u2 1 integrins and thus maybe involved in cell signaling231. Thus,
roles of collagen VI include structural roles such as anchoring the basement membrane to
the connective tissue, and serving as scaffolds for formation of fibrillar collagen
networks; and signaling roles, such as mediating the interaction of cells with the ECM.
Therefore, it could be involved in roles ranging from wound healing to tissue
232
development and architecture 2. Thus, the positive synergism displayed between injury
and TNF-a for COL6A3 possibly represents an attempt to form collagen microfibrils to
repair damaged tissue.
Histone H4 replacement-like
protein also shows considerable
synergism
(27.03%). This protein resembles histone H4, which is one of the basic units of chromatin
architecture in the nucleosome. DNA wraps itself around the nucleosome composed of
histones. Because of this close association with DNA, histones are involved in DNA
transcription, replication, DNA damage and its repair, recombination and overall
chromosomal stability
233.
Histones are basic proteins with a globular domain and a
charged NH 2 - terminus (histone "tails") protruding from the nucleosome. These tails are
highly involved in modulation of chromatin architecture, either by serving as targets for
chromatin remodeling factors such as Swi/Snf and NURF; or by means of diverse post
translational modifications such as acetylation and phosphorylation (for review2 3 4).
Synthesis of histones is closely associated with DNA replication during S-phase of the
cell cycle, and hence histone gene expression can be used as a marker for
proliferation 235 ,236. Therefore, the synergistic up regulation by TNF-a and injury of
Histone H4 replacement-like protein possibly represents increased cell proliferation due
to repair of damaged tissue.
SPARC and IGFBP7 are the proteins which show reduced expression because of
negative feedback between TNF-a and injury. SPARC is a glycoprotein present in the
ECM which mediates cell-matrix interactions but does not serve structural roles. It is a
Ca** binding protein which acts as a counter-adhesive protein, a modulator of growth
factor activity and a cell cycle inhibitor. It is expressed in high levels in tissues
undergoing remodeling, whether naturally, due to wound healing or due to disease (for
2
1).
reviewm
SPARC is stimulated by several members of the TGF-
super family
including TGF-1, BMP-2 and activin, and inhibited by bFGF, IL-1,
TNF-ax,
dexamethasone and LPS 238 -24 0 .
SPARC has been shown to induce production of MMPs 1, 3 and 9241; and also to
activate MMP-22 4 2 It also binds to several collagens including collagen type I, III, IV
and V24 3 . SPARC synthesis is markedly enhanced in arthritic joints (OA or RA), and in
such joints has been hypothesized to induce MMP production in the surface zone, and
modulate chondrocyte proliferation, matrix synthesis and mineralization in the middle
and deep zones 240 . A proteomic study by Wu et al
205
found SPARC to be increased in
OA cartilage compared to normal. In this context, it is highly surprising that we observe
negative feedback between TNF-a and injury, resulting in down regulation of SPARC.
We would expect up regulation, both in view of the fact that SPARC elevation has been
reported in OA cartilage, and that IL-1 and TNF-a alone reduce SPARC mRNA levels.
A possible reason could be high levels of apoptosis with injury, leading to a less number
of SPARC producing cells. Increased transport of TNF-a due to increased permeability
caused by injury and subsequent inhibition by TNF-a could be another reason for the
negative feedback. However, it is unclear whether increased transport may suffice to
explain extremely high negative feedback, and further studies need to be carried out to
understand better the reason behind this behavior of SPARC.
IGFBP7 is the other protein which shows down regulation due to negative
feedback between TNF-a and injury. This protein is known to be expressed in OA
articular cartilage. The gene coding for IGFBP-7 is present in chromosome 4q which has
been associated with hip OA susceptibility by linkage mapping, but further studies
indicate that polymorphisms in IGFBP7 gene may not increase susceptibility to hip
OA 244. As IGFBP-7 only weakly inhibits IGF, this significant down regulation is
probably due to an as yet unknown function of IGFBP-7.
In our study, we have thus identified six proteins on which TNF-a and injury act
synergistically. A more complete way to analyze interactions and feedbacks between
Cytokine and injury would be to perform multiple mass spectrometry experiments using
the 8-plex iTRAQ experimental design, and analyze the data using a two way Anova.
However, this study makes a start in trying to understand where the synergism between
injury and cytokine in GAG loss comes from.
4.6 Partial Least Square Regression (PLSR) Analysis
4.6.1 Motivation behind the PLSR analysis and Methodology
Our next aim is to connect what happens inside the cell to variables which can be
observed outside. By performing this study, we hope to find out proteins which are most
relevant in changing some specific measurable phenotypes of cartilage. We thus have two
matrices, X composed of log-transformed protein ratios under different treatment
conditions, and Y composed of phenotypes under the same treatment conditions. We
want to find out how the underlying change in protein ratios manifests as observable
phenotypes, and hence want to find a relationship between Y and X. Since the number of
protein ratios is much higher than the number of phenotypes, we have a problem of multicollinearity (one or more protein ratios are highly correlated and hence, the matrix X
contains superfluous information). Ordinary multiple regression can hence not be used.
The commonly used Principal component regression (PCA) eliminates the problem of
multi-collinearity by choosing directions of maximum variance in X and using these to
develop a multiple regression model with Y. PLSR on the other hand chooses those
directions which maximize covariance between X and Y, projects X and Y data points on
these directions, and performs regression. Therefore, PLSR helps generate those
independent variables which contribute most to the relationship between X and Y (for
review
114).
In our situation, this means that amongst all the protein ratios we have, we
can identify proteins which contribute most to observed phenotype. From the 74 proteins
for which we have data for all 5 treatment conditions, CLEC3A and CD109 are not
chosen because they increase error in the model. We use log transformed data for the
remaining 72 proteins across 5 treatment conditions as the X matrix. The Y matrix is a
(4*5) matrix consisting of log transformed phenotype ratios with respect to control. The
four phenotypes we choose are sGAG loss to the medium during the five day treatment,
nitrite accumulation in the medium in five days, rate of proline incorporation (indicative
if protein synthesis) on the sixth day (the day after the five day treatment ends), and rate
of sulfate incorporation on the sixth day. The five treatments are the same as for the X
matrix.
It is important to note that any relevant phenotype for which data across all
treatments including (cytokine+injury) are available can be chosen, these four phenotypes
are chosen because of availability of data for all the five treatments including the
(cytokine + injury) treatments. Also, sGAG release and proline and sulfate incorporation
rates are biologically significant phenotypes, as they indicate catabolism (sGAG release)
and anabolism (proline and sulfate incorporation). Choosing these phenotypes therefore
helps understand which proteins contribute most to catabolic and anabolic activities.
4.6.2 Some Observations from the PLSR plots
Figures 8a-b show that four factors (principal components) are able to describe
100% of the variation in the X matrix (protein ratios) and Y matrix (phenotypes). Two
principal components describe 88% variation in the training data set and 70% of Y
variation in the validation data set. This difference may be due to the presence of outliers
in the protein data matrix.
However, for obtaining trends about how phenotypes and proteins relate to each
other, projection on the first two principal components are sufficient. Figure 8c shows
the projection of different treatment conditions on the first two principal components. We
see that the cytokines IL-1p and TNF-a are close to each other, the two cytokine+injury
treatments are close to each other, and the injury treatment is separate from the other two.
Figure 8d shows the projection of proteins and phenotypes on the first two principal
components. Proteins and phenotypes present close together in the same quadrant are
highly correlated to each other, and proteins and phenotypes present in opposite
quadrants and far from each other are inversely correlated. We immediately see that
proline and sulfate incorporation rates, both indicators of synthesis, are highly correlated
as expected. sGAG loss to medium, an indicator of cleavage of proteoglycans, and hence
catabolism, is diametrically opposite to proline and sulfate incorporation rates, again as
expected. Most cytoplasmic proteins cluster together tightly in the fourth quadrant. We
also observe that SPARC and IGFBP7, two proteins which we earlier observed to
synergistically decrease in the presence of TNF-a and injury are clustered close to sulfate
and proline incorporation rates. Therefore, SPARC and IGFBP7 both seem to be
indicators of synthesis or repair, which is seen to reduce on treatment with cytokine and
injury. If the only role for IGFBP7 were in binding IGF, we would expect a negative
correlation of IGFBP7 with synthesis, with an increase in IGFBP7 decreasing free IGF,
and hence decrease anabolism. Therefore, this positive correlation of IGFBP7 with
synthesis suggests a new role for IGFBP7 in repair and merits investigation.
4.6.3 Identification of proteins which contribute most to observed phenotype
From Table 5, we see that the top five positive protein-phenotype correlations are
perlecan, pleiotropin, serum amyloid A3, CILP-2 and complement factor B, all with
sGAG. Since sGAG loss to the medium is an indicator of catabolism, these five proteins
are either key causative agents or key markers of catabolism. Also, the top five negative
correlations are pleiotropin with proline and sulfate incorporation, perlecan with proline
and sulfate incorporation and cathepsin B with sGAG release to the medium.
Perlecan is a large, multi-domain ECM proteoglycan expressed in almost all
basement membranes, and also in mesenchymal organs and connective tissue. It is
involved in a diverse range of developmental and biological processes, including
chondrogenesis, endochondral ossification, angiogenesis and wound healing. Perlecan is
known to interact with and sequester several growth factors, including FGF, VEGF,
CTGF, etc. Knox and Whitelock 30 hypothesize that perlecan performs such a diverse
range of functions by interacting with, and altering distributions of growth factors and
morphogens in developing tissue. Perlecan also interacts with several ECM molecules
including fibronectin, nidogen-1, nidogen-2, PRELP, etc and thus is involved in ECM
organization (for review 29 ). Perlecan deficient mouse embryos die at embryonic day (E)
245
10.5 with defective cephalic development, or die just after birth with skeletal dysplasia .
Late stage OA tissue samples showed elevated perlecan mRNA and protein levels in
cartilage adjacent to OA defects246, this suggests that perlecan is either involved in OA
pathogenesis, or is synthesized in OA cartilage in an attempt to repair tissue. Our study
however, shows that perlecan is highly correlated with sGAG release and highly anticorrelated with both proline and sulfate synthesis, showing that an increase in perlecan
released to the medium is an indication of catabolism, and reduced anabolism. Therefore,
it is probable that the increase in perlecan observed in late stage OA is not due to an
attempt at tissue repair, but rather due to worsening catabolism.
Pleiotropin is a cytokine implicated in tumor cell growth and angiogenesis,
apoptosis and mitogenesis (for review 67 ). It is undetectable in normal adult cartilage, but
is elevated in the synovial fluid of OA patients in early stages and found rarely in late
stages247.
In cultured chondrocytes, pleiotropin reduced MMP-1 and MMP-13, and
increased TIMP-1 and TIMP-2 mRNA levels, indicating a role in repair 248. However, a
study by Tare et al 249 found that over expression of pleiotropin in transgenic mice led to
increased type I collagen synthesis (associated with osteoblasts), and ossification within
articular cartilage. Our result shows high correlation of pleiotropin with sGAG loss, and
high anti-correlation with proline and sulfate incorporation, and suggests a catabolic role
for pleiotropin, contradicting the possibility of a role for pleiotropin in repair. However,
since cartilage catabolism is accompanied by endochondral ossification in OA, the
association of pleiotropin with catabolism might actually imply the role of pleiotropin in
ossification.
Complement factor B and Serum Amyloid A3 are both involved in the innate
immune response. Complement factor B has been discussed earlier, and is implicated in
cartilage cell death and endochondral bone formation20 1 . Serum Amyloid A3 (SAA3) is a
member of the Serum amyloid A family, apolipoproteins associated with HDL. The
SAA3 gene is not expressed in humans 2s; however, LPS stimulated macrophages secrete
SAA3 in a murine model, suggesting a role for SAA3 in local inflammation apart from a
general acute-phase response
. The high association of these innate immunity proteins
with sGAG release suggests that catabolism is accompanied by inflammation and innate
immune response.
CILP-2 has been discussed previously earlier in the context of TNF-a and injury
acting synergistically to increase CILP-2 levels. This protein, whose function is as yet
unknown, also appears to be a marker of catabolism.
It is, however, surprising that cathepsin-B, a cysteine protease which cleaves
collagens252 and aggrecan 2 2 and hence plays a part in catabolism, shows such a strong
negative correlation with sGAG release. One possible reason is that in our (injury +
cytokine) model, cathepsin is not the primary collagenase/aggrecanase and does not
contribute to sGAG release, therefore, the anti-correlation of sGAG release with
cathepsin B is an artifact.
4.7 Limitations of this study
To identify the proteins on which injury and cytokine act synergistically, we
combined data from two 4-plex iTRAQ experiments to obtain values for all the
treatments (IL-1,
TNF-a, injury, IL-1p and injury and TNF-ax and injury with respect to
control). Because there exists a variation of only -10.5% between the two experiments,
which is less than that typically seen between biological replicates, the data from both
experiments are combined as such without correcting for any experimental bias.
Additionally, when combining data between the two 4-plex experiments, to ensure that
there are no false positives identified, we first shortlist those proteins which are identified
by both iTRAQ experiments, and which have all ratios across both experiments with an
FDR<0. 1. This gives us a list of only 74 proteins, and in this final list we check for
proteins on which cytokine and injury exhibit synergism, ending with only 6 proteins on
which TNF-a and injury show positive or negative synergism, and none for IL-1IP and
injury. This methodology is exceedingly stringent, and may miss out several proteins on
which cytokine and injury actually exhibit synergism, but are not counted because one or
more of their FDR>O. 1. In other words, we may end up with several "false negatives".
An 8-plex iTRAQ experiment can circumvent both these problems, as we could
label samples from all the treatment conditions simultaneously. This would firstly
eliminate the need to combine data from two 4-plex experiments, and thus ensure no inter
experimental variation. Secondly, conducting an 8-plex experiment would ensure lesser
false negatives, and ensure more than just the 6 proteins we obtained on which cytokine
and injury exhibited synergism.
When we performed the PLSR analysis, we considered only the first two latent
vectors, to enable easy interpretation of how proteins and phenotypes relate to each other.
The first two latent vectors of our PLSR model, however, capture only -88% variation in
the training set, and 70% variation in the validation set. The addition of more latent
vectors would capture more information, but may complicate the interpretation of the
PLSR plots.
CONCLUSION
In conclusion, we have used 4-plex iTRAQ labeling followed by 2D-LC/MS/MS
to study the effect of the combination of cytokine and injury on cartilage explants. A
global analysis of the proteins released revealed a high correlation between (IL-13 +
injury) and (TNF-a + injury) treatment conditions. A k-means cluster performed on the
protein ratios revealed three distinct clusters, cluster 1 containing proteins highly down
regulated, cluster 2 mildly up regulated and cluster 3 highly up regulated with (cytokine +
injury). Most cytoplasmic proteins are present cluster 3 and are highly elevated with
(cytokine + injury), possibly due to mechanical lysis of cells due to injury. ECM proteins
are present in either cluster 1 or cluster 2. Looking at specific protein groups, C-terminal
collagen propeptides of some fibrillar collagen chains are down regulated with (cytokine
+ injury), indicating reduced synthesis of new collagens, and a decrease in the anabolic
ability of cartilage. ECM molecules such as COMP and matrilin-3 were down regulated,
and members of the SLRP family including chondroadherin, PRELP, biglycan and
lumican were highly up regulated with (TNF-a + injury). The elevation of lumican and
biglycan, known to be expressed in early stages of collagen fibril development possibly
indicates signs of repair. Proteases MMP-2, MMP-3 and MMP-9 levels don't change
much with (cytokine + injury), but MMP-13 is highly down regulated and ADAMTS-1
highly elevated with TNF-a + injury. This suggests that ADAMTS-1 may be responsible
for the synergistic sGAG release in the presence of cytokine + injury. An increase in
TGF-62 and activin, accompanied by a decrease in LTBP1 suggests attempts to repair
cartilage. CTGF, known to be induced by TGF-D is highly down regulated with TNF-a +
injury, a behavior similar to what happens in a sheep model of OA at the mRNA level,
although it is unclear why. Proenkephalin, AIMP1 and annexins A2 and A5 show high
elevation with TNF-a + injury and/or IL-1 + injury. The up regulation of proenkephalin
possibly helps in repair and in apoptosis caused by injury, the elevation of annexins
indicates terminal differentiation and onset of apoptosis and AIMP1 may play a yet
undetermined role in this situation.
Next, we combined the results from two iTRAQ experiments to obtain data from
injury alone, cytokine alone and cytokine + injury to find out proteins on which injury
and cytokine acted synergistically. From data combined from both experiments, we find a
very high correlation between (IL-1p + injury) and (TNF-a + injury) (-95.7%), much
higher than that between injury and (cytokine + injury), indicating that the domination of
injury over cytokine is not the reason for the high correlation between (IL-13 + injury)
and (TNF-a + injury). Also, the correlation between the two (injury + cytokine)
conditions is higher than the correlation between the two cytokine conditions (-90.3%),
indicating that while both cytokines act through similar pathways in chondrocytes, the
addition of injury further adds new common pathways. From the 74 proteins identified in
common between both the experiments and quantified well across ratios, we find that
TNF-a and injury exert positive synergism on CILP-2, COL6A3, Histone H4
replacement-like and aggrecan and negative synergism on SPARC and IGFBP7. While
increased transport of TNF-a caused due to mechanical damage might cause a slight
increase in aggrecan cleavage resulting in the small synergism shown for aggrecan,
increase in COL6A3 and Histone H4 replacement-like proteins are much higher and may
result from cellular pathways seeking to repair damaged cartilage.
100
A PLSR analysis performed between phenotypes and protein ratios removes
superfluous information and generates interesting associations between protein ratios and
phenotypes. Perlecan and pleiotropin are both highly correlated with sGAG release and
highly anti-correlated with proline and sulfate incorporation rates, indicating that both
these proteins are markers of catabolism. Complement factor B and serum amyloid A3,
proteins associated with innate immunity are both highly correlated with sGAG release,
indicating that local inflammation plays a role in catabolism. SPARC and IGFBP7 are
correlated with proline and sulfate incorporation, suggesting that the negative synergism
between TNF-a and injury for these proteins indicates a reduction in the ability of
cartilage to repair itself. High correlation between CILP-2 and sGAG release suggests
that this protein can also serve as a marker for catabolism. PLSR can thus help find
unique protein-phenotype associations, which may help discover unknown functions of
proteins.
Thus, our study indicates that our in vitro model of injury + cytokine affects
cartilage differently from either injury alone or cytokine alone, and causes both
catabolism and attempts to repair damaged cartilage. This model thus mimics OA
cartilage and may serve as a useful model system to study OA under controlled
conditions in vitro.
Future Work
While bovine cartilage serves as a useful in vitro model system to mimic OA like
conditions using a combination of injury and cytokines, using human cartilage explants in
our in vitro model will help avoid bovine-human differences in response to treatment, and
101
will be closer to human in vivo conditions. Also, as mentioned before, an 8-plex iTRAQ
experimental system would yield more proteins on which injury and cytokine exert
synergism, and could provide a better picture of metabolic pathways in the chondrocytes
which are affected by injury and cytokine.
102
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APPENDIX
Appendix A.1 and Appendix A.2: Profile of proteins released to media
from the first and second biological replicates respectively. Only
proteins identified by greater than 2 peptides with confidence > 90%
and which are well quantified across all ratios are shown here. Protein
ratios are normalized using the internal standard chondroitinase ABC.
Profile obtained using iTRAQ 2D-LC/MS/MS with ProteinPilotTM
Software 2.0.1 (ABI). Matlab scripts are used to calculate the weighted
means and standard deviations. For more detailed information on how
this list was obtained, refer to Chapter 2
Column 1: GI Number of Protein
Column 2: Name of Protein
Column 3: Percent of proteins covered by identified peptides
Column 4: Geometric mean of (TNF-a/ IL- 1p)
Column 5: Standard deviation of (TNF-a/ IL- 1f) in log space
Column 6: p value for the null hypothesis that (TNF-a/ IL-1p) is unity. Obtained by
Wilcoxon Sign rank test or paired t test.
Column 7: Geometric mean of (IL- 1f +inj/ IL- 1p)
Column 8: Standard deviation of (IL-1P +inj / IL-1 ) in log space
Column 9: p value for the null hypothesis that (IL-iI1 +inj / IL- 1f) is unity. Obtained by
Wilcoxon Sign rank test or paired t test.
Column 10: Geometric mean of (TNF-a +inj/ IL- 13)
Column 11: Standard deviation of (TNF-a +inj / IL- IfP) in log space
Column 12: p value for the null hypothesis that TNF-x +inj / IL- 1P) is unity. Obtained by
Wilcoxon Sign rank test or paired t test.
Column 13: Number of peptides used in calculating (TNF-a/ IL-1 3)ratios
Column 14: Number of peptides used in calculating (IL- 1 +inj / IL- 1p) ratios
Column 15: Number of peptides used in calculating (TNF-a +inj / IL- 1P) ratios
124
6
Accession 9 Name
gil11988897 PREDICTED:similarto heparansulfateproteoglycan2 (pedecan)[Bostaurus]
isoform12 [Bostaurus]
similarto fibronectin1 isoform4 preproprotein
giJ76610126
4 4 44 25 PREDICTED:
collagen,type 11,alpha1 isoform 1 [Bostaurus]
gi|16 1
gi|119900891 PREDICTED:hypotheticalproteinLOC540664[Bostaurus]
gi|119890645 PREDICTED:similarto alpha3 type VI collagenisoform5 precursorisoform1, partial[Bostaurus]
1 [Bostaurus]
gi]41386685 thrombospondin
gir30794280 albumin[Bostaurus]
1)(ChondroitnaseABC)(Chondroitinsulfateendolyase)(ChondrotinABCelminase)
ABC endolyase1 precursor(ChondroitinABC
gi|34098396 Chondroitin
4 [Bostaurus]
chondroitinsulfateproteoglycan
gi|119913814PREDICTEDsimilarto melanoma-associated
gi|119901095 PREDICTED:similarto collagen,typeXII, alpha 1 isoform1 [Bostaurus]
gil119887130 PREDICTED:similarto Collagen,typeVI, alpha1 isoform1 [Bos taurus]
gi]27806761 aggrecan1 [Bostaurus]
gi]115495027heat shock70kDaprotein5 [Bos taurus]
gij1 19889734PREDICTED:similarto Collagenalpha-1(XI)chainisoform1 [Bostaurus]
nidogen2 [Bostaurus]
gi|156120777
gill10347570vimentin [Bostaurus}
PREDICTED:similarto P63protein[Bostaurus]
gi|119892686
gil1
19890597PREDICTED:similarto alpha3 type VI collagen,partial[Bostaurus]
gill14051163collagen.typeXI, alpha2 [Bos taurus]
B, beta(actin bindingprotein278)isoform7, partial[Bostaurus]
gil1
7 19914559
4 1 2 PREDICTED:similartofilamin
giJ 740 8 laminA/C [Bostaurus]
gil148238040actnin, alpha4 [Bos taurus]
gi|95147674 complementfactor B [Bostaurus]
cartilageoligomericmatrixproteinisoform1 [Bostaurus]
gi|119894520
9 PREDICTED:similarto
gir9902896 complementcomponent3 [Bostaurus]
gi]148230374proteindisulfideisomerase-associated 3 {Bostaurus]
gill15497814nucleobindin1 [Bostaurus]
collagen,type Ill, alpha1 [Bos taurus]
gi|116003881
PREDICTED:similarto AHNAKnucleoprotein[Bos taurus]
gi|119919153
gi|77404252 collagen,type alpha 1 [Bos taurus]
gil119888668 PREDICTED:similarto Cartilage matrixproteinprecursor(Matrilin-1)[Bostaurus
gi]164452943gelsolina [Bos taurus]
gi]60592792 heatshock 9OkDprotein1, alpha [Bostaurus]
layer protein2 precursor(CILP-2)[Bos taurus]
gij76621011 PREDICTED:similarto Cartilageintermediate
beta subunit[Bos taurus]
gil27806501 prolyl4-hydroxylase,
14050715 moesin[Bos taurus]
gil1
matrixprotein1 [Bostaurus]
gil153791660 extracellular
4 [Bos taurus]
gi]114326226proteindisulfideisomerase-associated
antigen p97) ]CD228antgen) [Bootaurus]
gi]76607158 PREDICTED:similarto Melanotransferrinprecursor(Melanoma-associated
gi|156120795 quiescinQ6 sutfhydryloxidase1 [Bos taurus]
taurus]
A2
[Bos
annexin
gi|27807289
7
7 807
2 [Bos taurus]
gi2
44 matrixmetalloproteinase
[Bostaurus]
similarto matrix metalloproteinase-3
gi|119906908
63 4 3 PREDICTED:
PREDICTED:similarto matrilin-3[Bostaurus]
gi|76 01
gi|122692297 chitinase3-like1 [Bos taurus]
kinase1 [Bos taurus]
gi]77735551 phosphoglycerate
gil119921375 PREDICTED:similarto RP11-525G3.1,partial [Bos taurus]
gil1
19901238PREDICTED:similartolaminin alpha4 [Bostaurus]
gi]87196501 enolase1 [Bos taurus]
gil27807263 heat shockprotein90kDa beta,member1 [Bos taurus]
gi|1 15497900eukaryotictranslation elongationfactor 2 [Bos taurus]
gi|156718112latent transforming growthfactor beta bindingprotein1 (Bos taurus]
[Bos taurus]
complementcomponent1, s subcomponent
gi|115497210
3
gil15652 1687 matrilin2 [Bostaurus]
gil11991915 PREDICTED:hypothetical protein[Bostaurus]
gi]61845535 PREDICTED:similarto emilin[Bos taurus]
gil164450489AE bindingprotein1 [Bostaurus)
gil27805853 cartilagelinkingprotein1 [Bos taurus]
19902010 PREDICTED:similarto LOC512571protein[Bos taurus]
gil1
[Bos taurus]
gi|94966763 haptoglobin
gi|62988316 follistatin-like1 [Bos taurus]
gi]76253709 heat shock70kDa protein8 [Bos taurus]
gil147906412 hypotheticalprotein LOC615490[Bostaurus]
gi|27805991 latenttransforminggrowthfactor betabindingprotein2 [Bos taurus]
isoform1 [Bos taurus]
to vinculinisoformmeta-VCL
gi1 19918578
0 7 1 PREDICTEDsimilar
gi|278 6 8 vitrin[Bos taurus]
2 [Bos taurus]
gi]41386780 UDP-glucosepyrophosphorylase
gi]119903231PREDICTED:similarto lysyl oxidase-like3 protein [Bostaurus]
19903133PREDICTED:hypothetical protein [Bos taurus]
gil1
precursor [Bos taurus]
hydroxylase
gi27806477
gi|61888856 triosephosphate isomerase1 [Bos taurus]
gil27806257 collagen, type alpha2 [Bos taurus]
oxidase-like2 [Bos taurus]
gi|154707890
proteinNRP isoform1 [Bostaurus]
gi]76682880 PREDICTED:smilar to nucleolin-related
[Bos taurus)
phosphorylase,
glycogen
gill
15498012
2
9
2
[Bos
taurus]
VI,
alpha
gi|1154670 collagen, type
gir120474983annexin AS[Bos taurus]
gil114052136
37 2 3 FK506bindingprotein9 [Bs taurus]
5-dioxygenase2 [Bos taurus]
2-oxoglutarate
gil155 20
99 3 procollagen-lysine,
gil11991 5 PREDICTED:similartofilamin [Bostaurus]
9i)75832054 actin,beta [Bostaurus]
lyase
1,
lysyl
lysyl
I,
liver
mean115:std115:11pvalue 115:mean 116 std 116:11 pvalue 116 mean117:std 117 11 pvalue 117: Nopepll5hopepll Nopepll7
%Cov
370
348
346
3.39E-15
34.79845 1.005643 0.421889 0.5458818 0.961879 0.315976 0.0001377 1.253084 0.844274
857
682
628
49.32261 0.435739 0.496027 3.61E-102 1.047484 0.294038 1.60E-05 0.473026 0.639835 1.87E-103
719
719
717
0 0.461606 0.860192 1.25E-114
69.60323 0.826267 0.712588 3.87E-24 0.654007 0.542659
4
4
4
42.53996 0.542485 0.260546 0.0065573 0.894213 0.218261 0.2160647 1.252597 0.216957 0.05788702
123
120
114
4.86E-05
0.763684
1.222695
1.10E-06
0.385784
53.2872 0.65229 0.494509 6.39E-19 1.192888
368
357
347
3.49E-14
50.8547 0.815516 0.520715 3.35E-33 1.005309 0.288304 0.7150054 1.347252 0.921421
705
688
707
85.99671 3.023079 0.45549 3.63E-117 0.867879 0.434132 5.71E-22 1.198459 1.138088 0.00159624
250
244
238
1 0.342961 0.64236811
1 0.323575 0.6563822
1 0.358784 0.0385808
56.51322
83
81
80
2.86E-06
0 1.002979 0.413166 0.9146439 1.274493 0.578736
32.16904 1.34625 0.355638
79
69
87
0
23.52753 0.852151 0.343306 1.93E-05 1.067136 0.297717 0.021252 1.754255 0.357203
378
371
362
5.72E-28
53.74878 0.649362 0.44438 1.60E-50 1.015564 0.284613 0.5096392 1.224062 0.499061
731
728
717
5.48E-15
0.3414 2.97E-21 0.813244 0.913111
0 0.922274
18.99441 0.784089 0.472729
112
103
83
2.95E-19
62.13741 1.247723 0.528367 2.90E-05 2.974593 0.72733 2.24E-18 3.541618 0.842243
182
190
188
42.26747 0.919981 0.486381 0.0339878 0.864302 0.345823 9.03E-10 0.875004 0.656286 0.53356804
114
107
103
3.27E-08
0.787659
1.422424
0.0007069
0.360826
0.924994
0.210661
37.70115 0.96593 0.410477
82
81
75
0
0 4.166193 0.508689
4.74E-09 3.405984 0.487717
72.31759 1.432939 0459791
83
83
76
0
0 4.295576 0.831835
69.43973 0.999488 0.61657 0.6375414 3.582926 0.769483
84
79
78
0
39.15441 0.689371 0.432873 1.74E-10 1.20587 0.286836 1.99E-07 1.41886 0.289956
382
368
356
1.94E-44
45.50691 0.637727 0.635712 1.77E-28 1.108789 0.321063 1.06E-11 1.849601 0.658567
32
32
31
0
0.388416
2.725651
1.27E-13
0.379839
1.798648
0.0588728
0.423167
1.109722
27.42947
49
49
47
0
0 3.48788 0.529756
74.12587 1.221441 0.41052 1.69E-05 2.590192 0.482542
39
39
33
0
0 3.617987 0.673506
52.68935 1.304351 0.603734 0.0009554 2.917892 0.651177
120
105
110
0.0006317
0.635898
1.076385
0
0.33502
0.563283
2.95E-11
52.69383 0.83096 0.356832
132
134
134
9.27E-21
1.228831
63.62434 0.782051 0.332165 7.52E-18 0.939958 0.336537 0.0009198
29
29
30
8.84E-13
34.31668 1.69058 0.440162 2.55E-10 0.554469 0.379337 9.89E-13 0.318933 0.72373
73
74
84
0
0.643657
4.920994
0
0.535
4.180471
61.18812 1.161431 0.486953 0.003473
91
88
88
58.86076 1.344723 0.34105 4.95E-12 0.98958 0.329008 0.187172 1.061875 0.290694 0.00554071
80
78
77
7.71 E-09
42.63301 0.848729 0.569953 0.0004671 1.543071 0.529033 4.44E-16 2.050639 0.922916
14
14
8
1.53E-08
0.687947
4.804789
2.14E-07
0.721279
45.49967 1.006412 0.402019 0.952236 3.754362
28
29
28
526E-06
46.27478 0.491396 0.389364 7.44E-14 0.849487 0.354394 0.0036338 0.316493 0.800289
151
138
137
6.42E-11
0 1.395893 0.59142
68.56618 0.849852 0.480011 5.72E-08 0.753928 0.355448
70
67
86
1.18E-08
45.3265 0.797719 0.305244 1.66E-12 1.46885 0.463886 4.94E-07 1.61193 0.51147
36
35
31
0
0 4.315778 0.486888
42.97408 1.212443 0.455201 0.0019861 3.698287 0.533763
46
43
43
32.36515 0.858448 0.339405 0.0008235 0.944119 0.324471 0.0694573 0.899322 0.591975 0.52273553
60
60
60
0
0 3.828104 0.430247
62.94118 1.151039 0.371326 0.0200043 3.291955 0.423177
23
22
22
2.22E-16
49.74003 1.180499 0.28306 0.0003509 1.976554 0.297421 4.060-14 2.493254 0.284578
81
80
78
1.31 E-09
0 0571277 1.007268
43.13346 0.413906 0.377329 1.68E-14 0.76161 0.247638
36
35
28
0
60.34214 1.162803 0.53044 0.0396956 3.152702 0.775665 2.49E-14 4.033919 0.6537D6
31
31
31
42.21922 1.369667 0.406137 7.79E-07 1.176319 0.267303 5.62E-05 1.182175 0.58193 011694565
61
52
46
0
46.56085 0.895843 0.446222 0.0183304 1.744497 0.37099 1.850-09 3.435781 0.606333
41
40
38
2.618-08
0 3.638051 0.545126
67.55162 0.971116 0.528762 0.6173109 2.152968 0.440105
71
70
74
5.690-12
52.6475 1.116443 0.415104 0.0007673 0.746375 0.334907 2.25E-08 0.542329 0.732D67
95
97
93
4.440-18
0.314875
0.801977
321E-13
0.255386
0.864888
0
38.36478 0.260787 0.703519
125
122
123
8.990-05
56.79013 0.551502 0.430747 3.47E-19 0.057004 0.4602 4.960-19 1.171390 0.824810
087
805
851
56.26599 0.406649 0.421989 8.99E-141 0.82509 0.28308 1.89E-145 0.59098 0.300093 8.8-143
33
31
28
1.820-14
63.30935 1.224115 0.55838 0.012083 3.746138 0.742322 7.118-15 3.737121 0.626015
39
30
37
29.9373 1.048322 0.352778 0.2546114 0.995253 0.453871 0.8281887 1.218634 0.545084 0.2898089
17
17
17
0.03801338
0.532575
0.818708
2.26E-05
0.334845
0.722218
0.0165747
0.329199
0.862922
27.8481
53
52
44
0
0 4819707 0.576677
65.43779 0.960974 0.532531 0.4711225 2.95797 0.564104
25
20
25
2.448-15
39.42786 1.22407 0.40748 0.0015581 4.12454 0.572235 2440-15 5039212 0.65512
26
28
28
0
37.41259 1.220731 0.385121 0.0008322 3.683334 0.46267 8.300-56 4.192343 0.455185
24
24
24
8.200-05
26.23318 0.632534 0.381765 1.520-08 0369366 0.697547 4.640-10 0.372628 1.428103
45
45
45
2.020-07
0 0.754675 0.510241
32.08633 1.043111 0.262576 0.1327652 0.624724 0.328717
30
29
25
37.47391 0.766229 0.269485 2.200-07 0.873592 0.30648 0.005107 1.209664 0.452606 000295748
20
19
19
1.020-11
34.19483 1.117488 0.472504 0.1588925 2479094 0.453791 2.63E-10 2.841205 0.465213
29
30
26
31.42857 0.898998 0.440236 0.0529203 0.873563 0.298941 0.0008016 0851808 1.105543 0.26924046
30
30
30
25.04274 0.889162 0.35892 0.0144912 1.014537 0.279986 0.822161 1026874 0.520569 0.74987118
130
128
124
2.620-18
49.15254 0.855114 0.327271 1.648-09 0.945667 0.351833 0.2509707 1.694337 1039992
40
39
37
3.73E-14
56.15672 1.129493 0.474311 0.0001394 2.974565 0.867531 7.270-07 3229098 0.926168
33
33
33
6.66E-16
0 0.13D643 1130115
63.34165 0.381782 0.428035 5.390-07 0.141562 0.895019
54
54
54
2338-06
63.84365 1.240039 0.353315 3.64E-08 0774442 0.289031 7.25E-10 0494301 1,396514
11
11
9
3.928-06
0.649426
3.417283
00009766
0.852448
2.567655
49.07692 1.034633 0.448786 0.7556605
72
71
8
0570-07
42.47191 0.546603 0.490754 2.990-12 0.993797 0.449141 0.399628 1.588508 0.800243
17
17
17
0.4041 2.77E-05 0689328 0.904192 0.00193099
21.03923 0.340557 0.618178 00002931 0.679186
15
15
14
2670-07
36.33157 1.412648 0.572417 00063074 2431227 0.692558 5.210-56 3199324 0.707839
26
26
28
0518398 000035915
1.634551
10318040349224 0.8043618
33.74233 0.984274 0.502278 0.8109894
24
24
24
7.150-11
0.915289
4.325407
1.508-10
0.779516
3.337236
0.0864763
0.574226
1.109126
50.3937
17
18
18
35.23936 0.885171 0.377162 0.5629179 0.940327 0.321806 0.2134019 0.887358 0.529919 0.19849831
30
29
29
43.21678 0.876185 0.450525 0.1025633 0.711714 0.245211 5030-05 079728 0.921209 0.07190333
61
55
57
0.00256143
0.532095
1160271
0.0014157
33.47107 1.155461 0.325785 1188-05 0.900423 0.353866
49
43
35
0
77.51004 1.478887 0.521609 4.238-05 2.493635 0.608438 1.20E-08 3.112225 0.706996
20
21
21
7.368-08
49.4868 0.702918 0.62236 0.0012565 0.892258 0.21622 0.0014804 0.424701 0.653629
23
23
23
29.84496 0.811225 0.316853 0.0001454 0.834522 0.324703 0.0006167 0.971045 0.821046 0.42906726
13
13
13
8.81 0-09
48.33333 1.13597 0.366306 0.0968463 3.274319 0.4985 364E-08 4192086 0.533889
16
14
13
3.128-12
0.420101
4.293174
1.950-09
0.480869
3690009
0.0436801
43.36075 1.201128 0.421419
34
34
34
0
41.00327 0.724871 0.545296 1.990-05 1.436954 0.245008 8.398-14 1.584799 0.237539
33
33
32
0
0 4.17203 0.53276
69.47041 0.945781 0.398402 02568198 4.098743 0.506528
28
26
26
1468-12
1.000-12 4.007973 0790645
28.04878 1.023669 0.381426 0.6647152 4.020523 0775478
17
16
15
2.31 8-10
37.40157 1.053964 0.300144 03502808 2.113383 0.407388 2.590-08 2.550005 0.399806
15
15
15
4.418-09
0.4281 4.498-07 2936799 0.473249
18.23834 1.261767 0.382879 00044553 1.967561
54
54
46
0
0 4228356 0.848994
47746667 1.140169 0.450024 0.0056727 3.54527 0.593232
0.458171
gi|27806469 peptidylprolylisomerase B [Bos taurus]
gij61828365 PREDICTED:similarto Tropomyosin3 isoform1 [Bos taurus]
gil27806723 calreticulin[Bos taurus]
gi|119887291PREDICTED:similarto peroxidasin homolog [Bos taurus)
gill 19895885PREDICTED:similarto EGF-blkerepeats and discoidinI-like domains 3 [Bos taurus]
gi|27807437 matrixmetalloproteinase 9 [Bos taurus]
gi|114051908FK506bindingprotein 10, 65 kDa [Bos taurus]
gil38566696 serum amyloidA 3 [Bos taurus]
gij77736401 thrombospondin4 [Bos taurus]
gil27805949 inhibinbeta A [Bos taurus]
gil27807207 plasminogen activator inhibitortype 1. member 2 [Bos taurus]
gil27807367 tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein,zeta polypeptide [Bos taurus]
gil155371895sema domain, immunoglobulindomain(1g),short basic domain,secreted. (semaphorin) 3C [Bostaurus]
gi|27806697 chondroadherin [Bostaurus]
gi|27806147 secreted protein, acidic,cysteine-rich [Bos taurus]
gi|119888620PREDICTED:similarto alpha 1 type XVIcollagen [Bostaurus]
gi|41386719 milkfat globule-EGFfactor 8 protein [Bostaurus]
gi|76253900 GDP dissociationinhibitor2 [Bos taurus]
gil27806351 ezrin [Bos taurus]
(LDH-A)(LDHmuscle subunit) (LDH-M)[Bostaurus]
gil119920080
9 PREDICTED:similarto L-lactate dehydrogenase A chain
gil14822350 osteoprotegerin [Bos taurus]
gil110350683 biglycan[Bostaurus)
gi|156120479aldolase A [Bos taurus)
gi|134085671histone cluster 1, Hic [Bos taurus]
gil156121245insulin-likegrowthfactor binding protein 7 [Bos taurus]
gil27806317 annexin A8[Bos taurus]
gil115495019 procollagen-proline.2-oxoglutarate4-dioxygenase (proline 4-hydroxylase),alpha polypeptide I[Bos taurus]
gi|99028973 transcobalamin |1[Bos taurus]
gil116004023 phosphoglucomutase 1 [Bos taurus]
gil77404273 glyceraldehyde-3-phosphatedehydrogenase [Bos taurus]
gi]40254806 heat shock 70kDa protein 1A[Bos taurus]
gil164420731 transaldolase 1 [Bostaurus]
gi|27806907 clusterin[Bos taurus]
gill
14051756 heterogeneous nuclear ribonucleoproteinA2/B1 [Bostaurus)
gi]51491841 transketolase [Bos taurus]
gi|77404217 phosphogtycerate mutase 1 (brain) [Bostaurus]
gi|119901059 PREDICTED:similarto COL9A1protein, partial [Bos taurus]
gil30794358 versican [Bos taurus]
gil168804008 tubulin,alpha, ubiquitous[Bos taurus]
gil27806941 serine (or cysteine) proteinase inhibitor,clade A (alpha-1 antiproteinase, antitrypsin),member 1 [Bos taurus]
gi]73853762 annexin I(Bos taurus]
gil119331178 SPARC related modularcalcium binding 1 [Bostaurus]
gi|41386727 protein kinase C substrate 80K-H[Bos taurus]
gil 19903961PREDICTED:similarto Protein disulfideisomerase associated 6 isoform7 [Bos taurus]
gil27806739 apolipoproteinE[Bostaurus]
gi]77736275 calumenin [Bos taurus]
gill 19900887PREDICTED:similarto collagen XXVIIproalpha 1 chain precursor; preproprotein[Bos taurus]
gi|l 19908661PREDICTED:similarto Laminingamma-i chain precursor (LamininB2 chain) [Bostaurus]
gil41386707 vascular celladhesion molecule 1 [Bostaurus]
gil1 19912531PREDICTED:similarto mannose receptor, C type 2 [Bos taurus]
gil157074106 lamin B1 [Bos taurus]
gi]27806489 proenkephalin [Bostaurus]
gi|27805823 dermatan sulfate proteoglycan3 [Bos taurus]
gil149773543 steroid-sensitive protein 1 [Bos taurus]
protein [Bos taurus]
gi|119900517PREDICTED:similarto KIAA11027
gi|1 15496928EGF-containingfibulin-likeextracellularmatrixprotein 2 [Bostaurus]
gi|27806623 fibromodulin[Bos taurus]
gi|148230364heterogeneous nuclear nbonucleoproteinD [Bos taurus]
gi|l 15497628reticulocalbin2, EF-hand calcium bindingdomain [Bostaurus]
gil119908681PREDICTED:similartosuperficialzone protein [Bos taurus]
gil156121327galectin 3 [Bos taurus]
gil66792902 vanin 1 [Bostaurus]
gill 14053121reticulocalbin3, EF-hand calcium bindingdomain [Bos taurus]
gij76253701 decorin (Bostaurus]
2 [Bos taurus]
gil115496400dihydropyrimidinase-bike
gil27806853 lumican [Bostaurus]
gir76670918 PREDICTED:similarto histone H4 [Bos taurus}
gi|115496892non-metastatic cells 2, protein (NM23B)expressed in [Bostaurus]
gi|114051505serpin peptdase inhibitor,clade H (heat shock protein 47), member 1, (collagen bindingprotein 1(Boo taurus]
gi|114052731tubulin, beta polypeptide [Bos taurus]
gi|119903031 PREDICTEDhypothetical protein[Bos taurus]
gi|119913640 PREDICTED:similarto KIAA0051isoform 1 [Bos taurus]
gi]78369684 platelet-denvedgrowth factor receptor-likeprotein [Bos taurus]
gi|78369242 alpha-actinn [Bos taurus]
gi]129277510superoxidedismutase 3, exracellular [Bostaurus]
giJ68299807 eukaryotictranslationelongationfactor 1 alpha 1 [Bostaurus]
gi|115497328 procollagenC-endopeptidase enhancer 2 [Bos taurus]
gi|115495641 aldo-ketoreductase family1, memberAl (aldehyde reductase) [Bos taurus]
gil76635416 PREDICTED:similarto coat protein delta-cop isotorm 1 [Bos taurus]
gi]76613946 PREDICTED:similarto Histone H1.5[Bos taurus}
gi|31341666 mannosidase, alpha, class 2B, member 1 [Bos taurus]
gi|119912215 PREDICTED:hypothetical proteinisoform 2 [Bostaurus]
0.3585 03520491 1.441514 0390379
77.40384 0.966386
78.62903 1.216501 0.259127 0.0281599 3321663 0.353157
36.45084 1.33925 0.388971 1.50E-05 3.942023 0671703
27.04115 0.821375 0.287995 00014648 0764914 0.254156
36.77419 0.865865 0.172443 0.0003491 0.859876 0.224315
37.2191 0.971629 0.373629 0.7172178 0.785828 0307669
33.27616 1.15534 0.39642 0.046568 4.541796 0872852
79.38932 0.511158 0.325391 00001318 0.658872 0.296719
28.72008 1.482349 0.771648 0.0160671 0920643 0226488
47.05882 1.108032 0.432033 0.1934376 3.759034 1190221
52.14106 0.58862 0.400078 9.840-06 1.051048 0312012
61.63265 1.814499 0.540997 6.97E-08 3.974334 0515406
36.08522 0.626223 0.552839 7.390-05 1.833769 0469972
0 1.113816 0373196
51.24654 0.578176 0.522216
48.02631 1.268557 0.360931 4340-14 0.477958 0414947
21.38872 1.133376 0.690707 0.3307672 0.796239 0337696
37.00234 0.791304 0.273901 0.0001075 0.619551 0354467
52.58427 1.455453 0.665949 0008821 2.596066 0.420476
46.64372 1.091578 0.252855 00988308 2241708 0.281875
57.22892 1.178704 0.390814 00287841 2.637623 049076
44.27861 0.635828 0.924922 00159708 1.665717 0.451871
59.62059 1.09486 0.399052 00115285 2.228327 0.503143
45.87912 1.713039 0.466647 6.290-05 2.469162 0060426
64.78873 0.97495 0.404706 0.8458203 1.650374 0481195
57.80142 1.094136 0.426337 0.1213981 0.538502 0559197
68.19572 1.438976 0.344794 00018496 2.485153 0432846
48.50187 0.882886 0.367268 00679135 2.495708 0.831659
40.74074 0.555233 0.459658 0002449 0.89322 0.31755
53.20285 1.19411 0.353038 00482391 3.098721 0.547299
51.65165 1.349932 0.412958 5.060-05 469257 0.709112
41.65367 1.218518 0.660476 02067183 3.891821 0.221504
53.41246 1.647513 0.78044 0.0085615 4.19667 0.48062
0 0.532673 0.499506
32.57403 1.370788 0.365556
58.94428 1.269216 0.423061 0.0054606 4.265354 0899902
33.22632 1.001777 042484 06601456 2.824623 0490068
53.1496 1.420449 0.457248 1.680-05 2.984226 0.47117
27.92608 0.624194 0.419346 8.106-05 0.064839 0.225205
10.85478 1.341758 0.270482 0.0081088 0.71699 0.204008
38.13747 1.128293 0.426549 0.125152 3.777497 0.530255
38.94231 0.626197 0.408455 1.03E-06 1.087044 0.370509
57.80347 0.688388 0.559084 0.801079 1.263143 0.685786
33.41014 0.729588 0.323378 4.740-06 0.731745 0.242091
21.95122 1.283851 0.412294 0.0045111 3.884186 0.617272
40.67796 1.533899 1.239035 0.0981442 3.564787 0911481
56.64557 1.054537 0.307699 0.3506565 0628845 0.339228
45.07937 0.883407 0.436466 00494385 2.731974 0.342826
25.86558 0.755179 0.415235 0.00621 0888638 0.36141
1 0.827303 0.389165
28.74016 1.08494 0.340788
27.0636 1.583077 0.40522 0.0002906 0.532135 0528182
17.45602 1.25875 0.477965 0.0562236 1.172813 0.242125
43.00341 1.07196 0.454305 0.8505681 1.065838 0.308361
1 7.706637 1.083169
64.25856 0.952292 0.418135
26.47975 0.869887 0.219256 0.0008093 1.87483 0.286394
29.24528 0.940334 0.471483 0.5422245 1.337722 0.359504
21.95986 0.828445 0.994186 0.496875 2.306418 0.605184
0.8642 0.253817 0.0109751 0.84039 0295555
37.47178
35.2 0.961092 0.294838 0.006021 1.173788 0304319
41.17647 1.313532 0.470427 0.0984023 4581735 0.403819
30.91483 1.01621 0.59814 08954841 3.288107 0.808578
24.15186 0.752636 0.778006 0.129217 1.654838 0303415
38.86792 1.084874 0.500628 0.1772784 25512 064266
26.66667 0.388952 0.930631 00001055 1.220544 0.252873
34.14634 1.847044 0.718336 0.0013333 2.905404 0.856876
49.72222 0.730415 0.440608 4.230-06 1.108696 0413871
32.16783 1.377001 0.288088 00027255 3.100286 0.478821
35.67252 0.972895 0.301185 0.4589595 1338501 0257497
83.49515 0.82703 0.405577 1.880-09 1.37621 025284
61.18421 1.105545 0.210209 00468387 3.00549 0607591
36,1244 0.848087 0.362412 0.0351232 1481406 0432926
36.93694 1.032283 0.352411 0.7898694 3.855163 0380602
25.41322 1.712802 0.342161 2520-12 3.081876 0.488811
20.07481 0.958993 0.426455 06774568 2.536098 0.811613
35.2 0.84085 0.295958 0.0089347 0747438 0.240368
38.90134 1.389165 0.49615 0.0307261 2.214585 0481167
37.3444 0.909759 0.26464 00382466 0.687629 0.32273
29.87013 1.047109 0.340916 00670238 277265 0.850019
36.29808 0.689359 0.234566 2.890-06 0922919 0292597
49.53846 1.22208 0.379178 0.0395591 3.068088 0579483
29.7456 1.08162 0.421572 0.5838214 3.853607 0483844
57.52212 0.819695 0.450096 0.0325495 1149782 059554
23.72372 0.886321 0.446803 0.222994 1.14973 0304272
23.12704 1.378899 0.582837 0.0605531 2.492031 0.596562
4560-08 1.719177 0423647
1.33E-05 3.62064 0.443649
1780-14 3.62686 0771161
00001114 1.27309 0.37747
00001221 0.928577 0.17773
8.210-05 1147188 0.631454
2.480-09 4595993 0879052
4.570-06 0.835857 022354
0.0535164 047494 0922694
6.270-06 6055416 1.259887
0.3067599 1.561361 0.35485
4.440-16 4638462 0480175
00001064 2.611883 0591251
0.0002071 206508 0494707
0 0 179124 1.014901
00016794 0754229 0614367
8.520-08 0.628954 0422447
1.02E-05 2.653669 0575952
1.980-09 2642202 0.270054
9960-10 3.071962 0.59957
2.200-05 4.118681 1045629
3650-14 1.816831 0.487552
1.700-06 3072349 0.599473
0.0294921 8610595 0783553
2.720-08 0.397764 1046779
1.8005 4.042242 0555698
00002931 2.80955 0794449
0.1206319 2.086042 0497411
6.110-06 3.580732 0574067
4220-14 5369332 0.734137
5150-11 4.338255 0.207845
00002441 4465234 0.648436
0 0.603443 0.479738
2.780-08 53571 0.784822
1110-06 3972913 0.60178
3930-14 3.274677 0388514
0.0215454 0.752822 0.528793
1.8006 1.023065 0.351439
3.47E-10 5.599331 0.713884
0.1216249 0.957381 0.350759
0.0385797 1.638153 0.77381
6.800-08 0.616842 0729459
6.300-10 3596102 0.706315
3.030-08 4.920584 1.044945
3080-08 0.648509 0.475921
7.710-10 3.035601 0310606
08164648 2.123001 0.409484
0.0384872 0.742105 1166543
0.0001742 0.882593 0341505
00180407 1.253979 0.746504
00021871 3.308644 0.312812
4330-07 12.92838 1.420231
1.560-09 1.079035 0.830543
0.0035578 4.364235 0730059
0.0008231 3.343639 0.889053
0.008025 0.639841 1.00329
4.840-14 1.285537 0509777
2070-07 5790203 0.396794
1.050-06 4.260653 0.758722
3790-05 2.538553 0.63436
1.330-15 3928305 0.670743
0003363 0881697 0.433523
2290-06 4.103241 0.641637
00897178 0.694329 0.522041
2820-05 3.50074 0.456305
6490-10 2.23113 0309532
9610-12 2707767 0451848
2100-06 0408232 0.943364
0.0007492 2208003 0.34101
2.720-06 4.388894 0533753
0 3.107097 0.490403
00002813 3.587219 0.567402
3.220-05 1154384 028933
0.0002806 2.756921 0533143
7010-07 0.544909 0.462822
354E-08 3.486767 0.884485
1.7825 0.651812
0.1495701
3380-06 3.802311 056824
0.0001858 4.440202 0538426
0.2488871 4845611 0.88057
07043
00627517 1343001
0.0001733 2746881 0582638
2.22E-16
3.21E-05
1.62E-12
0.00602867
0.03530605
0.18830764
3.OOE-1
0
8.44E-05
0.00077837
0.00029305
7.36E-10
1.23E-05
5.30E-05
6.65E-20
3.73E-16
0.02244257
1.87E-06
8. 12E-05
3.02E-10
6.87E-10
1.15E-06
4.16E-13
4.76E-07
0.00089462
3.22E-07
2.86E-07
8.57E-07
3.87E-07
3.24E-06
1.31 E-14
1.22E-11
4.82E-08
0
8.79E-10
1.44E-08
8.30E-06
0.01025391
0.74156451
1.06E-11
0.39952894
0.00058879
0.00040001
4.30E-08
1.831E-10
1.18E-05
6.01 E-11
1.71E-06
1
0.11070289
0.1999653
0.00024721
6.07E-06
0.17895611
6.78E-07
2.23E-05
0.03911204
9.00E-13
5.72E-08
4.14E-08
8.91E-05
0
0.05794988
0.00012207
4.45E-05
1.01E-05
8.75E-07
0
7.28E-08
1.23E-08
9.47E-06
0
3.54E-06
0.02406251
0.0001105
1.58E-07
1.84E1-09
0.00059758
5.36E-07
0.00018981
2.64E-07
0.06036566
6.89E-05
gi|62751849 Parkinsondisease (autosomalrecessive, early onset)7 [Bostaurus]
9i[139949116
slit-like2 [Boslooms]
gi[1561205Dl
4 05 26 53 heat shock70kDproteinbindingprotein[Bostaurus]
procollagen
C-endopeptidase enhancer[Bostaurus]
gill1
component1 inhibitor[Bostaurus]
gi27807349 complement
6 [Bos taurus]
gi|27807167peroxiredoxin
gi|77735541valosin-containingprotein[Bostaurus]
gil148238309 aldolaseC, fructose-bisphosphate[Bostaurus[
gil27807007 insulin-likegrowthfactor bindingprotein3 [Bostaurus[
gi75812940 phosphabdylethanolaminebindingprotein[Bostaurus]
gil27806671 cathepsinB [Bostaurus]
PREDICTEDsimilarto collagen,typeIX, alpha2 [Bostaurus]
gi|119920777
similarto Collagen,type IX,alpha1, partial[Bostaurus]
PREDICTED:
gi|119901068
13 [Bostaurus]
gi27805999 matrixmetalloproteinase
gil27806637 fibrillin1 [Bostaurus]
gil27806617 GDP dissociationinhibitor1 [Bostaurus]
recognitionprotein1 [Bostaurus]
gil27808640 peptidoglycan
gi|157427782 insulin-likegrowthfactorbindingprotein5 [Bostaurus]
similarto cytokine-likeproteinZCYTO7isoform 1 [Bostaurus]
PREDICTED:
gi|119895711
gi|78369298stromalcell derivedfactor 4 [Bostaurus]
alphapolypeptide|| [Bostaurus]
(proline4-hydroxylase),
2-oxoglutarate4-dioxygenase
gi|77735539procollagen-proline,
[Bostaurus]
activationprotein,epsilon polypeptide
5-monooxygenase
gi|27806197tyrosine3-monooxygenase/typtophan
protein[Bostaurus}
gi|119919662
PREDICTED:similarto LOC539445
chemotaxin2 [Bostaurus
gi|27805983
leukocytecell-derived
gi|116003813amyloidbeta A4 protein[Bostaurus]
gremlin-1[Bostaurus]
gi|129270171
gil148228060 stem cell growthfactor[Bostaurus]
p465
pEndoplasmic
gi|119915902 PREDICTED:similartoThioredoxindomain-containing proei5precursorThioredoin-keprotein
1 [Bostaurus]
gi|27806401 secretedphosphoprotein
gi|76613223 PREDICTED:similartochitinase[Bostaurus]
gi|77404209 eukaryotictranslationinitiationfactor 4A2[Bostaurus]
PREDICTED:similarto serin proteasewithIGF-bindingmotif[Bostaurus]
gil119917871
X [Bostaurus]
gil164448628carboxypeptidase
glialfibrillaryacidic protein[Bostaurus]
gil113205970
32 family,memberB [Bostaurus)
gi178045555 acidic (leucine-rich)nuclearphosphoprotein
similarto LaminB2 [Bostaurus]
gi|119895049PREDICTED:
gi]27806829osteoglycin[Bostaurus]
protein[Bostaurus]
gi]78045491fascin homolog1, actin-bundling
largesubunitisoform3 [Bostaurus]
similarto Calpain2, (m/11)
gil119908208PREDICTED:
gi[62751777cofilin 1 (non-muscle)[Bostaurus]
gi|78042524 thioredoxindomaincontaining4 (endoplasmic reticulum)[Bostaurus]
gil27806449 dystroglycanI [Bostaurus]
gi]94966781 eukaryotictranslationelongationfactor 1 gamma[Bostaurus]
gi|164420791transforming growthfactor,beta2 [Bostaurus]
gil 14053333fibrinogen-ike 2 [Bostaurus]
gi[95006989 ribonuclease, RNaseA family,4 [Bostaurus]
1 [Bostaurus]
gi]27806081peroxiredoxin
B [Bostaurus]
gi|27806561lactatedehydrogenase
gi|77735425proteasomealpha7 subunit[Bostaurus]
factor 1 (macrophage) [Bostaurus]
gi|27806679colonystimulating
similarto tolloid-like2 proteinisoform2 [Bostaurus]
gi|119917542PREDICTED:
1 (splicingfactor2, alternatesplicingfactor)[Bostaurus]
gi|115497078splicingfactor, arginine/serine-rich
aminopeptdase 3 [Bostaurus]
gi|165905571
gill4051129 retinoicacidreceptorresponder(tazarotene induced)2 [Bos taurus]
gil157073966 reticulocalbin1,EF-hand calciumbindingdomain[Bostaurus]
1 [Bostaurus]
gi]27806541 matrixmetalloproteinase
gir41386760 CD14anbgen[Bostaurus]
similarto CUB andEGFcontainingprotein[Bostaurus]
gill 19915070PREDICTED:
2) [Bostaurus]
(aspartate aminotransferase
transaminase 2. mitochondrial
gil27807377 glutamic-oxaloacetic
0 ribonuclease/angiogenininhibitor1 [Bostaurus]
gi|7836951
gi|118601868heatshock90kDaprotein1,beta [Bostaurus]
[Bostaurus]
monooxygenase
alpha-amidating
gi|27806825pepbdylglycine
gi|82697375 histoneH2B-like[Bostaurus)
synthase2 [Bostaurus]
5'-phosphosultate
gi|1154971743'-phosphoadenosine
[Bostaurus]
similarto Man9-mannosidase
gi|119901198 PREDICTED:
giJ28603774 RhoGDP dissociationinhibitor(GDI)alpha[Bostaurus]
giJ71037405 heat shock27kDaprotein1 [Bostaurus]
[Bostaurus]
giJ27806751alpha-2-HS-glycoprotein
[Bostaurus]
gi|77735769complementcomponent1. r subcomponent
gil 19894807PREDICTED:interleukinenhancerbindingfactor 3 isoform2 [Bostaurus]
gi[94966765glucosephosphateisomerase[Bostaurus]
nucleobindn2 [Bostaurus]
gi[115496067
gi]27805977keratin10 [Bostaurus]
PREDICTEDsimilarto PDNP1[Bos taurus]
gil119901449
1, NAD(soluble)[Bostaurus]
gi[77736203malatedehydrogenase
gil41386798poly(A)bindingprotein,cytoplasmic1 [Bostaurus]
gil27806881epididymalsecretoryproteinE1 [Bos taurus]
gil27806415scinderin[Bostaurus]
gi|76610312 PREDICTED:similarto neuropilin2 isoform8 [Bostaurus]
activationprotein,gammapolypephde[Bootaurus]
5-monooxygenase
gil157744500
tyrosine3-monooxygenase/tryptophan
gir27806673 cathepsinL2 [Bostaurus]
[Bootauras4
gi]30794292 lactotransarrin
leucine
7089947 1.612388 0.31221 2.578-05 3.98492 0400179 2.81E-09 3.820856 0.679266 2.75E-08
28.52897 1.338105 0429092 00002926 0.904302 0.27225 0.0308789 1.227725 0.591427 0.13297131
28.18429 1243113 0.239059 0.0227445 2.67794 0.884008 0.0013486 3.156577 0.779729 0.00134692
41.24188 0.747768 039671 0.0010757 0.907298 0240483 0.0277299 0.975545 0.645648 0.8278468
2284957 1.934870408844 3.788-10 028844 0.842258 0.00023321.154815 0.379542 0.01799357
58.25 1.5748580.471361 00005459 3.539818 0.728845 1.10E-064.112762 0.687028 4.97E-09
3325082 1155745 1.015324 0.578125 1.982845 0.741708 0.015625 3.081692 0.629709 0.015625
4835185 1.292778 0.681553 0.193125 3.157838 0.899738 0.0002856 3.731659 1.033639 0.015625
371134 0.788581 0.778124 0.0288555 1761299 0.338075 1.65E-06 1.708408 1.506558
6258885 1.289117 030763 0.00282 3.01298 0.489092 3.13E-09 3.746912 0.524693 1.19E-09
31.34329 0.943841 0205782 0.0327489 1.389304 0.258249 0.0081208 0.504166 0.829374 0.0330881
2981104 0.54183 0.958949 0.00289950.822455 0.288062 0.0019401 0.609309 0.603253 0.00108429
54.0148 0.482305 0,458428 0.00032640.758333 0.193313 0.0005563 0.499832 0.555497 0.00140861
2590234 0449574 0.439799 0004883 0.398849 0.248278 1.14E-090.58289 0.213158 6.72E-08
0.0080701 0691041 1.667035
1605712 1158511 0.333925 0.31250850119 0.140244
3534878 1.814872 0.9995 0.0098531 3508770 0.548211 0.0005312 3.6348620.593508 2.34E1-09
51.05263 045803 0.326152 1.70E-080.808319 0.257445 0.003467 0.400714 0.724958 0.00027455
50.92251 1181072 0354524 00280809 0.906282 0.48406 0.9013177 2.080596 1.098557 0.00157819
1.077054
2748781 0507187 0.877454 3.35E-06 1.17427 0.37824 0.0084102 1.935233
42.25352 0778141 0.33739 0012125 0.85096 0.159828 0.0034501 1.083191 0.272478 0.24000671
24.22998 077955 0452039 005827 2.820207 0.308505 4.25E-062.514681 0.302407 4.98E-06
1.44E-11
72.94118 1.358303 0.443855 00002031 3.00073 0.552971 3.00E-11 3.319728 0.62026
0.5854520780314 0.0452885 4.897555 0.831237 9.61E-05 5.847062 0.859128 2.02E-05
189
642341 0.409485 0331874 7]74E-08 0.990859 0.311931 0.6551496 1.049618 1.145164 0.70533302
29.064751.538547 0.230885 00014379 0.840781 0.298271 0.086367 1.247608 0.218664 0.01595411
51.83044 0793555 0.87352 0141028 1.001231 0.337648 0.8968756 1.140038 1.154018 0.01479114
3240741 0848954 0.267988 00194377 1.002715 0.27112 0.9519144 1.23396 0.252748 0.00390625
1 [Bar 3947389 1.258969 0.559088 0.05354583.870127 0.817854 4.28E-13 4.664796 0.711726 4.55E-14
protein Epp446iso)otm
re(dculuo
37.78979 0.903294 0.221566 0.0022755 1.915212 0.235771 4.44E-16 1.401041 0.245955 2.70E-10
39.01809 030445 0.493303 00004883 0.209851 0.404558 7.54E-10 0.07019 0.829902 5.78E-09
3438803 1.2337720388759 0.21812943.109795 0.252134 0.0010031 3.010434 0.426075 0.00498072
2020849 0.771777 0.243891 8.73E-050.081847 0.202598 0.224032 0.981734 0.433694 0.29577637
19.883010.788564 0268784 0.001439 1.008874 0.249295 0.9681949 1.339499 0.394484 0.19335938
0.03125
48.83178 1.027088 0483528 0.8502459 2.292389 0.6884970.0082859 3.098982 0.936698
1.97E-05
28438 1.143311 0510882 0.3222514 3344078 0.476514 1.78E-05 4.841424 0.63541
32.781481.343139 0.667012 0.1147527 1.874172 0.482114 0.00117 2.5220160.602373 0.00041745
34.78281 0.858085 0.220385 0.00211511.021554 0.284641 0.7655521 1.061728 0.3270.34082302
23.32857 1.3344580.4889 0.01083013.119513 0.442908 0.0024413.158849 0.428223 8.73E-09
2.089055 0.255707 0.00015682
23.83014 0.777731 0.805924 0.25078992.057831 0.459223 0.0027078
59.03814 1.249729 0.371271 0.08799523.522417 0.42509 2.95E-05 4.929116 0.442864 9.21E-06
2508207 0.995942 0.314542 0.833009 2.889886 0.573365 0.03125 2.574683 0.61755 0.00110559
18.32402 1114704 0.428948 0.12392920.989739 0.21681 0.6524901 1.604039 0.364926 0.00193099
26.59091 1.182179 0847073 0.45095113.044482 0.592818 0.0011996 3.21599 0.638340.00131691
30.43478 0742424 0.259859 00022421 1.87391 g.261033 9.46E-05 2.987197 0.773021 0.00067956
3850784 1.17009 0309784 0.04671230.427778 0.195793 9.33E-09 0.4443880.340456 1.78E-06
57.1428 0804356 0.385366 2.98E-09 0.917848 0.249674 0.0003852 0.639449 0.405761 1.40E-07
8.24E-05
4.558172 0.460837
47.7389 1.1456550.251919 0.11600143.559536 0.494199 0.0802523
47.30539 1.217427 0.422007 0.09978333.073825 0.570874 0.000102 3.6255480.586296 4.38E-05
1.3595 0.250214 0.00751124.311514 0.535948 4.73E-05 5.37498 0.447148 7.15E-06
4508452
21.94115 1.5128 0.285552 0.015625 0.008248 0.535002 0.0208893 1.487397 0.40853 0.00297115
18.33333 0.558517 0.413338 0.00183580.932274 0.44272 0.1525279 1.677954 0.435032 0.00392804
54.83871 1.197937 0.333368 0.03353993.03228 0.520847 1.40E-06 3.276995 0.605293 2.84E-06
29.28709 1.483983 0514998 0.04256542.252881 0.725022 0.0111017 2.875392 0.698814 0.00308652
5123457 0668587 0341849 2.548-09 0.705825 0.385304 2.47E-07 0.775408 1.0322070.08804999
33.83686 110571 0.39971800947473 3.231942 0.338122 1.31E-08 3.505302 0.382501 2.27E1-08
31.5565 0.272697 0491877 1.30E-050.887994 0.145805 8.60E-06 0.954822 0.11586 0.7421875
3646113 0.952161 019569 0.25332950.378479 0.350338 1.01E-07 0.619293 0.556359 0.00207345
14.501510 78g134 0409551 0.0356442 0.90833 0.309445 0.4955312 1.431923 0.783978 0.08278948
36.74419 1.406094 0270802 00067711 3.928337 0.298463 1.67E-05 4.352382 0.349685 2.49E-05
2939597 1.501005 0325888 0.00704174.131356 0.247004 0.03125 3.17242 0.74366 0.00274529
3991713 1.179283 0338089 0.1518089 3.748732 0.368913 5.46E-05 4.628471 0.337131 1.70E-05
17.90123 066916 0.256285 0.00261280.921522 0.1398 0.0751198 1.025040.244148 0.73498679
73.015g7 0701006 0.441253 00220058 1.387118 0.449981 0.0345741 3.2462320.402594 3.08E-05
299197 1.099204 0395566 0.3926138 3408799 0.393327 4.44E-06 4.956252 0.403776 8.39E-07
27.6176 0669272 0337197 00039794 1.227779 042194 0.122205 1.555083 0.420027 0.0070192
0.633009 0.00056677
46583 1693909 0506794 0.015072 3518611 0682599 0.0013047 4.018423
50 0.99641 0.516659 0.975981 4.504843 0.777647 0.000323 5.119911 0.722672 0.00013365
25.90529 0280017 0351435 4939-08 0.44843 0.269395 2.37E-07 0.211971 0.516017 2.45E-07
0.0093022 0.608092 0477975 0.0267146 0.404853 0.967008 0.03929655
2994924 0559876 0.399
0.0625
2095075 1.456753 0582043 0.1058396 2103822 0.172287 0.0001595 2.924806 0.509752
29.08438 1.019204 0.584551 09159895 2.748777 0.93774 0.0082061 3.1795290.941867 0.00742862
2843373 0.9779950519511 0.8450852 1.283333 0.328989 0.0082484 1.505560.279417 9.05E-05
23.19392 0794292 0.417656 0.0875888 0.9189270.357075 0.368511 D.926095 0.310298 0.38139633
20.31873 1122001 0.570995 0.3842895 2.127114 1.082098 0.0116401 2.076167 1.308587 0.03135567
4.334892 0.225396 2.75E-08
30.23952 1620919 0254319 00001127 4.000058 0.345863 8.00E-07
0.1875 2.9200830.559438 0.001099 4.082206 0.496975 0.00017076
29.24528 1.241022 0.516059
0002295 1.695337 0.153522 0.000394 1.4882690.099131 0.00020578
3087249 0.454079 0.389
2293708 1.300330.289782 00434031 2.595421 0.60842 0.007284 3.324911 0.661368 0.00423199
2915767 1.3179350334412001207330.7882580.331260.0126559 0.965049 0.22483 0.53905575
51 01215 1.1957100515928 0.327959 2888529 0.338074 0.0005647 3.56837 0.347956 0.00029579
0.625
3502994 1190907 0253439 0.1028135 1.025708 0.10081 0.6850116 1.0336170.762904
2387006 0.69982 0.444285 0.0310855 0.538608 0.978791 0.0738598 0.571186 1.170383 0.15162747
0.08921194
0.46885429
0.00245713
gi|76620378 PREDICTED:similarto cytokine-likeproteinC17 [Bos taurus]
gi|125991942 Sec23 homologA [Bostaurus]
gi|845798534 lipopolysaccharidebindingprotein [Bos taurus]
[Bos taurus]
gi|11990114 PREDICTED:similarto malate dehydrogenasedecarboxylase(NADP+)
gill14051157 complement component 1, q subcomponent, 8 chain [Bos taurus]
gi115497294 canopy 2 homolog [Bos taurus]
gi|76651183 PREDICTED:similarto histone H3 [Bostaurus]
gi|27807523 ribosomalprotein P2 [Bos taurus]
gi|156121049 dimethylargininedimethylaminohydrolase1 [Bos taurus]
gi|75832065 TIMPmetallopeptidaseinhibitor2 [Bos taurus]
gi]61888850 adenylate kinase 1 [Bostaurus]
gi|28875793 thrombospondin2 [Bos taurus]
gi|119923009PREDICTED similarto put. HMG-17protein [Bostaurus]
gi|148232266fibulin1 [Bos taurus]
gi|119890932PREDICTED:similarto Lamininbeta-1 chain precursor (LamininB1 chain) [Bos taurus]
gi|119896006PREDICTED:similarto mannosidase, alpha, class 2A, member 1 [Bos taurus]
gl155372035 nidogen 1 [Bostaurus]
gi]62460514 angropoetin-ike 7 [Bostaurus]
gil164414726thymosin beta 4 [Bos taurus]
gi|27806095 protein S, alpha [Bos taurus
gi|45429977 staphylococcal nuclease and tudor domaincontaining 1 [Bos taurus]
gi|27807209 alpha-2-plasmininhibitor[Bos taurus]
gill 16004039peptidylprolylisomerase C (cyclophilinC)[Bos taurus]
gi|119914274PREDICTED:hypothetical proteinisoform 1 [Bos taurus]
gil27806553 microtubule-associatedprotein4 [Bostaurus]
gi45430019 ribosomal proteinL12 [Bostaurus]
nucleoprotein isoform1 [Bostaurus]
gil119919151 PREDICTED:similarto AHNAK
gi|119919898 PREDICTED:hypothetical protein[Bos taurus]
gi|119908402 PREDICTED:similarto Calsyntenin-1,partial [Bos taurus]
gl157785567 calnexin [Bostaurus]
giJ78369456 nucleophosmin(nucleolar phosphoproteinB23, numatrin) [Bos taurus]
gil119331192 H2Ahistone family,member X [Bos taurus]
gil27807469 peroxiredoxin2 [Bostaurus]
gi[77736471 SH3 domainbindingglutamic acid-rich proteinlike 3 [Bos taurus]
gir76611607 PREDICTED:similarto C1QC proteinisoform 1 [Bos taurus]
gill18151330 tyrosine 3-monooxygenaseltryptophan5-monooxygenase activation protein, theta polypepsue 005 taurus]
gi|119892706 PREDICTED:similarto chondroitin4-O-sulfotransferase[Bostaurus]
gil60592767 ribosomalprotein, large, PO[Bos taurus]
gi|62751593 proflin 1 [Bos taurus]
gi|78042564 CNDPdipeptidase 2 [Bos taurus]
gi27806625 frizzled-relatedprotein [Bos taurus]
gil119901416 PREDICTED:similarto Syncrip protein [Bostaurus]
gil29135295 aspartate aminotransferase I [Bos taurus]
gil110626119 insulin-likegrowth factor bindingprotein 6 {Bostaurus]
gi|119907521 PREDICTED:similarto NuMAprotein[Bos taurus]
gi|155371853 thrombospondinrepeat containing1 [Bos taurus]
gi}114052384 heterogeneous nuclear ribonucleoproteinA1 [Bos taurus
gi|115495555 endoplasmic reticulumprotein29 [Bos taurus]
gij27806279 caldesmon 1 [Bos taurus]
giJ78042498 phosphoglycerate dehydrogenase [Bos taurus]
gi|27806447 prosaporsin[Bos taurus]
gil27805981 ribosomalprotein SA [Bostaurus]
gil155372057 cysteine-rich secretory protein LCCLdomaincontaining 1 [Bos taurus]
gil125630701 collagentriple helixrepeat containing1 [Bos taurus]
gill18151356 angiogenin, ribonuclease. RNase A family,5 [Bos taurus]
gil28461187 myosin,lightchain 6, alkali,smooth muscle and non-muscle[Bos taurus]
gir27805805 fatty acid bindingprotein 5 [Bos taurus]
gi56119114 purine nucleoside phosphorylase [Bos taurus]
gi|61097917 cystatin E/M[Bos taurus]
gi|1 19888578PREDICTED:hypotheticalprotein [Bos taurus]
gi|56710317 keratin 5 (epidermolysisbullosa simplex, Dowing-Meara/Kobner/Weber-Cockaynetypes] [Bostaurus]
gil119919488 PREDICTED:similarto cathepsin D isoform 1 [Bos taurus]
poly(rC)bindingprotein 1 [Bos taurus]
gi]62751650
hemoglobin,gamma [Bostaurus]
gi|62460494
gi|116004251 granulin [Bos taurus]
[Bos taurus]
gi|27806813 pleiotrophir
gi|30466254 gelsolin-likecapping protein[Bos taurus]
gil28461287 hepatoma-derived growthfactor (high-mobilitygroup protein 1-like)[Bostaurus]
subunit,alpha type. 1 [Bos taurus]
gil78369458
7 proteasome (prosome, macropain)
gil2780714 C-type lectin domainfamily3 member A [Bos taurus]
gil27806139 secreted frizzled-relatedprotein 1 [Bos taurus]
gi|115497736offactomedin-like3 [Bos taurus]
gi|134085635glyoxalase I{Bos taurus]
gi|119919689 PREDICTED:similarto RNAbinding motifprotein, X-linkedisoform 5 [Bos taurus
similarto myosin,heavy polypeptide 9, non-muscle [Bos taurus]
gil119892775
7 PREDICTED:
gi]6275184 ribosomalproteinLlOa [Bos taurus)
gi|164518978 diazepam bindinginhibitor[Bos taurus]
gi|77735725 cathepsin H [Bos taurus]
gil119926480 PREDICTED:similarto Meteorin,glialcell differentiationregulator-like[Bos taurus]
gil119907290 PREDICTED:hypothetical protein[Bos taurus}
gi]77735597 FK506-bindingprotein 4 [Bos taurus]
gi|157074098 leprecan 1 [Bos taurus]
52.55474 0.772804 0.448173 00201767 0.914237 0365116 02366615 0617444 0571456
16.92708 1.40908 0.550793 0.1706566 4321113 0.756614 0.011496 4706377 0.624164
23.28482 0.195757 0.44865 0.000302 0.406175 034643 0.0002543 0.329361 0,643969
20.62147 1.467496 0.340693 00106179 4.224115 0.462441 1.671-07 4.233192 0493512
0.3034 0.0002543 1.784666 0.272691
31.17409 1.103934 0.2474 00117622 0.631504
5.045-07 3777664 0.363764
57.14286 1.031638 0.370242 0.7466377 3.754973 0393293
55.14706 0.842391 0.226061 00045065 1.355866 0233258 0.0001135 2.695744 0069296
57.3913 1.883075 0.8785 00516846 4409362 0.981136 0.0012072 6.355423 0687346
55.08772 1.236429 0.555719 0.4416299 2.976156 0.624865 0049293 2773325 0.600857
40.90909 0.867925 0.293951 0.0170916 1.479176 1.09336 0.0412596 1.025353 0.241434
38.14433 1.136216 0.430822 0.3033108 3023313 0565108 0.000306 4901766 0.59492
13.24786 1.005457 0.336821 0.9553344 1109125 0210213 0.1260952 1.450622 0313212
1.110-05 4168457 0659649
42.22222 0.646482 0.829652 0.0304121 2144563 0499891
0625 0605337 0.326422 00923653 0.664477 0377662
15.58074 0.887858 0.247168
15.35245 1.354883 0.234493 00060169 1148729 0.449674 0.3464434 1.046109 0.20613
0.4375 0993192 0.234221 0.6643037 1.267403 0479661
16.59389 1.243177 0.281497
16.6801 1.231962 0.36954 0.1034256 0877971 0.561423 0.4293699 1.070923 0.456856
20.93023 0.968973 0.470794 0.6187797 0.373047 0497327 0000686 0300522 0743839
81.81818 1.30961 0.482918 01060539 6.545266 0.647427 0.000154 9.062092 0597922
18.66667 1.17304 0.332141 0296675 1.146996 1368479 0.296675 1250631 1.242364
28.24176 0.986197 0.378524 0.6972496 3.137771 0.444296 00002761 4365947 0.341663
0.942 0.386282 0.6624672 1.042147 0.242856 0763677 3.120632 0.202312
18.90244
44.33962 0.746961 0.398982 444E-05 0.90611 0322438 0.0274032 1.201796 0.446562
20.91429 1.114657 0.268242 03297717 3.595856 0387989 0.0056739 3.135764 0308311
25.74627 0.784726 0.556233 02964631 1.941072 0453118 0024877 2.856925 047348
00625 4.543992 0804589
43.0303 1.051228 0.153254 0.3578261 3.399142 0.621972
36.92494 1.238706 0.309334 0.0900441 2.705998 0.997999 00059426 3.486668 0.71653
24.85714 0.696563 0.354593 0.0002441 0873866 0.394239 00651016 1113116 0.402894
17.28135 0.971916 0.307732 07515049 0.937539 0.153447 0.1634666 1306979 0.201769
19.56155 0.981782 0.4997D2 09194103 3.107429 0.510515 00078239 3.498451 0343663
33.67347 1.151309 0.476669 0.4582779 2.610443 0.613626 0.0154491 3.539745 0.916422
62.23776 0.721476 0.046847 0.0032687 1.331159 0389176 0.2144017 3.195369 0.528406
46.23116 1.335529 0.376182 0.0165782 4.071044 0.517156 112E-05 4.946965 0579555
3.9735 0.602612 0.0009766 4.226144 0.6916
53.76344 1.523003 0.241733 0.0005666
21.69118 1.196257 0.499483 0.1373665 0.713353 0360547 0.0018497 1715993 0.691086
2.885-05 4.270728 0.235607
53.46939 1.666969 0.181633 00001768 4.070146 0340142
33.28197 1.328366 0.280622 0.0160601 0.750151 0.456758 0.1076629 1.084646 0.033707
38.67925 2.339525 1.705012 0494 4967163 0752382 00006669 5.276421 06488
47.85714 0.984943 0.229807 0.7732803 2.827048 0.262665 2.065-07 3961251 0.95482
28 0.759355 0.657534 0.3126366 1393516 0.063048 00613242 2.115963 0.493526
27.69231 0.611032 0.397021 270E-07 0.947817 0.181766 0.0071035 1.868062 0.433897
30.4078 0.978475 0.537585 0.8965113 245728 0.593261 0.0083154 2.944606 0.662037
1.4351 0.81286 0.5316989 4.332814 0.399891 0.0649655 4.030831 0.157902
29.05569
39.28692 0.515588 0.843656 0.0021144 0.6635 .30365 0.0002922 0.787142 0.499883
24.79962 1.310653 0.767904 0.4724615 1.570161 0.931907 0.3025977 1.900436 0.772411
16.07477 0.609054 1.186849 0.246469 0.692773 0.161206 0.0026575 0.600518 0.842992
32.5 1.401466 0.278818 0.0398761 2.349795 0.462218 0.0101637 3.707999 0.375063
40.31008 1.209341 0.469429 0.2132029 5887609 0.59221 5635-08 6.924352 0.649171
0.655 0,0466966 5.043265 0771672
27.66355 1.521052 0.861412 0.3484215 3.234318
29.26829 1.091927 0.23322 0.293771 1.915942 0.338410 00049064 2.520743 0.607506
15.42857 0.780148 0.148658 0.0169044 0.994096 0,250669 0.8962366 0597473 0.756674
30.50847 2.017567 1.029917 0.1442065 6706982 0.714377 00002276 7.159304 0659366
23.2 0.718178 0.660928 0.1807677 1.217623 0.210773 0.3434273 1.521064 0.491197
46.50206 0.939134 0.253669 0.3765814 0.866708 0.194334 00261033 1.124384 0.360969
0.3125 0.770496 0.740227
42.56757 0.561035 0.269208 5.04E-05 0.904662 0.266371
44.37086 1.189206 0.334906 0.0966763 2.372D42 0.481176 0.0076125 2.789196 0.525725
53.33334 1.143116 0.588056 0.5057353 2.664728 0.465376 0.0007116 3.373152 0.539626
22.83737 1.077707 0.16977 0.386629 2.49569 0.480878 0.0420877 328739 0.453214
41.61074 1.588616 0.617223 0.0730954 2.46493 0470962 0.0035763 1023269 0645059
33,33333 1.305207 0.579915 00531219 5.156013 1.36457 4.395-05 5291402 1.331999
28.1198 0.568797 0.044792 0.0247297 0755315 0.62039 05163629 0796984 0.22566
25.97087 1.343171 0.029007 0.0015513 2.009387 0265239 00227652 1591036 043976
33.98876 1.676168 1786418 0.5455095 3122778 0.520259 00021674 4.541486 0514743
70.34483 2.79713 0.550535 00125265 1.199678 0402669 02966016 3962602 0.365669
16.18399 0.937838 0.30186 0.5792656 1.237269 021475 0.0693237 0351941 1.002473
42.2619 0.334023 0.63773 0.0002441 1.375822 0.39724 0001486 1.622514 0.624073
19.19771 1.580628 0.619966 02067068 2.304159 0.90919 0.150557 2794152 0.666304
51.88285 1.156699 0.776058 0.627433 274657 0.75906 00130126 3205741 0.596991
25.47528 1.348022 0.391818 0.015625 3.217466 0590741 0.0002686 3.529642 0.839054
38.07107 D.487568 0.137961 0.0006384 0.713065 0.236462 0.0251242 0728636 0.403697
35.71429 0.600276 0.353855 4.14E-05 1.010426 0.232691 0936477 1.375163 0.703576
19.21182 0.461024 0.437169 0.0046268 115529 0.332775 02520667 076319 0.569631
0.03125 3.196009 0.427377 0.0002126 4.158412 0.723263
35.86957 1.343524 0.194285
5.9913 063796
48.59335 0.957059 0.484712 0.6080712 3.306448 0.533767 00073567
17.04835 1.130043 0.300774 0.3276844 1.40175 0.407355 00565616 2.059305 0453447
37.78802 1.390803 0.337679 0.067115 5065662 0.510943 00005196 5632550 0.559054
144E-09 5756974 0.420637
72.41379 1.865069 0.402858 910E-06 4.57462 0415176
29.55224 1.142346 0.205271 0.1595653 1.684237 0.456006 00477095 2.935988 0.451667
25.24272 0.59365 0.376463 00018407 0.64177 0328059 00629004 0692403 043771
20.61856 1.765132 0.057721 0.0016523 0322906 0.194399 00041666 0.476651 1.69626
22.22222 1.183397 0.220437 0.1976026 2.247146 1.107326 0.0793661 3.249727 0.986715
14.94565 1.197811 0.733036 0.6022613 2.296675 0.279972 0017423 2.512035 376722
0.10564933
0.01229541
0.00172116
6.82E-08
7.49E-13
0.00390625
4.41E-06
0.00020993
0.05132816
0.57150453
5. 18E-05
0.00186412
3.26E-07
0.28146037
0.46117897
1
0.58778549
0.00229874
4.75E-05
0.296875
2.20E-05
0.00050626
0.00667512
0.00174715
0.00772229
0.00372183
0.00164087
0.15939404
0.00539029
0.00183715
0.02854164
0.03157637
9.74E-06
0.00195313
0.00976563
3.79E-06
0.61365111
1.61E-05
5.02E-06
0.02201728
2.04E-14
0.00694147
0.03530835
0.06738705
0.17339178
0.12274193
0.00207823
5.67E-08
0.009058
0.00418842
0.14392217
0.00013219
0.05113466
0.26137904
0.7421875
0.0078125
0.00050396
0.00476912
0.91396118
2.91 E-05
0.28874226
0.11860294
0.00014232
0.00195396
0.05740508
0.00165727
0.09747653
0.00329443
0.00121962
0.1087656
0.05549302
0.20064388
0.00093924
0.00393705
0.00679523
0.000056759
3.51E-10
0.00628833
0.01847104
0.38921172
0.01837422
0.02575885
38
[Bostaurus]
gi]8885 16 superoxidedismutase2, mitochondrial
[Bostaurus]
activationprotein,beta polypeptide
5-monooxygenase
gill15432055tyrosine 3-monooxygenaseltryptophan
[Bos taurus]
alpha
subunit
protein
complex,
coatomer
gil157785569
4
protein(809taurus)
gil1199050DPREDICTEDsimilartogrowth-anest-specioc
oncostatn Mreceptor[Bostaurus]
gil122692559
gi76613071 PREDICTEDsimilarto class mu glutathione S-transferase isoform 1 [Bostaurus]
gi]83715992coatomerproteincomplex,subunitgamma1 [Bos taurus]
tract bindingprotein1 [Bos taurus]
polypyrimidine
gi|27806103
specificfactor [Bostaurus]
periostin,osteoblast
gi|95147666
gi|77736071heterogeneousnuclear rbonucleoprotein K [Bos taurus]
PREDICTEDsimilarto histoneHix[Bostaurus]
gi|119914936
1,q subcomponent, A chain [Bos taurus]
component
gil6246D582 complement
similarto peptidyl-Procistrans isomeraseisoform 1[Bos taurus]
PREDICTED:
gil119902257
[Bostaurus]
alpha-1-microglobulin/bikunin
gil27806743
42
similarto collagentype 5 alpha1 {Bostaurus]
gil1990 80PREDICTED:
D [Bostaurus]
gill
15494984apolipoprotein
gi|77735583 S-adenosylhomocysteinehydrolase[Bos taurus]
2A, regulatorysubunitB [Bos taurus]
gill 14051445proteinphosphatase
3 (Bos taurus]
gi|114052264phosphoglucomutase
group box1 {Bostaurus]
gi41386729 high-mobility
IsA NovelBacteriostacAgentThat nereresith Siderophore-MediatedIronL
p6
gil119904118PREDICTEDsimilarto ChainA,NeutrophilGelatnase-Associated tipocalin
similarto latenttransforming growthfactor beta bindingprotein3 [Bostaurus]
gil119919315 PREDICTED:
similarto adlican[Bostaurus]
PREDICTED:
gil119920682
gi|164452935SWAP-70protein[Bos taurus]
hypothetcalproteinLOC535277[Bostaurus)
gil155372051
4 {Bostaurus]
gil27806085peroxiredoxin
gi]27806087probneatgirine-nchandleuome-nchrepaatprotem]Bostaurus
enzymeE1 [Bostaurus]
ubiquitin-activating
gil156523068
6-sulohansterase]Bostaurus]
gil76631783PREDICTEDsimilarto chondrorho
cell growthregulatorwithEF-handdomain1 [Bos taurus]
gil114051730
isotorm1]Bos taurus]
gil119884990PREDICTEDsimilarto ceruloplasmin
glyoxalasedomaincontaining4 {Bostaurus]
gi|156120555
gij114326274crystallin,zeta(quinonereductase)[Bostaurus]
gil27806677 connective tissue growthfactor [Bostaurus]
subunit,alphatype,6 {Bostaurus]
gill14053135
49 proteasome(prosome, macropain)
gil278068 melanomainhibitoryactivity[Bos taurus]
WD repeatdomain1 [Bostaurus]
gil114052350
6
gil2780611 tissue inhibitorof metalloproteinase1 [Bostaurus]
gil119905546 PREDICTED:similarto Cu/Zn superoxide dismutase [Bostaurus]
gi]94966811 alpha-iacidglycoprotein(Bos taurus]
matrin3 [Bostaurus]
gil156523084
gi]990289635'nucleotidase,ecto[Bostaurus]
gi]27807517settee(orosisteine]proteinaseinhibitor,dade8 (ocatbumin], member9][Bostaurus]
gil62460568 eukatyobotranslatonelongatonfactor 1 beta 2 [Sostautus]
synthetase{Bostaurus]
glycyl-tRNA
gil147902079
gi77735461 canopy4 homolog[Bostaurus]
gi29135329 glutathioneS-transferase pi{Bostaurus]
betaA, lysosomal [Bostaurus]
gi27805995 mannosidase,
gil114052595galectin3 bindingprotein[Bostaurus]
1 [Bos taurus]
phosphonbosyltransferase
hypoxanthine
gij77404434
7
hypothetical proteinisoform6 [Bostaurus]
gil11991101 PREDICTED:
chemokine[Bos taurus]
similarto IL-10-inducible
gi[76642911 PREDICTED:
similarto spectrin,beta,non-erythrocytic isoform1 [Bos taurus]
gil119903493 PREDICTED:
protein 2 homolog (Bos taurus]
ARP2actin-related
gil156121073
gil77735683S100 calciumbindingproteinA2 [Bostaurus]
[Bos taurus]
gill15497428calcyphosine
proteinSMT3B[Bostaurus]
gi|27807481 ubiquitin-like
Protein Sostaurus]
gi|116003917ar upstreamelement-binding
18 [Bostaurus]
gi77736560
77 8 stathminl/oncoprotein
gi|11549 2 aconitase1,soluble [Bostaurus]
gi]77736371actin relatedprotein203complexsubunit2 [Bos taurus
gi[29135293farnesyldiphosphatesynthase[Bostaurus]
gi]51036244 eukaryotictranslationinitiationfactor 5A[Bostaurus]
gi]84000347 interleukinenhancerbindingfactor 2 (Bostaurus]
von WillebrandfactorA domaincontaining1 Bos taurus]
gil157073982
pyrophosphorylase 1 [Bos taurus]
UDP-N-acteylglucosamine
gil114051892
[Sostaurus]
5 class111)chi polppepride
alcohol
dehydrogenuse
giJ77735453
similarto GalNAc-T5[Bostaurus]
gi]76609798PREDICTED:
gil77735537ephrin-Al[Bostaurus]
gil1
14052973glypican1 [Bostaurus]
similarto mammaryserumamyloidA3.2[Bos taurus]
gi[76691395PREDICTED:
giJ77735939proliferatingcell nuclearantigen[Bos taurus]
gi|27807275matrixGla protein[Bostaurus]
gil27806703
7 CD44antigen[Bostaurus]
[Bos taurus]
gil15727990
9 parathymosin
gil15612105biotinidase[Bostaurus)
gi[91680535proteasomeactivator subunit1 [Bostaurus]
gi|75812956GTP bindingproteinRabla [Bos taurus]
retinolbindingprotein4. plasma[Bostaurus]
gil164420709
1,cytosulic 85 taurus]
gill 39948301branchedchainaminotransferase
dipeptidylpeptidaseIll [Bostaurus]
gil164420725
isotorm12 [Bootaurus]
similarto ubpiguihon
gi[76638716PREDICTED:
1
7.18E-07
2747748 1.235636 0.493475 00829534 0.734209 0.227854 00007784 1.999054 0261175
319E-06
55.69106 1.422587 0.077752 174E-05 3.501318 0192254 2.94E-06 3.858543 0211649
0.3975730089731726100250.473913000291173.1876850.565521000271062
129085 1.316966
7765276 0721107 0376862 0.04859110.697037 0.093336 0.03125 0.548286 0.769348 0.02458663
31.17284 1.280483 0.106645 0.0001005 0.6362 0270181 8.02E-05 1.042406 0.240358 0.47596106
0.0625
0.125 3909715 0.427273 00005115 4.55274 0.491575
5183486 1.338978 0.684998
18.7643 1.383876 0.198783 00552539 2835368 0.600945 00156205 3.367195 0.788878 002123441
18.39736 1.071288 0.177561 043833034928856 0.459518000214357.5342880.258851000018219
0.7413240.575318023030411
21.822800667162 0.55327012488160941027 00817 0.1268117
3189655 08924170420267 0858325624682580.657117002915133.4616170.613798001001715
0.498831016888725.6339820.740719000166614
0.701122083050651.304308
4252336 0.902183
0.505138000198304
40.5737712139770.2851660008303 07348110.25533700001522 1.549626
2.5496730.608262006165172
-1 2222372 0.781190.1267682
1
57.31707-1.00065
0.1203970.00293502
04552212 1.202043
0.191366080858570.9703770.158274
33.230641.014587
00354010 0.83402074081 0.98111659
0.3021130180258 0927065 0.216521
22.426470.831915
0.452896 0.449429 3216-07
0.3487150.5554434
0.444943 1 19E-081,046545
34.391530.354994
-1
1
-1
-1 2728891 0.533151 0.1631087
1
37.037D4 -1.00065
19.1356 0.808148 0.109661 03252149 2717641 0.488088 0.0365571 4.41776 0.505432 0.01803671
2121771 1.213355 0270846 02173425 2.652676 0.096038 0.001358 2.75446 0308063 0.0145299
29.76744 0934584 0016017 0.0726401 3357907 0.782882 0.1962704 5.707625 0.760044 0.13755545
219182 0o31c24300001014 0387581 0363147 0.0010616 0.18633 0.793478 000174783
17.73504 0.754775 0406388 02250735 1112067 0.066451 0.0642077 0.708965 1.331028 0.5846554
15.83567 1.099632 0.772836 0789192 1131677 0.328484 04663631 1.191522 0.470687 045043484
20.51282 1.125470.509696 0622627 3064104 0.674581 0.054061 3.790778 0.758144 004812702
52.8168 1.476054 0685859 0.45391873102647 0.331506 0.0912452 3.180255 0.245301 00659195
4708029 0.886062 0.381269 01790651 1.576858 0.295458 5.216-05 2.110987 0.276311 6.08E-07
19.98504 1.028341 0.225889 0.791198 1766713 0.430917 0.082431 2.247018 0.285457 00193342
14.27221 1835553 0.961293 0.4203646 5.673488 0.316458 0.057302 8.848527 0.510027 0.01141375
29.64509 0.989903 0230848 0.901174 0.870173 0.15354 0.0717274 1.118471 0.131443 0.0914539
30.15267 1.374686 0564551 0.2030251 1.244621 0.3458 0.1742345 1.469283 0.338737 0.04651187
14.24528 0.510658 0.346303 0.0112042 0.773488 0.184435 0.0209447 0.459270 0.556732 002741702
34.56376 1.361714 0.523666 0.2793152 4272346 0.430063 7.496-05 4.233143 0.449098 9.27E-05
34.84848 1.415376 0.253623 0.0760381 3465257 0.09736 0.0818 3.899744 0.13153 0.08149262
20.3434 0.557302 0.359124 0.000204 0.996824 0.101397 0.7113753 0.134078 0.741618 1.10-05
40.2439 0.99902 0.37092 0.99438833.892598 0.253461 0.00507584.470833 0.273579 0.00530201
39.23077 0.93442 0413171 0.55020780.74613 0.394438 0.0275114 1.870968 0.317091 844188343
24.91748 1.328011 0.317688 0.08196092.835062 0.8448 0.0144696 3.231304 0.589377 0.01099513
32.36719 1.30685 0.251035 01004857 0.847312 0.272364 0.567031 0.540126 1.701831 0.46123532
36.17021 1.751774 0.020838 0.00632993.824468 0.549542 6.88-05 3.578872 0.383382 0.019625
37.12871 0.640945 0.68266 0.68488920.238141 0.21781 0.04784 0.180854 8.18886 0.03443773
0.120
0.125 3.118349 0.901589
23.81017 1.160861 0.498817 0.37806072.281601 0.88777
0.9642980.151043 0.6027264 1.523897 0.150462 8.1120271 1.975657 8.391831 8.04815696
17.07317
33.06878 1.568132 1.134077 0.56727582.791528 1.382881 0.3732823 3.194459 1.677478 0.39216793
27.11111 1.042784 0.568523 0.90645353.033238 0.42681 0.0010695 4.09589 0.615496 1.016-05
21.92102 1.411195 0.087404 0.07892224.581574 0.30509 0.00842674.982354 0.274885 0.0046598
24.68619 1.163013 0494403 0.3315886 3.65057 065318 8.766-05 4.676185 0.764743 00078125
4285714 1.26077 0.144679 00182182 2.979707 0.11706 7.976-06 3.837103 0.287837 0.00610483
14.90338392585 0239537 00103341 0.768706 0.076163 0.0129617 0.490066 0.483761 00913343
16.21622 1163616 0312811 0.258239 0855052 0.088827 0.01318830.725326 0.38382 009888402
0.4512 0.00110554.128777 0.525803 0.08096119
20.6422 1.736061 0.481642 00224143 3252737
2056834 0.879581 0.624866 0.5835043 1.916642 0333636 0.01451052.200812 0.459368 0.01577774
99 0.875858 0123182 0.27094020.800304 0299641 0.1833167 0.829947 1.195581 0.43601883
14.47313 0.713101 059002 0.4498234 1.833073 0.596717 0.2888106 2.515698 0.597666 0.1807333
23.09645 1.001828 0.697862 0.98818462.304177 0.029813 0.011229 2.067647 0.874D45 0.27182784
-1 2.466222 0.120242 0.0028888 2.563626 0.158206 0.06438019
1
36.08247 -1.00065
28.54233 1.388068 0421027 03516481 3.522336 0.11175 0.0278185 4.282173 0.328754 0.00812933
00625
0.625 4.313778 0.401973
38.94737 1.348473 0.36111 00560946 3.350866 0.477124
0-3266460.05383599
8.61e61t8
19.44012 2.11329 0269365 011127844.6547710.3677080.0745735
48.99029 1.092151 0325027 05704541 0995114 0342775 6.4828973 1496251 0.214272 004242202
12.26097 1.240157 0.17276 02382912 3423462 0.386158 0.0155636 4.481167 0.439657 0.01348308
27.66667 1.018386 0.197026 0.8136051 2258753 0622534 0.0331859 296798 0.588514 0.01288204
1898017 1.203639 0435333 03082513 3.539508 0.239562 00096248 3.779224 0.267713 0.00073658
24.02597 1453199 1.286417 0.5439189 2.954515 0170368 0.003964 3.754468 0.132889 0.00161227
27.4359 1.828959 0.671820 01530618 2537814 0.533873 0.0154008 3.62311 0.465778 005411404
1684915 1.099628 0.131237 03810268 0807840 0.102784 0.1945011 1.090683 0.050353 017938155
0.240058 0.0158773
0.4591730.4016492.107073
0282574 021363952.447533
20.114941.226566
14.70588 1.048268 0.260757 0.6424549 2.946919 0.36427 0.0035499 3.528374 0.332388 0.00163491
17882290.273644014437637
143617 1.02627 0.201030840112913033420.4151650.4245001
20.97061 113559 0.365597 0.327316 0.839321 0362003 0.1809834 1.18891 0.33226 0.1682474
0.122 0.011785 0.986551 0.435525 093417054
254025 1.210355 0.77746 053101 0.797344
76.33588 0416971 0.352713 2.02E-08 0408124 0.374995 8.01-09 096142 0.234995 038254998
33.33333 0.761819 0420592 02466368 1.739169 0431616 0.0869073 2.034908 0.234062 00694241
4466019 1.287522 0396059 00016206 0.754217 063986 0.0183724 1.280808 0662351 0.04100272
17.75956 1100475 0.406985 0406822 040386 0.32497 3.886-05 0.68014 0.502869 0.02643126
28.43137 1744462 0.642378 0.0492541 3952378 0628303 0.0021777 4150371 0.681311 0.00252709
8 0.814699 0455306 05260767 1.140688 0236245 6.3163806 1.005905 0.151722 0.94970084
35.34136 1.091096 0.459176 0.6840743 2735559 0.288460 0.0130874 4546808 0.418258 0.01200284
28.01106 0667297 0.163042 0.0853354 4.239819 0.35D6830.0012976 5.530162 0.377976 0.00097051
40.29851 0593539 0.486594 D.2726927 0637269 0.156723 0.2517599 0300645 0.06204 0.0183279
17.61696 1.334924 0.373477 0.04141872335966 0576351 0.00141222.805883 0.64513 000088134
20.13699 1.801793 0048435 0.02560064.015375 0217609 0.048973 4672917 0.390478 0.07862271
94.63760 1.204583 0.27885 0.06551012.015621 0.246148 0.0001837 2.392524 0.159873 6.926-06
10
6
5
5
9
5
3
3
4
4
5
11
1
6
17
14
1
2
3
2
5
3
3
3
2
10
3
2
4
4
4
3
3
8
2
7
4
3
7
2
5
3
2
2
2
6
4
3
4
5
4
2
2
2
1
2
5
2
3
2
4
4
3
3
2
3
4
2
5
4
14
3
17
7
5
2
3
4
2
6
2
6
8
6
5
6
9
5
4
4
4
4
5
11
3
6
17
14
2
3
3
2
5
3
3
3
2
10
3
2
4
4
4
6
3
8
3
7
4
4
7
2
5
2
2
5
3
8
5
3
4
5
4
3
2
2
3
2
5
2
3
3
4
4
3
4
2
3
4
2
5
4
14
3
17
7
5
3
3
4
2
7
2
6
10
6
5
7
8
5
4
4
4
4
5
11
3
6
17
14
1
3
3
2
5
3
3
3
2
10
3
3
4
4
4
6
3
8
3
7
4
3
7
2
5
3
2
9
3
8
5
3
4
5
4
3
2
2
3
3
5
2
3
3
4
4
3
4
2
3
4
2
5
4
14
3
17
7
5
2
3
4
2
7
2
6
gil122692375 hypotheticalprotein LOC783871[Bos taurus]
gi|114052248 plastin 3 [Bos taurus]
gi|119906762 PREDICTED:carilage-derived morphogeneticprotein 2 [Bos taurus
gil115495441 hexosaminidaseA [Bos taurus]
PREDICTEDsimilarto ventroptin[Bos taurus]
gi119920091
tumorprotein, translationally-controlled1 [Bostaurus]
gil62177164
gi]62751339 proteasome (prosome, macropain) subunit, beta type, 2 [Bostaurus]
protein) [Bos taurus]
gi78369310 stress-nduced-phosphoprotein 1 (Hsp70/Hsp9O-organizing
gi|27806555 legumain [Bostaurus]
g|87080799 small induciblecytokine subfamilyE. member 1 [Bos taurus]
gil77735431 ornithineaminotransferase [Bos taurus]
gill 15497922hypotheticalprotein LOC505636[Bos taurus]
gi]157279995dipeptdyl-peptdase 7 [Bos taurus]
gil155372073 cullin-associatedand neddylation-dissociated1 [Bos taurus]
gil119917901 PREDICTED:similarto Carboxypeptdase X 2 (M14family) [Bos taurus]
gil27806887 fusion (involvedin t(1216) in malignantliposarcoma) [Bostaurus]
thioredoxin[Bostaurus]
gil27806783
gi|155372333 lectin, mannose-binding2 [Bos taurus]
6-phosphogiuconolactonase [Bos taurus]
gi|84370199
4
gi|783696 5 SH3 domainbinding glutamicacid-rich proteinlike [Bos taurus]
gi|27805945 inositol(myo)-1(or4)-monophosphatase 1 [Bostaurus]
giJ77735429 proteasome alpha 3 subunit [Bos taurus]
gi|77735823 ribosomalprotein S20 [Bos taurus)
kinase 1, cytosolic[Bos taurus)
gil1 14051347
7 69 29 cytidinemonophosphate (UMP-CMP)
spectrin, alpha, non-erythrocytic1 (alpha-fodrin)[Bos taurus]
gil66 0
gi|1 14052270
9 chordin-like2 [Bos taurus]
gi|12672282 BCL2-associated athanogene 3 [Bostaurus]
gi|114051291tissue specifictransplantation antigen P35B [Bos taurus]
gi]78042510 haloacid dehalogenase-likehydrolase domaincontaining2 Bos taurus]
gil149642873hyaluronan and proteoglycanlink protein3 {Bostaurus]
1600002004 gene, partal [Bostaurus]
gil119930148
97 PREDICTED:similarto RIKENcDNA
gil278064 plasminogen activator inhibitor-1[Bostaurus]
gil115497482zinc binding alcoholdehydrogenase, domain containing1 [Bostaurus]
gil119879173PREDICTED:similarto putativeMAPKactivating proteinisoform 2 [Bos taurus
cyclohydrolase [Bos taurus]
gill 154974425-aminoimidazole-4-carboxamideribonucleotideformyltransferase/IMP
gil134085825carboxypeptdase Z[Bos taurus]
gi|75832090 isocitrate dehydrogenase 1 (NADP+),soluble [Bos taurus]
gi27807129 thioredoxinreductase 1 [Bos taurus]
gil119908206PREDICTED:hypothetcal protein[Bos taurus]
gil114052667RAD23 homolog B [Bos taurus]
(calgizzarin)[Bostaurus]
gil149642987
89 S100 calcium bindingprotein Al1
gi|284611 lectin, galactoside-binding,soluble, 1 (galectin 1) [Bos taurus]
gi27807075 S100 calcium-bindingprotein A4 [Bos taurus]
gi27806067 plasminogen activator, urokinase receptor [Bos taurus]
gil27806725 cathelicidinantimicrobialpeptide [Bos taurus]
gi|27806273 chemokine (C-C motif)ligand 2D [Bostaurus]
gi|l
14052322 alpha-N-acetylgalactosaminidase[Bos taurus]
gil114050905 SERPINE1mRNA bindingprotein 1 [Bos taurus]
gi]76669880 PREDICTED:similarto LRTS841 [Bos taurus]
gi|119915943 PREDICTED:similarto serine (or cysteine) proteinase inhibitor,clade B (ovalbumin),member 1 isoform 1 [Bos taurus]
gi119912825 PREDICTED:solute carrier family9 (sodium/hydrogenexchanger), member 3 regulator1 {Bostaurus]
gi76608584 PREDICTED:similarto KIAA0653protein isoform 1 (Bostaurus]
gi|27807445 peroxiredoxin5 precursor [Bostaurus)
gi|155371881 alanyl-tRNAsynthetase [Bostaurus]
gi|1 15496898Hi histone family,member D [Bos taurus]
gil119924945 PREDICTED similarto agrin [Bos taurus]
gil119922764 PREDICTED:similarto MSTP006[Bos taurus]
gi61888866tropomyosin1 alpha chain [Bos taurus]
giJ60592784 hexokinase 1 [Bos taurus]
gi[76649536 PREDICTED:similarto fibulin2 precursor, [Bostaurus]
gi27806923brain ribonuclease [Bos taurus]
gi|119909757 PREDICTED:similarto E-selectinligand-1 [Bostaurus]
glutamine-fhuctose-6-phosphatetransaminase 1 [Bos taurus]
gi158262747
gi|27807193 platelet-activatingfactor acetylhydrolase, isoform lb, beta subunit 30kDa [Bostaurus]
protein [Bos taurus]
gi|119907132
7 3 PREDICTED:similarto Hyoul
gi|8269 34 cystatin B (stefin B) [Bos taurus]
retinoblastoma bindingprotein 4 [Bos taurus]
gi|116004245
33
gil14823 12 dermatan 4 sulfotransferase 1 [Bostaurus]
19915491PREDICTED:similarto Complement C4-Aprecursor (Acidiccomplement C4) [Bos taurus]
gil1
gi78042520 eukaryotictranslation initiationfactor 4B [Bos taurus]
gil1
19894607PREDICTED:similarto peptidoglycanrecognition proteinL [Bostaurus]
gi|157427940activated RNApolymerase 11transcriptioncofactor 4 [Bos taurus
gi|77735427 protein phosphatase 1, regulatory subunit 7 [Bos taurus]
gij77735717 proteasome (prosome, macropain) subunit,alpha type, 4 [Bos taurus]
gil29135303 glutaminyl-peptidecyclotransferase (glutaminylcyclase) [Bostaurus]
gil117935057 dihydroxyacetonekinase 2 [Bos taurus]
gi|122692601 hypotheticalprotein LOC526913[Bos taurus]
gil157151716 annexin A6(Bos taurus]
gil27807521 prostaglandinH2 D-isomerase [Bos taurus]
gi|114051526 coactosin-like1 [Bos taurus]
gi|76615127 PREDICTED:similarto Dihydrolipoamidedehydrogenase isoform2 [Bos taurus]
gi|76671278 PREDICTEDsmiar to vesicle aminetransport protein 1 isoform 1 [Bos taurus]
27926 0300125 0.00220895
44.76744 0.552975 0.604769 00662065 1244118 0.095675 00077462
25.03912 1.323663 0.526258 0.3153689 2.232197 0.526929 0.0634759 3044657 059854 0.04329931
18.51064 0.567102 0.548921 0083639 1.350388 0.033151 00001309 0.654959 035796 0.2957879
-1 2.0671330.9895010.37747031.873520.796051D035399461
1
16.44613 -1.00065
23.39956 0.937338 0.048121 0.0769204068945 0.5151650.2079753087366 1.4475480.96743644
0.62196 2,340446 0.412916 0.1498302 3.121972 0.566442 0.15279323
35.46512 1.158834 0.443541
29.35323 1.330207 0.274933 0.1225159 3.127191 0.379439 00006479 4.246609 0.660994 0.00237663
32.22836 1.341008 0.807716 03724710 3.140066 0.564662 00101359 3.704626 0525602
24.01848 0.768093 0.152944 0.1750179 0.975966 0.08125 05616646 0979913 0205985
37.57764 0.978151 0.51457 0.9427495 3.248512 1.009726 0.0440782 450022 0.875604 0.0157647
15.03417 1.395334 0.98313 04870478 2.029195 1041695 0.2346279 1919908 1320611 0.34246752
-1 1.581124 0400711 0.2610647 226249 0236818 0.08990153
1
28.51711 -1.00065
23.97541 0.857851 0.549094 06691034 1304726 0.197209 0.2269135 1.089601 0.096418 0.31989787
18.86179 0.695073 0.378225 02896027 2.907305 0.49621 01440313 3759088 0843245 0.1926009
18.60158 0.797696 0.095673 01288634 0.730217 0224571 02104108 0.684151 0102243 0.24606708
32.61719 1.181835 0.481151 0.606781 4238665 0.550486 01180872 5.516432 054895 0.09948294
42.85714 1.444023 0.440707 0.054952 3509945 0.475087 00010569 419445 0.412321 0.00035965
23.11978 1.153124 0.410591 0.4790391 127617 0.343735 0226436 1493062 0.605143 0.23972742
30.23256 0.576743 0.018861 0.0106797 1.205925 0040074 0.0688709 1254963 0.811092 0.55656123
0.0625
0.4042 00038984 4378125 0516322
47.36842 1.577395 0.086695 0059265 3.14094
27.43682 1.400499 0.331543 0.1288063 2.957581 0692891 00602492 3363121 0.681528 0.04702865
38.82353 0.932204 0.22374 06348077 2.281576 0313706 0.1182173 2.446468 0.00794 0.00276909
31.09244 0.790159 0.17284 0.0290852 1.737038 0.268753 00099104 3.188097 0.532714 0.0080911
25.87719 0.927323 0.676895 0.8051482 2611393 0.755215 0.0874872 3160965 0.680789 0.05172991
16.58576 1.184876 0.388282 0537734 2947246 0.371875 01068734 3840887 0.388681 0.08957329
19.76744 0.884934 1.269861 0.798205 0.41275 0.65961 0.0776335 0.487832 0.680651 027704591
27.0855 2.802923 1.289169 03502997 3.458467 0048328 0.0122142 4.271478 0.13829 0.02969692
23.98754 1.654949 0.535261 01433048 4.137355 0278018 0.0061372 4.214638 0.236823 0.00431224
24.71042 0.94649 0.757689 0.9053114 3.266997 0.791617 00033087 4.125668 0317018 0.00013437
0.625 1.241183 0.2373 0.04251385
41.38889 1.158435 0.537488 0.4291358 1.01502 0.217778
38.37209 0.609115 0.452781 0.1114128 0.931329 050876 0.8898663 1754287 0.451165 0.03690212
16.41791 2.200635 0.29896 0.004716 4.486546 0380705 0.0014754 8.368697 0.330522 0.00034211
-1 1.893992 0.327402 0.1582271 2.316179 0.902675 0.07474413
1
13.10541 -1.00065
15.89649 1.426563 0.799612 0.5317789 3.512832 0.665082 0.0123022 5.059416 0.032401 0.00310552
13.85135 0.945259 0.440325 0.8357586 3.088379 0.800938 0.2143968 3.857588 0.686000 0.15539029
18.47826 0.559446 0.166333 0.0128532 0.856328 0.130455 0.0905337 1.188414 0.289823 0.26980953
24.63768 1.045861 0.344877 0.8372108 2.802093 0.576889 0.1841776 2.929273 0.27283 0.07874667
19.23848 1.282764 0.3450D6 0.3805389 2.327132 0.981346 0.3316377 25314 0.715079 0.22974867
2.6914 1.083964 0.15213374
17.80303 1.214952 0.224236 0.1830811 2.43674 0.887958 00547898
-1 2757201 0.625255 0.1879261 3.188905 0.658176 0.17267951
1
19.36274 -1.00065
29.12621 1.030253 0.247403 0.8497925 2.568362 0.112691 0.0374885 4.252827 0.107625 0.02318819
30.37037 1.224775 0.175201 0.0447682 220260 0.339462 0.8069073 2.735369 0.439069 0.00704158
42.57426 0.879406 0.024022 0.0878722 2.602672 0493875 0.1588096 3.749839 0.737501 0.16996106
16.36364 1.121041 0.414642 0.6754922 1.833391 0.813034 0.3733367 2.407823 1.479507 0.43922794
17.89474 0.1597 0.784296 0.0066379 1.138599 0.187596 0.1580967 0.329782 1.259486 0.08457753
19.79167 0.194847 0.555875 0.0179878 0.419872 0.220179 0.0100069 0.482231 0.146586 0.00572903
12.89538 1.139529 0.595913 0.8404207 1.51588 0.332239 0.0911832 1.883756 0.367761 0.07135406
-1 3.400362 0.220987 0.0584873 5.344381 0.284973 0.05477894
1
27.27273 -1.00065
22.9765 0.448547 0.945133 0.167938 0.759383 0.335878 0.1712008 1.831562 0.088643 0.00521001
24.41176 0.866515 0.14408 0.2903878 3.970274 0.119787 0.0272171 4866283 0.500872 0.0979879
29.61957 1.16955 0.347958 0.528402 1.937879 0.32838 0.1533487 1.906841 0.396522 0.1861921
12.87425 1.184635 0.264365 0.0080988 0.252437 0.804793 2.606-05 0.641921 082809 0.05737305
36.07306 1.113993 0.326673 0.4880402 4.136891 0171283 0.0023432 5.802393 0.11972 0.00074166
18.69835 1.087238 0.469583 0.7796875 3232799 0.34756 00922806 3.627258 0.063241
28.86598 0.849143 0.420366 0.5716697 2.006775 0033187 0.0150018 9.050744 0.149403 0.02115459
16.48301 0.866545 0.317657 05250638 0823302 1.139062 07801894 1.01548 0439552 0.95468536
25.0715 1.305384 0.165716 0.1887662 3.244213 0.394372 0.1041913 3.761606 0.031078 0.00731903
50.35211 0.833011 0.034991 0.0593724 1.718769 0.723139 0.3723729 1.984483 0.477072 0.2093176
2182267 1.0053760.19034862
0.3211840.1267448
2.195976
11.88659 0.685052 0.923112 0.5561625
9.444905 1.109473 0.049989 0.1483473 1.321788 0.510396 04727018 1.376373 0.520491 0.42900195
2.68E-05
41.31736 0.723807 0.332298 0.0029219 0.96977 0.130105 02522524 0.273223 0656186
16.37131 1.093871 0.410077 0.7344326 2.581782 0169757 0.0560792 1.506366 1.445697 0.66626145
0.138435
24.81645 1.231305 0.084642 0.1256687 2535182 0.377752 01259132 3.108689 0.51129
24.45415 1.027925 0.91947 09620578 2.814638 011982 00021668 4334730 0.218424 0.00353319
-1 2.035217 0816025 0.3268293 2365465 0.387398
1
27.17283 -1.00065
71.42857 1.478201 0.182256 0.0332131 2501006 0.120338 00030223 2.85596 0.131669 0.00251171
2447641 0.2575070.08917497
-1 1.9875140.6012590.260174
1
17.41176 -1.00065
17.81915 0.740922 0.011548 001198860.728226 0.348724 03094067 1246645 0.505891 0.38991166
17.96785 0.166893 0.97678 0.1662811 1082212 0.420537 0743752 0.316188 2.318201 0.4957697
-1 3.503268 0512061 0.1264097 2.816826 0333674 0.09976674
1
25.40984 -1.00065
20.13536 4.792911 0.487863 0.0004903 0395206 0.772723 00175063 1.139641 0.698255 0.57900678
41.73228 1.13341 0.21623 0.4487548 4.359615 0183893 0.0025105 5755411 0.17363 0.00156915
-1 3.783091 0.000171 4.036-05 5.134357 0.088936 0.01695884
1
25.83333 -1.00065
27.20307 1.218217 0.382513 0.3274525 3.009222 0.61398 0046819 3.822741 0.658926 0.03526476
-1 0.955946 0524784 0.8743152 0939491 0.711602 0.88726404
1
27.42382 -1.00065
23.18339 1.770718 0.856066 0.4035772 2710145 0.889998 02540701 2.181308 0.368757 0.14534993
-1 5440118 0.813041 0.1471004
1
-1 -1.00642
1
33.91813 -1.00065
1.94607 0.114224 0.05340423
0.2842 0.5159396 1.580233 0.112174 0077197
22.28826 0.875483
35.6021 2.053996 0.214395 0.0139374 0.468867 0.252016 0.0169907 0277285 1.052004 0.09295571
30.28169 1.478547 0.347001 0.2615507 1.981741 0.060627 02472422 3.391487 0498548 0.12571595
-1 2.645573 0.610104 0.23100851
1
-1 -1.00642
1
23.96857 -1.00065
12.93532 1.28624 0.582208 0.5406334 2.614265 0.289968 0.0940907 2701291 0.075577 0.02371975
0.00555204
0.87372895
0.01531197
0.13818516
gij76652407 PREDICTEDsimilarto ChainA,CrystalStructureOf HumanRangapl-Ubc9isoform2 [Bostaurus]
gi27806129 ribosomalproteinL24 [Bos taurus]
1, 127kDa [Bos taurus]
gil122692537
73 7 damage-specific DNAbinding protein
8 PREDICTEDsimilarto FAM20Cprotein[Bos taurus}
gill991
similarto CD109 [Bos taurus]
gi]119901084PREDICTED:
gi|41386699 heat shock 70kDa protein2 [Bos taurus]
gi|157074012 EF-hand domainfamily,member D2[Bos taurus]
gi|122692293 chlorideintracellularchannel 4 [Bos taurus]
gi]77735589 splicing factor,argininelserine-rich2 [Bos taurus]
g]155372143 arginine-rich,mutated in early stage tumors [Bos taurus]
gil27805833 chemokine (C-X-Cmotif)ligand 5 [Bostaurus]
gill18151144 tenascin C [Bos taurus]
phosphodiesterase delta 1 (Phosphoinositidephospholipase C] ]PLC-deha-l ]Phospholpase
gil119914306PREDICTEDsimilarto 1-phosphatidylinositol-4,5-bisphosphate
gi|77735633 cysteine-rich,angiogenicinducer, 61 [Bos taurus]
gi|158937293fibroblastgrowthfactor receptor 1 [Bos taurus]
gi|41386687 leukemia inhibitoryfactor (cholinergicdifferentiationfactor)[Bos taurus]
gil115495701 pentraxin3 [Bos taurus]
gill 15497506LIMand cysteine-rich domains 1 [Bos taurus]
gi|27806365 UDP-glucosedehydrogenase [Bostaurus]
gi|155372327tumornecrosis factor receptor superfamily,member 6b [Bostaurus]
gi|169658384non-metastatic cells 1, protein(NM23A)expressed in[Bos taurus]
gi|119888815PREDICTED:similarto GALEprotein isoform 2 [Bos taurus]
gi|76607797 PREDICTED similartocis Golgi-localizedcalcium-bindingproteinisoform 2 [Bostaurus]
gi|62752D12 ribosomalprotein L18[Bos taurus]
gi|147902328thymopoietin[Bos taurus]
gil27807361 nuclease sensitive element bindingprotein 1 [Bostaurus]
gil119895504PREDICTED:similarto heterogeneous ribonucleoproteinAO[Bos taurus]
gir77736367 poly(rC)-bindingprotein2 [Bos taurus]
gi]77736353 dimethylargininedimethylaminohydrolase2 [Bostaurus]
gi|114051796glucosidase, beta, acid (includes glucosylceramidase)[Bos taurus]
gi62751960 nbosomal proteinS26 [Bos taurus]
gil119879493 PREDICTED:similarto limbicsystem-associated membrane protein6c- [Bos taurus]
gil145279649tumor necrosis factor alpha [Bos taurus]
nucleoprotein, partial {Bostaurus)
similarto AHNAK
gil119938155
7 PREDICTED:
7 77 3
540 eukaryotictranslation initiationfactor 4A isoform 1 [Bos taurus]
gil
ATPase, He transporting, lysosomal accessory protein1 [Bostaurus)
gil28461231
7
32 PREDICTED:similarto synaptopodin[Bos taurus]
gil119895
114 05 42 3
ubiquitincarboxyl-terminalesterase Li [Bos taurus]
1
gi|
gir76633778 PREDICTED:similarto syndecan 4 [Bos taurus]
gl1 54707892 Clq and tumornecrosis factor related protein 5 [Bos taurus]
gil153791208 immunoglobulinsuperfamilycontainingleucine-richrepeat [Bos taurus]
gil119890624 PREDICTED:similarto insulin responsive sequence DNA bindingprotein- [Bos taurus]
gil27806675 cystatin C [Bos taurus]
gil164259354 reticulon 4 isoform 2 [Bos taurus]
gil115497820 parvalbumin[Bos taurus)
gir84000233 hypotheticalprotein LOC517857[Bos taurus]
gi|27806297 biliverdinreductase B (flavinreductase (NADPH))[Bos taurus]
gi|77736489 high-mobilitygroup nucleosome bindingdomain 1 [Bos taurus]
gil1 15497074esterase D/formylglutathionehydrolase [Bos taurus]
gil77735391 regakine 1 precursor[Bos taurus]
gil62460528 nascent polypeptide-associatedcomplex alpha subunit [Bos taurus]
gill 15497050isopentenyl-diphosphatedelta isomerase [Bos taurus]
gil77735825 cathepsin K [Bos taurus]
gi77735687 proteasome (prosome, macropain) subunit, beta type,6 [Bos taurus]
gi|27806823 osteomodulin[Bos taurus]
gij82697405 LSM3homolog,U6small nuclear RNAassociated [Bos taurus]
gil115497398neuron derivedneurotrophic factor [Bos taurus]
gil119879765 PREDICTED:similartoeukaryotic translationinitiationfactor 4 gamma, 1 [Bos taurus]
ribosomalprotein S19 [Bos taurus]
gi|82697365
7
gil15 954420 angiopoietin-like2 [Bostaurus]
gir84000197 FK506bindingprotein 3, 25kDa [Bos taurus]
gi|41386683 beta-2-microglobulin[Bos taurus]
chain 2 [Bos taurus]
giI62751407 fast skeletal myosin
gill 19919943 PREDICTED:similarto methyl-CpG-bindingprotein2 isoform 1 [Bos taurus]
gi]77736355 RAN,member RAS oncogene family[Bostaurus]
gi|149642675 ribosomalproteinS17 [Bos taurus]
gi|45430009 fumarylacetoacetate hydrolase domain containing2A [Bostaurus]
gi|154152039 scrapie responsive protein 1 [Bos taurus]
gi|119907227 PREDICTED:hypothetical proteinLOC535533isoform 2 [Bos taurus]
gi|84000371 H2Ahistone family,member V [Bos taurus]
giJ27807173 S100 calcium bindingprotein A10 [Bos taurus]
gill 19927320 PREDICTEDsimilarto lamininB2, partial [Bos taurus]
gil7662D955 PREDICTED:
50hypothetical protein [Bostaurus]
giJ77736063 dynactin2 (p ) [Bos taurus]
gi|149642591 0100calcium bindingprotein Al [Bostaurus]
giJ78369246 acireductone dioxygenase 1 [Bos taurus]
gi]77735889 mesoderm development candidate 2 [Bostaurus]
gill 14052282nuclear casein kinase and cyclin-dependent kinase substrate 1 [Bos taurus]
7 [Bos taurus]
gil1 19888616PREDICTED:similarto beta 1,3-N-acetylglucosaminyltransferase
gil115497034ubiquitinspecificpeptidase 14 [Bos taurus]
gil27806277 calpain, small subunit 1 [Bos taurus]
gi|27806265 leukocytecell derived chemotaxin 1 precursor [Bostaurus]
light
27.8481 0.838683 0.297707 076523 1.8926480.828082 023114 2775658 0.9621350.11767174
31.84713 0.299926 0.348412 0.0896313 1.559483 0.354058 0.240169 6.89452 0.560858 0.09003547
3.0876720.318510.01254228
12.45614 1.110984 0.044019 0.027213 2.4122750.4071360.0330273
24.71783 0.76106 0.07298 0.0827319 0.747602 0043851 0.045987 0.604188 0.095755 0.05912638
0.94923 0409485 0.6351143 1.113193 0.444676 0.43302788
36.22449 1.019964 0.426654 0.680915
34.54834 0.843417 0.785543 0.5746141 1769436 0913078 0.078125 2786537 0.968469 0.00180243
24.38017 1.793432 0.112593 00600325 2865644 041333 0.1217369 4361099 0846208 0.17475981
27.27273 1.17274 0.328963 01946439 2.710524 0261827 0.0002585 3.301008 0318316 0.00026735
33.48416 1.270996 0.145926 00547751 7.86308 0443527 0.0073721 8.410554 0.399028 0.00557626
18.43575 0.848986 1.101397 0.9118423 3.28157 1175707 0.2887952 3.836271 1819622 0.3728619
23.21429 0.179465 0.263135 00476897 0670318 0245752 0.1034332 0.27815 0.496713 0.11969159
1
50.91638 D.770316 0.294486 00256867 0602405 0.356736 00089668 1431962 1.303577
15.621 1.035261 0.323216 08653991 2.394548 0.291299 0.103929 2.95617 0.266369 0.07687783
0.67666383
2.306675
053295
02821737
0533266
1745372
0.7074747
10.20942 0.883789 0.51206
12.56098 1.753452 0.47468 0.0421556 0559447 0521762 0.2614632 1122839 0.125486 0.07607451
-1 0.963503 0190164 0.6378362 0.331646 0.726595 0.0629296
1
17.82178 -1.00065
29.58115 1.670089 0.348366 00667453 1.193437 0490979 04771401 2.573371 0.426845 0.03114173
27.27273 1.321589 0.146172 0.1604673 2.747591 0.409624 00206214 2530716 0.39146 0.02730964
12.75304 1.648265 0.581918 0.3305285 4.526757 016537 0.0342841 5.402224 0.035946 0.00664913
30.06757 0.677278 0.157293 0025035 1.419267 0240222 00700653 1.789695 0.171036 0.01362673
51.97368 1.599096 0.496178 01421363 3221894 0487525 00066466 5113621 0.525556 0.00293428
14.39394 1.003638 0.243785 0.9764014 367117 1123597 0.1024996 4.28586 0.971872 0.06457963
13.68876 0.873921 0.473286 05486248 1459335 0521968 02172251 2.065455 0.464456 0.06451703
-1 4234364 0.124859 00270982 5.293795 0.185178 0.03090209
1
45.74468 -1.00065
24.86911 1.629695 0.244807 01535627 1171980 0.032739 0.0668263 1.915535 0.39931 0.18618914
28.7037 0.913776 0.424419 0.580643 257372 0.355249 00046603 4.140374 0.414895 0.00220445
18.72131 1.612215 0.242052 0.0388583 2692241 0.504924 0.0396778 4.291759 0.545594 0.02173644
6.46E-05
-1 2838597 0370689 0.0228783 4.3053 0.029314
1
23.20917 -1.00065
-1 4.459772 0.743368 0.15217439
1
-1 -1.00642
1
23.85965 -1.00065
15.67164 0.764494 0.202429 02246044 1.254877 0.384624 04501166 1.678967 0.156796 0.09394594
42.6087 0.485585 0.151816 00652849 1.730674 0.56823 0.3025819 3.148392 0.620932 0.16512648
16.17977 1.159068 0.19806 0.204879 0798589 011301 0.0361716 1.088801 0.253514 0.48996056
6.837607 14.20984 1.293349 0.0096325 0911562 0.678383 0.8157827 14.54193 1.193093 0.00747859
66.79587 0.849069 0.201162 0.3441443 3.886245 0.072836 0.0168176 4.959856 0.14599 0.02842712
40.64039 1.16605 0.353513 0.3926128 4233965 0.197121 0.0030011 4.463149 0.121149 0.00104883
16.88034 0.960254 0.077019 0.3130112 1.005032 0.197032 0987851 1.422343 0.092712 0.01090967
-1 2.424683 1.728491 0.4877888 3.240050 1.513455 0.35835129
1
12.58503 -1.00065
13.88889 0.962528 0.00119 00690604 1.689114 0.859838 0.3554163 2.089131 0.997176 0.37288398
17.08543 0.683179 0.248639 0.0617444 0.418593 0.797848 0.1108239 0.821749 0.63134 0.20072435
21.39918 0.847331 0.101863 0.1856581 1.392054 0.068518 0.0658721 0.832781 0.214649 0.33214533
11.91589 0.83523 0.581162 0.2152827 0.591791 0.597903 0.0273438 0.929361 0.662288 0.677246D9
10.12048 0.901494 0.025889 0.0770335 0.820672 0.319856 0.4170883 0.784367 0.215142 0.26075991
35.81081 0.311661 0.505464 0.1332276 040507 0.032231 00110491 0.347844 0.310227 0.09103958
1.6576 0.8083492 1.871928 1.281245 0.5037745 0.998028 2.136357 0.9987981
9.49868 0.777559
36.36364 1.014075 0.188671 0.9088892 4.286481 0.434321 00928578 2.549938 0.347009 0.11458395
23.75 1.171824 0.508378 0.6403982 3306598 0281712 0.073567 4.85265 0.352999 0.06945593
18.93204 1.564585 0.63012 01331828 2.125048 0.58417 0.0344776 3.034521 0.605806 0.01319904
32.67327 D.444938 0.555178 0.2062116 1.943734 1161423 0.4539377 2.284475 1.615261 0.48644602
-1
1
-1
-1 4369147 0504538 0.1362306
1
12.76596 -1.00065
18.47826 0.451793 0.098061 0.0384317 0667916 0.53123 0.3602041 0.388718 1.36881 0.39305904
13.02326 1.830435 0.724095 0.3382421 4.504433 0.378398 0.0783849 5.934570 0.025173 0.00441075
27.1777 0.877947 0.778639 0.7897649 1.459049 0.325729 0.256461 2.013007 1.231721 0.45322823
35.62874 0.934242 0.047902 0.2097279 2.224044 0.048229 0.0189731 4.633014 0.04837 0.06984299
32.21757 1.333942 0.047645 0.0515986 3050473 032389 0.0905222 3.477498 0.235773 0.05885961
14.45498 0.774023 0.249999 00593557 1.07641 0153721 02961305 1.225383 0056163 0.00187667
31.37255 1.468764 0.902448 05450427 3.680527 0607336 0.1436591 4.276493 0.263572 0.05644745
39.05326 0.670412 0.278612 020918070997721 0.16174093058591548858 0.5022670.32627669
0.01968 7244449 0.08588 0.01349983
18.13773 1.564344 0.75853 04416059 5483087 0106958
-1 1.692251 0.653953 03518414 6243338 0.798797 0.13406917
1
35.17241 -1.00065
16.22718 1.028046 0.593377 0.9198322 1139332 0241889 0.3285328 1.277148 0.47329 0.32563774
24.55357 0.812919 0.853254 0.7084508 1.916825 0.544387 0.2499603 2.67173 0.989421 0.29183193
39.83051 1.416328 0.47085 0.0759663 083683 0.320825 0.1371149 2.344783 0.035110 0.00680338
19.18605 1.240037 0.337093 02527990 3.986864 0.165871 00023171 4.063859 0.358809 0.00813073
-1 8006162 0.471435 0.08121488
1
-1 -1.00642
1
25.71943 -1.00065
22.68519 0.739306 0.430781 0307744 2.028151 0204308 0.0903641 2.630704 0.148516 0.04781956
049007392.4549420.0303630.17932944
1.235928
32.59259 0.542396 0.748698 0.3436696 1.800149
0837969 0.47235345
061909903311537 1.565225
26.11465 1.274767 0.170997 021215551.709406
45.91837 0.738699 0.312672 001862760421795 0.242594 003125 028535 0.6809720.00127969
013582672750132 0.031820.00981346
1 2202432 0.346186
1
13.22141 -1.00065
0324879 002513154.1789970.122533 5.76E-05
48.4375 0.760566 0458467 0182695 1.609169
38.14433 D.839503 0.497589 026758712623931 0360347 9788-093993368 0.422779 8.99E-10
13.90729 1.083504 0.254515 0.6386092 0965657 0045925 0.317017 1.357422 0.088716 0.08998154
59.52381 0.694827 0.419084 0.3265113 1135705 0147721 0.3436365 2.488608 0.160546 0.05480918
-1 2.792355 0.054156 0.0165552 3.203302 0.807278 0.15935018
1
16.62531 -1.00065
17.02128 0.991839 0.070916 0.6413485 3.244039 0.087176 00232301 4.796569 0.248085 0.04927254
27.93296 1.213911 0.401982 03527036 255191 0.258428 00121312 2.915009 0.114788 0.00183849
28.87931 1.077581 0.23601 0.6374274 3.852189 0.986506 0.2201164 4.933039 0.775939 0.14892666
-1 1.82684 0.790179 0.170527 4.057833 0.901211 0.19448039
1
32.09876 -1.00065
0.0527240.05168312
0.8926640.34249050503191.373841
0.1970440.1436533
14.250611.524025
0460966 0.10132238
4.085951
3.1782160.414290.1112683
0.057796 0.074474
19.028341.273419
0.1746570.03414982
1 4.924903
1
-1 -10642
1
18.63116-1.00065
10259280563429 0.90400419
0.4152580.0620404
1.3531021.003270.4493391.529699
15.22308
similarto MGC84382protein(Bos taurus]
gi|119909677PREDICTED:
giI122692323ysozyme [Bostaurus]
factor)[Bostaurus]
migration inhibitoryfactor (glycosylation-inhibiting
gil75812914 macrophage
gill 15496488SEC22 vesicletraffickingproteinhomologB [Bostaurus]
proteincomplex2, beta 1 subunit[Bostaurus]
gill 15497346adaptor-related
9i|76629502 PREDICTED:simiartoTGOLN2protein[Bostaurus]
gi|76635454 PREDICTED:similarto OAF homolog(Drosophila)[Bos taurus]
beta [Bostaurus]
gi|134085842glucuronidase,
28.4
55.10204
20.86957
16.33466
10.30494
16.77632
28.36879
4.907975
0.804016 1.044613
0.742951 0.688586
1.296906 0.428584
1.291398 0.488746
1.497709 0.414254
0.927459 0.279476
0.958118 0.023639
0.704209 0.263444
2.026587
0.7429104
1.22306 0.4508602 2.810786 1.083154
0.53975271.160912 0.324298 0.5340143 2.758603 0.059322
0.0186749 3.646303 0.546461 8.86E-05 3.957525 0.598624
0.4800242 3.392663 0.687048 0.1720918 4.493164 0.881982
0.2969266 1.574513 0.063904 0.0444805 2.047706 0.037289
0.677674 1.543475 0.159946 0.1154222 1.40067 0.171665
0.1659524 0.974144 0.157536 0.7457215 2.663787 0.08286
0.224216 2.083319 0.001541 0.0006607 2.831372 0.086064
0.30210375
0.01823637
8.86E-05
0.17833888
0.01623032
0.15578092
0.02637374
0.02578849
Accession Name
2 (perlecan)[Bos taurus]
gi|119888979PREDICTED:similartoheparansulfateproteoglycan
isoform12 [Bostaurus]
giJ766101267 PREDICTEDsimilartofibronectin1 isoform4 preproprotein
collagen,type11,alpha1 isoform2 [Bostaurus]
gil16441442
8
gil307942 D albumin[Bostaurus]
gil34098396 ChondroitnABC endolyase1 precursor(ChondroitinABC lyase1)(ChondroitinaseABC)(Chondroitinsulfateendolyase)(ChondroitsABCelroinase)
gil41386685 thrombospondin 1 [Bostaurus]
PREDICTEDsimilartoalpha3 typeVI collagenisoform5 precursorisoform1, partial[Bostaurus]
gil119890645
proteinLOC540664[Bostaurus]
gil1 19900891PREDICTED:hypothetical
gil27806761 aggrecan1 [Bostaurus]
gi|119901095
PREDICTEDsimilartocollagen, typeXII, alpha 1 isoform 1 [Bostaurus]
gi|l 15495027heat shock7DkDaprotein5 [Bostaurus]
PREDICTEDsimilartoCollagen,type VI,alpha 1 isoform1 [Bostaurus]
gi|119887130
chondroitinsulfateproteoglycan4 [Bostaurus]
PREDICTEDsimilarto melanoma-associated
gi|119913814
PREDICTEDsimilartoalpha3 typeVI collagen,partial[Bostaurus]
gi|119890597
[Bostaurus]
PREDICTED:similartoAHNAKnucleoprotein
gi|119919153
gill10347570vimentin [Bostaurus]
gi|114051163collagen,typeXI, alpha2 [Bostaurus]
gi|119892686
PREDICTEDsimilarto P63protein[Bostaurus]
gi|119914559
PREDICTED:similartofilamin B. beta(actin bindingprotein278)isoform7, partial[Bostaurus]
gil148238040
actinin,alpha4 [Bostaurus]
gi[77404182laminA/C[Bostaurus]
gi|l56120777nidogen2 [Bostaurus]
gil60592792
7 heat shock90kDprotein1,alpha[Bostaurus]
3 [Bostaurus]
gil14823034 proteindisulfideisomerase-associated
gil119894520 PREDICTEDsimilarto cartilageoligomericmatrix proteinisoform1 [Bos taurus]
gi|164452943gelsolina [Bos taurus]
gi|99028969complementcomponent3 [Bostaurus]
betasubunit[Bostaurus]
gi|27806501prolyl4-hydroxylase,
gi|153791660
extracellularmatrixprotein1 [Bostaurus]
gil119889734
PREDICTED:
similarto Collagenalpha-l(XI)chainisoform1 [Bostaurus]
gil115497814nucleobindin1 [Bostaurus]
similarto Cartilagematrix proteinprecursor(Matrilin-1)[Bos taurus]
gil119888668 PREDICTED:
2 [Bos taurus]
gil27807447 matrixmetalloproteinase
gi|87196501enolase1 [Bostaurus]
gil95147674 complementfactor B [Bostaurus]
[Bostaurus]
similarto matrixmetalloproteinase-3
gil119906908
4 3 PREDICTED:
PREDICTED:
similarto matrilin-3[Bos taurus]
gi|766301
gill14050715moesin[Bostaurus]
gi|114326226proteindisufideisomerase-associated 4 [Bostaurus]
gi]122692297
chibnase 3-like1 [Bos taurus]
2-oxoglutarate
5-dioxygenase 2 [Bos taurus]
gl155372023procollagen-lysine,
gi]27807289 annexinA2 [Bostaurus]
quiescinQ6 sulfhydryloxidase 1 [Bos taurus]
gil156120795
gil27805853 cartilagelinkingprotein1 [Bos taurus]
partial [Bostaurus]
similarto RP11-525G3.1,
gi|119921375PREDICTED:
2 [Bos taurus]
gi|41386780 UDP-glucosepyrophosphorylase
similarto lamininalpha4 [Bostaurus]
gil119901238 PREDICTED:
gi|115497900eukaryotctranslationelongationfactor 2 [Bostaurus]
giJ61888856 triosephosphate isomerase1 [Bos taurus]
gil27807263 heat shockprotein90kDabeta,member1 [Bos taurus]
[Bos taurus]
proteinLOC615490
gil147906412 hypothetical
similarto emilin(Bostaurus]
gir61845535 PREDICTED:
antigenp97)(CD228antgen) 905taurus]
precursor(Melanoma-associated
gi|76607158PREDICTEDsimilarto Melanotransferrin
isomeraseB {Bostaurus]
gi|27806469 peptidylprolyl
collagen,type 111,
alpha1 [Bostaurus]
gi]116003881
[Bostaurus]
gil115497210complementcomponent1.s subcomponent
kinase 1 [Bos taurus]
giJ77735551 phosphoglycerate
gil27806147 secretedprotein,acidic,cysteine-rich[Bostaurus]
gi|110350683 biglycan[Bostaurus]
gij75832054 actin,beta [Bostaurus]
gi|119919953 PREDICTEDsimilarto filamin[Bostaurus]
I [Bostaurus]
alphapolypeptide
4-dioxygenase(proline 4-hydroxylase),
2-oxoglutarate
gi|115495019 procollagen-proline,
A [Bostaurus]
gi|27806559 lactatedehydrogenase
gi|115496702
collagen,type VI,alpha2 [Bostaurus]
PREDICTED:
similarto LOC512571protein[Bostaurus]
gi|119902010
precursor[Bostaurus]
gi|27806477lysyl hydroxylase
gi|76253709heatshock70kDaprotein8 [Bos taurus]
hypotheticalprotein[Bostaurus]
gil119919157PREDICTED:
gi|77404252 collagen,type alpha1 [Bos taurus]
gi|62988316 follistatin-like1 [Bostaurus]
mutase1 (brain)[Bostaurus]
gi|77404217 phosphoglycerate
matrilin2 [Bostaurus]
gi|156523168
[Bos taurus]
activationprotein,zeta polypeptide
5-monooxygenase
gi|27807367 tyrosine3-monooxygenase/tryptophan
[Bos taurus]
gi|27806723 catreticulin
gi|148238309 aldolaseC,fructose-bisphosphate[Bostaurus]
dehydrogenase
[Bostaurus]
gir77404273 glyceraldehyde-3-phosphate
gill14052136FK506bindingprotein9 [Bos taurus]
layerprotein2 precursor(CILP-2)[Bos taurus]
similarto Cartilageintermediate
gi]76621011PREDICTED:
antitrypsin),member1 [Bos taurus]
gi]27806941 serine(or cysteine)proteinaseinhibitor,clade A (alpha-1 antiproteinase,
gil94966763haptoglobin[Bostaurus]
gi|27806697chondroadherin
[Bostaurus]
1,
%Cov
26.34935
44.75021
55.00705
85.66722
52.88932
47.69231
45.04037
32.81527
17.40438
22.98335
63.35878
42.64849
24.45019
36.76471
41.59497
62.23176
40.14977
67.06282
27.82132
47.63996
59.26573
32.64368
47.3397
62.57426
46.03175
40.97311
28.95846
66.47059
54.54546
31.97578
60.12658
50.73529
55.06808
60.36866
Nopep 16
1lluel161meanl17 stdl171lpcaluell7l4Nopepl11
115:11stl1
mean115 stdl15l1pualue
0.837063
0.463156
0.854572
2.45993
1
0.73053
0.582081
0.849801
0.752685
0.719242
1.109696
0.622398
1.040585
0.62676
1.039134
1.380347
0.686068
0.906607
1.194375
1.26428
1.217868
0.808751
1.205972
1.0893
0.818788
0.695187
1.063743
1.073538
0.338604
0.839092
1.121138
0.835415
0.761708
1.041078
43.88962
0.6658
37.10692
50.97002
52.513
44.32348
53.70844
36.08924
63.71682
45.14991
50.64746
23.35423
48.8189
21.79417
36.71329
72.69076
38.80597
37.52809
30.04926
33.55886
75.48077
42.90587
28.77698
65.94724
52.30263
53.11653
47.46667
18.10017
42.50936
61.44578
48.74591
44.40298
48.76033
33.69231
30.6163
42.72044
49.83713
61.02362
31.10647
61.22449
37.88969
42.85714
40.84084
32.92683
24.6473
39.18269
45.63591
48.75346
0.379018
0.56015
1.033309
1.173791
0.355438
1.008474
0.864522
0.885577
0.877402
0.810071
1.035136
1.092885
1.176607
1.374135
0.947458
0.610225
0.90724
1.171926
0.775224
0.728525
0.825658
1.014866
1.112631
0.90193
1.142032
1.119296
0.962406
1.095854
0.672606
1.167611
1.014552
0.941139
1.057389
0.588984
0.920442
1.290904
0.622527
1.352603
1.191075
1.040839
1.284868
0.952533
0897385
0.654795
0.480795
0.627942
0.403478
0.480728
0.495462
0.479478
0.419691
0.493838
0.538468
0.104069
0.512405
0.381935
0.480298
0.325312
0.249999
0.480739
0.370255
0492979
0.464923
0.321133
0.540658
0.394529
0.359493
0.343373
0.474195
0.446279
0.487005
0.386157
0.280662
0.345317
0.455412
0.38777
0.374925
0.390752
0.557064
0.58665
0.400061
0.633204
0.453447
0.317744
0.603892
0.422411
0.263717
0.449534
0.576208
0.365959
0.469326
0.322768
0.561388
0.423182
0.51317
0.348699
0.662467
0.350471
0.320153
0.405526
0.476838
0.276615
0.457414
0.432879
0.361646
0.488555
0.299691
0.370927
0.396579
0.454125
0.372884
0.303548
0.884305
0.447895
0.431925
0.311904
0.353771
0.379856
0.55751
0.373938
0.319976
0.298604
0.353901
0.315468
0.323534
0.458112
0.422765
1.9DE-i5 0.95259
1.36E-93 1.052178
6.13E-35 0.840488
2.04E-111 0883801
1
0.989830327
1.57E.31 1.057974
5.40E-19 1107817
0194181551 1.112003
0 0816278
1.72E-13 0982038
0024460731 2823067
721E-55 1.008858
0.047922995 0.928117
8.58E-11 1010198
06078493 3.092819
1.490-10 2.542251
4990-29 1113795
0000194084 3.247247
0.006403559 1.848702
0000114481 2578089
5030-06 2.17346
8150-12 0.908361
000537598 2.949889
0109789208 3.817216
4.580-07 0827782
1150-14 1.649112
0074060176 0.811825
0.008549401 3.010583
0 0815204
827E-13 0.847742
0.000237774 1.011852
1.870-11 0.803501
2.990-08 0739579
0393765163 2.791033
0 0.58171
0 1.006669
0 0.588751
0.761636645 1.700946
004049287 2.897887
0.5802
3010-117
0796128801 2.369590
002005348 1.620062
0.00094702 166031
3.3-07 0.935443
0.003648842 0.819274
0388958428 2.778324
0.304105828 0796267
0010127881 2.801989
3.290-07 3.010825
0.34980025 3010549
5.600-10 0.877448
0.008698904 0990708
0001172306 1.171209
1100-09 1.497094
3.680-08 1.435887
2.8-07 0681547
0.787327151 3.25846
0000437327 0.433091
3.30-05 1943041
0009011311 3.081512
0095124695 1617911
0510699563 2836113
013030691 2.451282
1.036-06 1.329379
0.001632762 3468224
0650464916 0.97276
0.206054688 1.833408
0423419308 2.443185
1.700-05 0838006
0006624735 0.892288
1800-06 3140921
1560-07 0764861
0000570644 3000337
0013218783 3.972464
1 2.175947
4.930-05 4767639
0700480746 3.459247
0.006546787 0.871611
6.920-11 0863964
1.130-11 0188748
0 1.134407
0.373037 00088365 0998097 0.883258 0000136975
2.82E-93
0.423887 0.0007858 0484351 0.840485
4.770-111
0.589894 3.08E-96 0.535849 0.830221
09846 1091581 0.974578433
0.495928 1.49E-18
1 0.320736 0436577998
0387833 0.3194828
0.33551 7.05E-06 0.951059 0.737226 0200718485
0,499157 0.0086458 0.957857 0.81187 0034312499
0.1262940.229409638
0.3771220.70602371.162073
552E-39
2.38E-49 0744753 0806282
0.43587
928E-06
0.345564 0.2847857 1153545 0388945
0
0 277909 0682487
0.642853
533E-17
0.301594 0.023367 0.874573 0379201
0283046 0.0002174 0.927416 0425555 0076052035
0.290596 0.857575 104808 0377368 0038908652
5700-07
0.743213 1310-07 2.638091 0.733315
8390-14
0 2.738
0.713441
0886741
3.730-36
0.347323 4410-08 1.629073 0.646425
0
0 2.653344 0.745841
0.734753
2430-12
0.363359 7.370-14 1.74755 0497287
0
0 2.664101 0694165
0.812227
0
0 2.624695 0.54318
0.466516
0.298581 5.870-08 0.955872 0.576384 0707942141
2560-06
0.538509 3330-15 3260966 0.579182
0
0 3.793069 0.691047
0.748355
1420-10
0.533429 7.250-09 0.551296 1.118423
1770-06
0.580126 7.88E-07 1348877 0.557911
2310-10
0.384343 2.840-12 0.356582 0.903084
0
06411
0 2.977922
0.583446
1.230-11
0.344116 1.88E-09 0.306434 0899426
6350-18
0.337052 222E-18 0.711882 0.505987
4.810-05
0.356226 0.5324485 0.883468 0.302727
0 0.959839 0552348 0.866595362
0.338838
7.55E-11
0.450868 6.17E-10 0.440702 0635573
0
0 3407778 0.805833
0.721168
2.101114
0.479446
0 0.717888 0.478212
1.76E-08
0.293237 0.739739 0.80568 0.306294
0 0.831069 0.762416 0.000940778
0.493742
1.60-06
0.484271 1.820-5 1.727707 0.454487
1.466-13
0.799682 2.77E-12 2.8862 0.777428
2.590-118
0.28437 1.070-119 0.322687 0.443066
4.44E-16
0.441774 2.22E-14 2.565107 0.42218
3.190-07
0.561806 1.300-06 1.719897 0.64929
2.43E-13
0 2.953609 0.547068
0.408226
0.383396 2.310-07 0.975970 0.305248 0.397689938
0.593539 0.0105995 0.902332 0.48741 0.013061608
1.740-12
0.534484 4.440-16 2.508068 0.654265
0.304242 3.800-05 0.832184 0.345183 0.004018315
1.35E-11
0.578151 1.320-12 2.58226 0.607902
0
0 2.84055 0.664001
0.60713
2.900-09
0.575034 5.790-10 3.428719 0.729066
0.350422 6.520-05 1.077281 0.858107 0807656019
0.313715 0.5207495 0.891227 0429387 0.040499183
0.318859 0.0142498 0.980021 0.53173 0.933285925
373E-10
0.508777 2.23E-08 1.26326 0.381398
0.461725 4.93E-06 1.348145 0.735978 0.00842754
2.220-16
0.260751 2.220-16 0.586669 0340813
3.190-11
0.813019 2.200-14 2.909108 0960754
0
0 0.267344 0.904945
0.455733
0
0 1.452485 0.547575
0496503
0
0 2.87937 0.85164
0.857538
1970-05
0.446145 0.0002908 2.246832 0578947
1.330-08
0.724586 3920-08 3161361 0.881849
6410-08
0.68869 6230-09 2.131829 0.74977
0.349184 2850-08 1142093 0302527 0.006284216
0
0 2.93251 0.500255
0.44042
0.340762 0.0345908 1.026278 0.288379 0.201064286
1.178599 0.0136719 1.933138 1.566201 0.088752455
1.23E-09
0.80538 2630-09 2.379748 0.58095
0.491821 0.0245361 0.423029 0958062 0.000610352
3.570-08
0271447
1.330-15 0.295712 1.0329
1.200-14
0 2.441112 0599982
0.546255
000068081
0.24757 5.710-07 0711202 0.52512
1.580-14
3.350-14 274531 0596008
0.625155
3.250-07
3020-11 3033952 0901157
0.640949
0.71009 00002261 2.069744 0.669801 0000177336
4.830-13
3.33E-14 4.94648 0.055547
0457855
2.930-13
0.833595
1.040-14 2866455 0.815351
0.355929 0.00078 0.904995 0.38123 0.047402976
1.01E-08
0.268318
9.840-05 0.688005 0.338425
7.970-14
0.806206
7.330-15 0.155989 0.921258
2.090-16
288-07 1.558928 0.401822
0.298218
247
598
743
674
219
275
110
2
671
59
108
334
74
79
12
65
307
80
37
40
43
81
28
67
88
58
33
81
59
146
78
125
55
65
258
591
739
650
220
280
110
2
879
64
116
343
74
81
17
71
323
87
37
43
43
63
29
86
86
59
33
89
60
146
77
125
56
68
98
00
81
104
15
31
706
27
27
52
113
27
31
21
24
45
19
51
38
28
63
54
39
31
103
102
62
12
21
22
26
32
40
11
20
14
40
32
20
30
17
10
19
34
35
29
28
99
81
105
16
33
729
28
27
63
113
28
32
21
26
70
19
53
38
26
63
54
39
36
103
116
67
12
22
24
20
35
43
11
22
14
40
35
20
30
20
12
19
36
36
28
26
103
upept1?
255
589
744
68
225
281
112
2
895
68
126
339
75
81
17
74
333
88
36
46
43
84
29
86
86
59
32
90
61
147
77
133
56
69
105
81
104
17
36
719
28
29
71
118
28
31
21
26
71
19
53
39
26
63
54
39
35
103
114
67
12
22
26
26
37
42
11
22
14
40
35
20
31
19
12
19
36
38
28
26
102
5.80E-05
4027304 1125927 0.427507 0.19047228 1.893274 0.499106 6.26E-05 2.384881 0.700173
gi|157074106laminB1 [Bostaurus]
1.66E-13
D 0.409857 0.63264
0.9942650459222 0.8912971140.516234 0560052
31.89006
gi|27806907 clusterin [Bostaurus]
42.00880.5723240.327987 8.68E-09 0.915573 0.473782 0.1909616 0.564658 0.721397 0.00012594
gi|27806257 collagen,type 1,alpha2 [Bostaurus]
0.0085296810.942108 0.340611 0.2022533 0.863529 0.450099 0.088978291
0.481149
0791002
22.82051
gi|164450489AEbindingprotein1 [Bostaurus]
0.533307 0.387538885 2.148625 1.114603 0.147246 1.902193 1.325119 0.247878468
6572581 1.203989
3 isoform1 [Bostaurus]
gi|61828365 PREDICTED:similarto Tropomyosin
5.91E-07
0 0.24743 1.262241
2.22E-16 0.30131 0.545653
22.34679 0477334 0.39839
transforming growthfactorbeta bindingprotein 1 [Bostaurus]
gi|156718112
6.77E-10
4.12E-05 1.330537 0.380719 0.0001744 1.727248 0.411099
0.406881
34.50882
0.747163
2
[Bos
taurus]
gi|27807207 plasminogen activator inhibitortype 1, member
4.00E-15
0.30045
1.664574
0.0003589
0.556375
1.44796
0.462619285
41.22807 0.957583 0.452189
gi|27806853 lumican [Bostaurus]
7.86E-08
3184499 0.935102 0.333769 0.341791907 3.125026 0.374674 2.44E-09 2.55356 0.381484
gil115498012 glycogenphosphorylase, liver[Bos taurus)
0.001018829
1.340117
2.221409
6.45E-07
0.825105
2.577366
0.051651999
0.482983
1167313
5978836
gi]82751849 Parkinsondisease (autosomal recessive, early onset) 7 [Bos taurus)
3244382 0845298 0.356417 0.024928977 D.797066 0.361873 0.0032635 0.788065 0.372537 0.006361088
gil27807437 matrixmetalloproteinase9 [Bos taurus]
0.93617784 3.215671 0.434908 5.39E-05 2.645173 0.599273 0.00075482
4874157 1 008134 0404379
gi|76253900 GDPdissociationinhibitor2 [Bostaurus]
1.49E-08
1.73E-06 1.498072 0.455662 9.49E-06 1.840324 0.475653
3475366 0.656269 041703
gil155371895 sema domain, immunoglobulindomain (1g),short basic domain. secreted, (semaphon) 3C [Bus taurus]
2.17E-D6
1.30E-06 3.288567 0.43516
3.295887 0.398104
3972222 1146393 0.255354
gir76682880 PREDICTEDsimilarto nucleolin-relatedprotein NRP isoform 1 [Bos taurus]
7.84E-10
7.94E-06 0.419281 0.36339
0.562113
0.513318
7.26E-07
0.570317
0448264
72.51908
gi]38566696 serum amyloidA 3 [Bos taurus]
32.97546 0799637 0423876 0.018105078 1.069017 0.473747 0.5745095 0.936048 0.411462 0.523185861
gi27806781 vitrin [Bos taurus]
0
6.68E-11 0.578059 0.483977 7.99E-15 0.417834 0.456215
5567376 0.774017 0.289178
gil156121245 insulin-likegrowthfactor bindingprotein 7 [Bos taurus]
5.79E-05
3733184 1.397087 0398414 0.006582678 2.490301 0.516419 0.0019531 2.664316 0.71645
gi|78369242 alpha-actinin[Bostaurus]
29.65426 0872476 0356122 0.060376673 0.965418 0.314061 0.5830078 0.789571 0.565253 0.053630741
gi|119903231 PREDICTEDsimilarto lysyl oxidase-like3 protein[Bos taurus]
4.00E-09 0.809814 0.284731 6.34E-05 0.611837 0.796064 0.000140134
37.06294 0.673846 0.235276
gi|119903133 PREDICTEDhypothetical protein [Bostaurus]
28.72008 0946067 0666918 0.668260819 0.770715 0.318171 0.0004471 0.596724 1.025448 0.029541016
gi|77736401 thrombospondin4 [Bos taurus]
9.48E-05
54.91713 091483 0.369929 0.174969615 2.546949 0.420705 0.0002764 1.367317 0.394717
gi|120474983 annexin A5 [Bos taurus]
7.93E-08
4.1447 0.734835 1.22E-07 5.027523 0.819795
3764706 1.07804 0.333489 0.279569061
inhibinbeta A [Bos taurus]
gi27805949
4
2.94E-08
3588144 1.09679 0438155 0.238874125 2.523834 0.644209 5.21E-08 2.603798 0.660478
gij4025 806 heat shock 70kDa protein 1A[Bos taurus]
3.06E-07
2645161 0.773023 0.352321 0.000231289 0.705205 0.297968 4.44E-07 0.715834 0.255707
gil119895885 PREDICTEDsimilarto EGF4ikerepeats and discoidinI-like domains3 [Bos taurus]
2.94E-06
0.552981
1.12E-06
3.411074
3.935189
0.538403
0.028022175
52.22552 1.238868 0.37494
gi|164420731 transaldolase 1 [Bostaurus]
6063349 0.851828 0.328314 0.500616253 1.433246 0.26743 0.2306782 4.001638 0.068226 0.015194624
gi|155371863 histone cluster 1, Hld [Bos taurus]
1.11E-08
42.79379 0.965033 0.308589 0.498755942 2.635921 0.538609 2.05E-10 2.528015 0.70501
gi|168804008 tubulin,alpha, ubiquitous[Bos taurus]
0.00643178 2.571771 0.845196 0.0003052 2.691735 0.926881 0.000427246
39.83516 1.456509 0.8686
gil156120479 aldolase A [Bostaurus]
2.D3E-08
43.10851 1.261221 0.429936 0.009074546 4.906097 0.859797 2.37E-08 3.846697 0.733993
gil1 14051756heterogeneous nuclear ribonucleoproteinA2/B1[Bos taurus]
1.79E-08
60.8365 0.900143 0.625208 0.350254639 4.301779 1.027594 5.73E-08 6.706334 1.185234
gil27806489 proenkephalin [Bostaurus]
4.67E-05
0.615649
1.948011
0.0001398
0.620444
2.056151
0.709051716
0.602992
0952057
36.89024
gil1 14053121reticulocalbin3, EF-handcalcium bindingdomain [Bostaurus]
8.43E-07
56.93431 0.88498 0258455 0.019177603 1.985809 0.399244 7.34E-08 1.849762 0.45305
gi]27806085 peroxiredoxin4 [Bos taurus]
39.06346 0.70963 0.290526 0.000212765 1.00785 0.357468 0.9441931 1.298553 0.788375 0.197172895
gil115497328 procollagen C-endopeptidase enhancer 2 [Bostaurus]
2.15E-05
0.416675
2.30525
5.13E-07
0.262848
2.440332
0.081894695
0.346696
1933682
1.171852
gi|119912215 PREDICTED:hypotheticalprotein isoform2 [Bos taurus]
5.61E-06
3.070175 0.768143
2888364 1.172907 0.475952 0.138293414 2.707686 0.693884 8.72E-06
gi]94966781 eukaryotictranslationelongation factor 1 gamma [Bostaurus]
4.36E-05
2.000045 0.382546
36.8881 0.965646 0.296138 0.630514667 2.090992 0.294809 5.40E-06
gi[27806351 ezrin [Bostaurus]
0.754362
0.001969849
2.768278
0.0015722
0.547593
2.561417
0.178795263
361552 1.206622 0.426321
gi|119918578PREDICTED:smilar to vinculinisoform meta-VCLisoform 1 [Bos taurus]
0.00116154
0.00692 0.633805 0.607659
1.75E-06 0.858276 0.295083
29.7235 0833238 0.325341
gi|119331178SPARC related modular calcium binding 1 [Bos taurus]
39.72222 0.909011 0.434115 0.075654317 1.10709 0.44731 0.1091174 1.120131 0.444784 0.021602435
gi[76253701 decorin [Bostaurus]
6.75E-05
8.63E-05 1.009105 0.279098 0.8959932 0.750986 0.408437
40.79823 0.727481 0.485901
gil114052653procollagen C-endopeptidase enhancer [Bos taurus]
3.25E-09
43.93939 0.903605 0.225695 0.00369553 2.955933 0.792807 8.33E-10 2.895901 0.857683
1
[Bos
taurus]
gil68299807 eukaryobc translation elongationfactor 1 alpha
6.51E-12
0.766198
3.928961
3.93E-12
0.649795
3.373809
1.B2E-05
5855263 1.066869 0.557906
gi]115496892non-metastatic cells 2, protein (NM23B)expressed in[Bos taurus]
2.76E-22
0 2.038513 0.349828
5.28E-14 1.333828 0.284532
70.87379 0.779528 0.307805
gi76670918 PREDICTED:similarto histone H4 [Bos taurus]
0.0002955 2.283279 0.802632 0.000532804
0.952032
2.978355
0.125970649
0.431983
1.181813
49.30789
[Bos
taurus]
reductase)
Al
(aldehyde
1,
member
family
reductase
aldo-keto
gil115495641
0.073378059
0.567645
0.81011
0.0604612
0.32636
0.006041276
0.900239
0.381171
2209302 0.801612
gil154707890lysyl oxidase-like2 [Bos taurus]
3.38E-06
6.D9E-05 1.3755 0.49172 0.0276241.4423270.30729
354067 0633137 0.478665
gill14051505 serpin peptdase inhibitor,clade H (heat shock protein 47), member 1, (collagen bindingprotein 1) [Bos taurus]
1.663922 0.977401 0.001238017
3859348 0.745777 0700688 0.010130054 2.053107 1.D43318 0.0001156
gil114051908 FK506bindingprotein 10,65 kDa [Bos taurus]
3.58E-06
4323204 1.179926 0432622 0.134949764 2.829475 0.442488 8.27E-06 2.952465 0.423224
gi|118601868 heat shock 90kDa protein 1, beta [Bos taurus
24.28717 0711018 0.35738 0.001865616 0.994869 0.329713 0.7952449 1.379617 0.351506 0.001825279
gi|119900887 PREDICTED:similarto collagenXXVilproalpha 1 chain precursor; preproprotein [Bostaurus]
8.15E-06
0.50656
3.198501
5.52E-05
0.677598
3.40858
0.013554497
0.39997
1.337168
26.75045
gi|94966765 glucose phosphate isomerase [Bos taurus]
0.0078125
8.97E-05 0.320559 0.720169 0.0003259 0.252591 0.802247
27.11864 0.372085 0.501949
gi|30794292 lactotransferrin[Bos taurus]
25.71042 1.018847 0.342963 0.83106789 1.279463 0.699551 0.1886494 1.072044 0.940242 0.710279484
gi|119917101 PREDICTED:hypothetical proteinisoform 6 [Bostaurus]
0.1953125 2.509343 0.626335 0.0078125 2.315977 0.56859 0.000477678
23.07692 1.099276 0.41174
2 [Bos taurus]
gil115496400 dthydropyrimidinase-like
7.12E-06
1.35E-07 2.089754 0.647019
37.16012 0.930906 0.492291 0.419250873 2.43038 0.584041
gil15707396 reticulocalbin1, EF-hand calcium binding domain[Bos taurus]
1.01E-08
2.992986 0.542725 2.48E-10 2.74771 0.649615
0.000136922
0.321317
06.47059
1.278383
taurus]
[Bos
epsilon
polypeptide
protein,
activation
5-monooxygenase
gil27806197 tyrosine3-monooxygenase/tryptophan
0.033521707
0.514091
0.791706
0.0447558
0.418005
0.740266
0.015955232
0606565
1477331 0.540961
gi|27805991
3 latenttransforming growthfactor beta binding protein2 [Bos taurus]
1.56E-05
3.58E-07 1.062949 0.426523
2.87E-10 1.139816 0384728
28 0859221 0.363144
gi|2780662 fibromodulin[Bostaurus]
175049 0770344 0.231358 0.001244457 0.957782 0.262863 0.3729791 1.10643 0.254901 0.086168407
gil119887291 PREDICTEDsimilarto peroxidasin homolog [Bostaurus]
3.63E-12
1.20E-12 3.144248 0.837481
gil119915902 PREDICTEDsimilarto Thioredoxindomain-containingprotein 5 precursor (Thioredoxin-likeproteinp46) (Endoplasmic reboutumprotein ERpd6) isoform 1 [Bust 3971292 0.997541 0.50088 0.981283756 3.006458 0.723948
2.20E-06
290976 1.506858 0.390306 0.001864326 2.846165 0.286288 2.84E-07 2.876172 0.387523
gil78369310 stress4nduced-phosphoprotein 1 (Hsp70/Hsp90-organizingprotein) [Bos taurus]
8.41E-05
0.380839
1.873962
0.033177
0.473816
1.343691
D.072756884
0.357698
1202307
22.79898
gil119892775 PREDICTEDsimilarto myosin. heavypolypeptide 9. non-muscle[Bos taurus]
7.47E-05
19.88743 1.219497 0.313394 0.074649923 3.827347 0.373161 5.64E-05 4.182297 0.430176
gi|41386727 protein kinase C substrate 80K-H[Bos taurus]
23.88721 0.632282 0.948914 0.004192246 2.049133 0.405612 2.42E-05 1.595401 0.43057 0.000324779
gi|1 19908681PREDICTED:similarto superficialzone protein[Bos taurus]
24.29907 0.778807 0270498 0.000254386 1.51588 0.304836 7.87E-06 1.198242 0.263457 0.001529606
gi27805823 dermatan sulfate proteoglycan3 [Bostaurus]
1.50E-07
1.79E-06
0.720128 0.197288
23.00062 0.761568 0.540382 0.019970875 0.757169 0.223901
gi|119888620 PREDICTED:similarto alpha 1 type XVIcollagen [Bos taurus]
2734027 1052909 0.806197 0.679115689 0.946403 0.281463 0.1907425 0.864673 0.63068 0.196108186
gil139949116 slit-like2 [Bos taurus]
1.73E-06 0.937929 0.310296 0.0517578 0.86595 0.935589 0.002545598
5761589 0.478727 0.264371
gi 27805983 leukocytecell-derivedchemotaxin 2 [Bos taurus]
7.88E-05
48.62385 1-333883 0.500675 0.050782434 1.393843 0.413415 0.0164323 2.288128 0.49468
gil27806317 annexin A6 [Bos taurus]
3706468 0765069 033217t 0.005260277 1.871569 0.445891 0.0001465 4.23411 0.759813 0.001953125
gil148223509 osteoprotegerin [Bostaurus]
2.25E-10 0.896397 0.249134 0.0019443 0.821616 0.430317 0.005111049
2507375 0.676009 0208364
gi|119917871 PREDICTED:similarto serin protease withIGF-bindingmotif [Bos taurus]
4.11 E-07
3395932 0.964435 0657748 0.752539626 2.195263 0.728227 2.96E-06 2.236703 0.799112
gi|119903961 PREDICTED similarto Protein disulfideisomerase associated 6 isoform 7 [Bos taurus]
2102102 082622 0.327152 0.049479839 1.230651 0.269044 0.0219369 0.940036 0.242164 0.447340792
gi|31341666 mannosidase, alpha, class 2B, member 1 [Bos taurus]
41.20482 0916578 0.272702 0.095089985 1.38818 0.367229 0.0001809 1.255011 0.272421 0.000128137
gil115496067nucleobindn 2 [Bos taurus]
12.18574 1.108672 0436888 0.333419449 0.743294 0.488374 0.0248765 0.793328 0.316939 0.017885709
gi|30794358 versican [8os taurus]
9.50E-10
3.44E-08 0.215502 0.273773 8.26E-09 0.128364 0.274317
3255814 0.396545 0195772
gi76613223 PREDICTED:similarto chitiase [Bos taurus}
39.36508 0.969705 0.682093 0.853675631 3.123114 0.769672 0.0002268 2.24359 0.507328 0.000111094
gi77736275 calumenin [Bos taurus]
0.009286548
1.124706
0.0016373
2.9947
0.914706
3.714805
0.105825187
0.483219
1.308305
55.9633
gil28461273 glutathione S-transferase M1[Bos taurus]
24.91103 1.026151 0.396244 0.792354392 2.236643 0.464371 0.0002214 2.64342 0.6928230.000656927
gil116004023phosphoglucomutase 1 [Bos taurus]
2.647829 0.944823 0.003718017
29653 1.162354 0.492613 0.285804312 3.610974 0.673524 0.0001169
gil115497628reticulocalbin2, EF-hand calcium bindingdomain [Bostaurus]
1.56E-11
22.86325 1132734 0.364022 0.017929343 0364105 0.705031 1.94E-10 0.58983 0.326639
gi]27807349 complement component 1 inhibitor[Bos taurus]
8.13E-07
1.81E-10 1.008216 0.255657 0.8784018 1.566374 0.435435
2.84615 0.627622 0.260391
gi]27806625 ftizled-retated protein Bus taurus]
latent
0.064194283
gi|78369510 ribonuclease/angiogenininhibitor1 [Bos taurus]
gill 18151330tyrosine3-monooxygenaseltryptophan5-monooxygenase activation protein,theta polypeptide[Bos taurus]
gi[41386719 milkfat globule-EGFfactor 8 protein [Bostaurus]
PREDICTED:hypotheticalprotein [Bos taurus]
gi[119919898
06
gi|278 561 lactate dehydrogenase B [Bos taurus]
gil156121049dimethylargininedimethylaminohydrolase1 [Bostaurus]
gil115497174 3-phosphoadenosine 5'-phosphosulfate synthase 2 [Bos taurus]
gi|119895049PREDICTED:similarto Lamin 92 [Bostaurus]
PREDICTED:similarto coat protein delta-cop isoform 1 [Bos taurus]
gi76635416
7
gil12927 510 superoxide dismutase 3, extracellular[Bos taurus]
gil27806401 secreted phosphoprotein1 [Bostaurus]
gi|149773543steroid-sensitive protein 1 [Bostaurus]
gi|27806679 colony stimulatingfactor 1 (macrophage) [Bos taurus]
gi|51491841 transketolase [Bostaurus]
gi|119913642 PREDICTED:similarto KIAA0051[Bos taurus]
gi]119908661PREDICTED:similarto Laminingamma-i chain precursor (Laminin62 chain) [Bos taurus]
stromalcell derivedfactor 4 [Bos taurus]
gi]78369298
94
gi|7581227 30 phosphatidylethanolaminebindingprotein [Bostaurus]
gill1405 1 tubulin,beta polypeptide[Bos taurus]
apolipoproteinE [Bostaurus]
gi|27806739
7
64
gi|2 808 D peptidoglycanrecognition protein1 [Bos taurus]
gil27807167 peroxiredoxin6 [Bos taurus]
gil114053333 fibrinogen-like2 [Bos taurus]
gi|82697375 histone H2B-like[Bos taurus]
hypothetical protein [Bos taurus]
gi|119903031
4 6 PREDICTED:
gil119901 1 PREDICTED:similarto Syncrip protein [Bos taurus]
gi]71037405 heat shock 27kDa protein 1 [Bostaurus]
gi|1 16003813amyloidbeta A4protein [Bos taurus)
gi|119901059 PREDICTED:similarto COL9A1protein,partial [Bos taurus]
giJ76611607 PREDICTED:similarto C1QC proteinisoform 1 [Bos taurus]
gi[75832065 TIMPmetatlopeptidaseinhibitor2 [Bos taurus]
gi|119896006PREDICTED:similarto mannosidase, alpha, class 2A,member 1 [Bos taurus]
gi|148230364heterogeneous nuclear ribonucleoproteinD [Bos taurus]
gi27805977 keratin 10 [Bostaurus]
gi]27806095 protein S, alpha [Bos taurus]
gil119900517PREDICTED:similarto KtAAl027protein[Bos taurus)
gil66792902 vanin 1 [Bostaurus]
gil1
19917901 PREDICTED:similarto CarboxypeptidaseX 2 (M14family)[Bos taurus]
[Bos taurus]
gil125991942
3 Sec23 homolog A
binding protein [Bostaurus]
gi|8457985
gil113205970 glialfibrillaryacidicprotein [Bos taurus]
gi[27806541 matrixmetalloproteinase1 [Bos taurus]
gi[78369684 platelet-derivedgrowth factor receptor-likeprotein [Bos taurus]
gi]27807517 serine (or cysteine) proteinase inhibitor,clade B (ovalbumin),member 6 [Bos taurus]
gil115496928 EGF-containingfibuhn-ike extracellularmatrixprotein2 [Bos taurus]
gi]95006989 ribonuclease, RNase A family,4 [Bos taurus]
gi|28875793 thrombospondin2 [Bos taurus]
gi|27806449 dystroglycan 1 [Bos taurus]
gi62751777 cofilo 1 (non-muscle)[Bos taurus]
giJ27807469
9 peroxiredoxin2 [Bos taurus)
gi|7740420 9 eukaryotictranslationinitiationfactor 4A2 [Bos taurus]
induced) 2 [Bostaurus]
gil11405112
3 27 retinoicacid receptor responder (tazarotene
galectin 3 [Bostaurus]
gil156121
gi27806813 pleiotrophin[Bostaurus]
gi|95147666 periostin, osteoblast specificfactor [Bos taurus]
gi|119919662 PREDICTED:similarto LOC539445protein [Bos taurus]
gi|77735541 valosin-containingprotein [Bos taurus]
gi|60592767 ribosomalprotein, large, PO [Bostaurus]
giJ77735461 canopy 4 homolog [Bos taurus]
gil27806829 osteoglycir [Bos taurus]
gi(99028973
9 transcobalamin |1[Bos taurus]
7
9i157 8556 coatomer protein complex, subunit alpha [Bos taurus]
gij61808545 PREDICTED:similarto histone H2Aisoform 1 [Bos taurus]
hepatoma-derivedgrowth factor (high-mobilitygroup protein 1-tike)[Bos taurus]
gi(28461287
3 2
6 44 eukaryotictranslation initiationfactor 5A [Bos taurus]
gil510
gi|75832045 HLA-Bassociated transcript 1 [Bos taurus]
gi|41386707 vascular celladhesion molecule 1 [Bos taurus]
gil119915070PREDICTED:similartoCUB and EGFcontainingprotein [Bos taurus]
gil134085613alpha 1 type XVIIIcollagen [Bostaurus]
gi|115497294canopy 2 homolog [Bos taurus]
gilo14053135proteasome (prosome, macropain) subunit,alpha type, 6 [Bostaurus]
factor 3 isoform2 [Bos taurus]
gil119894807
97 PREDICTED:interleukinenhancer binding
gil164518 8 diazepam bindinginhibitor[Bos taurus]
gi|73853762 annexin I[Bos taurus]
complement component 1, q subcomponent, B chain [Bos taurus]
gill14051157
9
gi|780454 1 fascin homolog 1, actin-bundlingprotein [Bos taurus]
gi|780455552 acidic(leucine-rich)nuclear phosphoprotein32 family,member B[Bos taurus]
gi|15742778 insulin-likegrowthfactor binding protein 5 [Bos taurus]
gi]27807007 insulin-likegrowthfactor binding protein 3 [Bos taurus]
gi|129270171 gremlin-1[Bos taurus]
gi|119926480 PREDICTED:similarto Meteorin,glialcell differentiationregulator-like[Bos taurus]
gi78042524 thioredoxindomain containing4 (endoplasmic reticulum)[Bos taurus]
lipopolysaccharide
29.38597
55.91837
33.48946
26.28571
47.60479
39.29825
31.21951
35.92715
27.0057
41.49378
45.68345
20.96436
27.07581
27.60835
22.83019
21.33858
32.67606
49.73262
22.2973
45.88608
46.31579
48.66071
44.89796
78.57143
19.6281
31.11702
41.66667
15.68345
19.71253
25.36765
33.18182
18.42795
35.62092
23.95437
22.66667
1.126192 0.324758
1.199329 0.39451
D.79112 0.202064
0.775134 0.46563
1.508136 0.342936
1.237758 0.3963
0.949886 0.251807
1.020326 0.212387
1.193124 0.247001
0.926996 0.387711
0.720812 0.458792
0.605496 0.607317
2.070581 0.802553
1.028374 0.531252
1.436444 0.503329
0.926587 0.285185
1.012411 0.517378
1.099546 0.353673
1.038783 0.49663
0.733549 0.252604
0.639251 0.33389
1.323234 0.296632
0.749614 0.318248
0.778357 0.309967
1.617291 0.439488
0.824457 0.443684
0.930182 0.428426
1.404063 0.335795
0.699285 0.338444
0.734554 0.67762
0.65191 0.497802
0.999529 0.28011
1.082545 0.371005
0.714804 0.141639
0.691925 0.320239
22.2747 0.848161 0.076552
17.64706 0.405888 0.843808
26.51715 0.686724 0.290136
1809896 1.12328 0.277486
23.07692 0.248446 0.43585
52.1028 1.142308 0.609279
26.01279 0.421773 0.29099
36.8 0.980503 0.400557
31.21693 1.448994 0.534954
31.37698 0.776944 0.283558
59.86394 0.470833 0.438507
13.84615 0.843256 0.307429
14.07821 0.874871 0.407648
71.08434 1.299646 0.549125
39.19598 1.246076 0.520799
21.86732 1.360362 0.492577
50 0.588901 0.517981
36.60377 1.050061 0.481141
38.69048 0.458214 0.616786
23.62003 0.620313 0.577812
16.21271 1.032594 0.352598
28.0397 1.600285 0.979663
46.22642 0.939665 0.34456
12.5523 0.961306 0.529227
34.11371 1.043169 0.513296
22.91667 0.68501 0.310794
0.276264
20.75163 1.102223
62.30769 0.694453 0.450515
42.67783 1.064069 0.347332
24.02597 0.993644 1.03353
28.27103 1.04593 0.454598
20.43302 0.896205 0.529789
10.2719 0.702824 0.509628
19.15456 1.108286 0.310159
33.51648 0.901592 0.387093
31.30081 1.161236 0.344706
17.3913 1.005649 0.464716
79.31035 1.461292 0.433004
36.12717 0.760038 0.297116
22.67206 0.661922 0.425196
22.10953 1.222581 0.434414
34.09962 1.171976 0.326434
49.07749 0.954689 0.404841
35.05155 0.746612 0.194963
0.895
63.58696 0.752901
22.3301 0.404822 0.399363
21.92116 1.009579 0.180837
0.300595249 2.622966 0.510622 0.0012207 1.77584 0.790103 0.046451449
01010121323005314 0.519199 5.93E-05 2.537452 0.70156 0.000908615
4.52E-06
1.05E-05 0.539079 0.500076
7.496-06 0.640309 0364921
0011395109 0.944692 0.50241 0.4270805 0.791509 0.5186360.027304536
9.61E-07 3.66979 0.501802 0.00390625
0.000392235 4.069971 0942095
1.72E-05 3.00167 0.674195 0.000267971
0.5117
0.156403106 3.70924
8.15E-06
0.571167 1.97E-05 2.843166 0.533737
2.902697
0.439653794
0.3111610.0003285 2.039338 0.733335 0009154083
1.944109
0.720053349
0.091502996 3.559594 0.440376 0.03125 3.951069 0.654932 0.00067055
0.376073329 0.701524 0.257354 443E-05 0.628839 0.397688 0.001953125
0.000291367 2.779999 0.444469 2.98E-11 0.861641 0.406777 0.051614392
7.53E-07
0056229617 1.622905 0.402909 0.0196995 3.790859 0.157842
0001457649 070331 0.456362 0.0032712 1.744261 0.580574 0.0000840259
1.93E-08
0.920956016 4.020656 0.641951 3.20E-08 3.845949 0.670613
3.84E-05
0.033195974 3797069 0.730459 5.136-05 3.948679 0.723811
0392433702 0903793 0363619 0.3132383 0.86015 0.154319 0.026026895
0.109375
0.92627127 1.250975 0444424 0.1217071 0.734348 0.350268
4.73E-09
0116220906 2.426716 0.401752 6.14E-11 2.189436 0.481435
6.69E-05
0.759217249 2.469264 0.572251 0.0003883 2.399148 0.432184
1.39E-05
0.000341695 0.704996 0.346392 0.0010251 0.47D103 0.39876
0.000409542 0792692 0.299915 0.0046462 0.71025 0.459951
1.67E-09
1.80E-09 5.140501 0.459027
000390625 4067222 0.399759
0.013672157 0.396909 0629676 0.0010998 0.340626 0.850908 0.003100862
1.82E-05
0.0359793 1.529371 0213163 0.0010414 2.604213 0.216527
2.22E-16
0.629535 6.51E-14 3.302432 0.463762
3.916-09 3.6169
0299776229 2.991697 0.520536 0.0008649 2.56434 0.543379 0001575162
8.87E-05
5.30E-05 2.496215 0.555573
0.517012213 3426523 0670677
0.002337023 0.975401 0.270109 0.5734683 0.826073 0.343311 0.056168721
9.736-05 1.053999 0.397699 0.4115302 0.95028 0.407553 0.026398981
0.054991937 1.132333 0.506994 0.3249347 0.683994 0.580632 0.013026282
0.2939 0.000226808
3.996-05 1.974962 1141225 0.0140974 0.79563
0.997929406 0.999099 0.355969 0.9104525 1.009918 0.288851 0.826414819
0.643733331 2.312701 0.562955 0.0672586 2.274411 0.987044 0.092233717
0.000401162 0.599967 0.423997 0.0071728 0.643632 0.3127 0.004795902
0.0625
0.125 0.496097 0.097204 1.21E-05 0.422755 0.718362
0.125
1.497581 0.6134
0033191021 1.644241 0.656075
5.84E-10
2.52E-05 0.552479 0.504984
1.756-09 1.392702 0.34499
1.0253 0.426367 0.782321414
0.014026424 0.973799 0.077667 0.0034577
4.91E-05 2.025963 0.324223
0.245595267 2.345791 0229969
1.79E-07
1.966-05 0.424693 0.313313 2.11E-06 0.314787 0.298409
0.517790539 3.051097 0.456294 0.0014673 3.277184 0.402737 0.000132843
0.000139795 0.947792 0.321252 0.4893589 0.904175 0.268745 0.306090759
0.299 0.9042234 0.975588 0.302229 0.882598401
0.842916695 1.023016
0.391170505 2.369377 0.703193 0.0946462 2.645973 0.754333 0.013462004
0.045796595 0.906221 0.29523 0.066161 0.473723 0.520026 0.010437137
3.88E-05
4.976-11 0.75013 0.403742 2.33E-05 0.505201 0.481114
0.059936619 1.339209 0.502969 0.0078125 0.989947 0.48982 0974569727
01159614 0947697 0.425033 0.4221623 1.059235 0.482058 0.454602142
0072047296 3.503711 0.532703 8.05E-06 2.868775 0.680483 0.000138879
0.156919757 3.716369 0.697934 0.0003912 3.177227 0.894847 0.002474373
2.34E-06
0.01996463 3.009031 0.617036 2.13E-05 2.942636 0.474047
0.00092 0.743661 0.792982 0.077022558
0.000100372 0.619703 0.599036
1.47E-12
0.473661304 2.929179 0.742356 4.99E-11 3.182978 0.698586
1236-06 1.57945 0.397929 6.18E-06 1.605253 0.868891 0.004185803
0.056077025 1.377917 0.137072 0.0019626 0.799599 0.164351 0.014688243
0.77993524 4.090764 0.960567 0.0037025 2.538432 1.212873 0.040063294
0015625 2.430097 0.402511 0.015625 2.910937 0.498942 0.000167836
5.79E-06
4.04E-06 2.839283 0.838384
0.521179669 2.692939 0.68635
0.971797147 2.619545 0.902403 0.0063833 2.659301 0.402291 0.000329245
1.0946490420726 0.2957764 1.210667 0.336312 0.005811501
0.659518694
0017260013 099271 0.2515140.1768685 1.310973 0.096146 0.000669569
0.315996319 2.719342 0416479 0.0004127 2.725266 0.373542 0.000206011
4.66E-15
0003090092 0973912 0.060754 0.1121356 2.588773 0.378437
0.637719162 2.676727 0.590959 0.0060942 3.409638 0.638057 0.003296838
0.990709602 2.223171 1.207639 0.0110729 2.175438 1.000295 0.003571663
0.675319797 3.090223 0959097 0.0004906 2.787343 1.069869 0.001644201
0.59039496 0.455245 0.197909 0.0011572 0.504189 0.487159 0.028514686
0.058640939 0.99664 0376304 0.8056481 0.955899 0.592788 0.860490913
03426475 0963963 0.209996 0.478133 0.970308 0.191641 0.80011816
0.439693459 2.414472 0.569202 0.007906 1.574095 0.628333 0.074235276
0147267129 2524294 0.693279 7.98E-05 2.336087 0.851779 0.000365813
D.0625
0.963199927 2.214517 052779 0.0033862 2.012049 0.722936
8.06E-08
0.000649227 4.737242 0.6969 6.58E-08 3.947945 0.629687
004109943 1193621 0.197493 0.024503 0.951472 0.337637 0.724154961
2.22E-07
5 576-07 1013925 0.390959 0.3313349 0.684714 0.355503
1.06E-06
0.02120626 2.319909 0.44932 6.38E-08 2.025029 0.491916
0.111557495 3.344199 0.549291 0.0001651 3.197614 0.676538 0.000564204
0.706974927 0.917127 0.419721 0.4342584 1.326475 0.856312 0.275477997
0.109375
0.000490913 1.504305 0.199503 8.6E-05 1.875692 0.823987
0.45527291 0.954642 0.354793 0.1841908 0.59448 0.86671 0.100457964
0.007304042 0.732573 0.274444 0.0418192 0.476848 0.369024 0.010811399
0.900093969 3.14199 0.510159 0.0077449 2.457633 0.748651 0.092871086
0.014955195
0.1236341
0.000538952
1, NAD(soluble)[Bostaurus]
gi|77736203 malatedehydrogenase
gi27806415 scinderin[Bostaurus]
[Bostaurus]
gi56119114 purinenucleoside phosphorylase
gi]77736471 SH3domainbindingglutamicacid-richproteinlike 3 (Bostaurus]
gi]45429977 staphylococcal nuclease andtudor domaincontaining1 {Bostaurus]
inhibitor(GDI)alpha[Bos taurus]
gil28603774 RhoGDP dissociation
gamma[Bos taurus]
gil62460494 hemoglobin,
gi|77735429 proteasomealpha3 subunit{Bostaurus]
A chain [Bos taurus]
component1.q subcomponent,
gi|62460582 complement
ubiquitin-acvatingenzymeE1 [Bostaurus]
gi|156523068
gi61888850 adenylatekinase1 (Bostaurus]
gi(119914274PREDICTEDhypotheticalproteinisoform1 [Bostaurus]
13[Bos taurus]
gi|27805999 matrixmetalloproteinase
gi157074098 leprecan1 [Bos taurus]
gi1156120501heatshock70kDprotein bindingprotein[Bos taurus]
gi115497728 aconitase 1, soluble(Bos taurus]
gil119907132 PREDICTEDsimilarto Hyout protein[Bos taurus]
alpha polypeptide il [Bos taurus]
4-dioxygenase (proline 4-hydroxylase),
2-oxoglutarate
gi77735539 procollagen-proline,
(NADP+)(Bostaurus]
gil119901144 PREDICTED:similarto malatedehydrogenasedecarboxylase
2 [Bos taurus]
gil157954420
3 angiopoietin-like
gi2780750 ribosomalproteinS27a [Bos taurus]
gill 14052264 phosphoglucomutase 3 [Bos taurus]
1, cytosolic[Bos taurus]
gi|139948301 branchedchain aminotransterase
gi28603770 capping protein (actin filament)muscleZ-line,beta (Bnstaurus]
gi]88853816 superoxidedismutase2, mitochondrial[Bostaurus]
partial [Bostaurus]
similarto Calsyntenin-1,
gi|119908402 PREDICTED:
gi|27806673 cathepsinL2 [Bos taurus]
gi|119919488 PREDICTED:similarto cathepsin 0 isoform1 [Bos taurus]
gil119894607 PREDICTED similarto peptidoglycanrecognitionproteinL (Bostaurus]
gi]77404434 hypoxanthinephosphoribosyltransferase 1 [Bos taurus]
gil27806553 microtubule-associatedprotein4 [Bos taurus]
gil27807445 peroxiredoxin5 precursor [Bos taurus]
gil27806671 cathepsin B [Bos taurus]
gi|27807523 ribosomalprotein P2 [Bos taurus]
gi|76610312 PREDICTED:similarto neuropilin 2 isoform 8 [Bos taurus]
gil27806637 fibrillin1 (Bos taurus]
gir27807209 alpha-2-plasmininhibitor[Bos taurus]
gi}83715992 coatomer proteincomplex,subunitgamma1 [Bostaurus]
gi|27806139 secreted frizzled-relatedprotein 1 [Bos taurus)
gil119919689 PREDICTED:similarto RNA bindingmotif protein, X-linkedisoform 5 [Bos taurus]
gill 15497086cortactin (Bos taurus]
gi|134085635glyoxalase I[Bos taurus]
gil119890932PREDICTEDsimilarto Lamininbeta-1 chain precursor (LamininB1 chain) [Bostaurus]
types) (Bos taurus]
gil56710317 keratin 5 (epidermolysisbullosa simplex,Dowling-Meara/Kobner/Weber-Cockayne
gi|76620378 PREDICTED:similartocytokine-likeproteinC17 [Bostaurus]
gi|155372035nidogen 1 [Bostaurus]
gi|75832090 isocitrate dehydrogenase 1 (NADP+),soluble [Bos taurus]
gi|27806447 prosaposin [Bos taurus]
gi|78369645 SH3 domainbinding glutamicacid-rich protein like[Bos taurus]
gi|115494984apolipoprotein0 [Bos taurus]
[Bos taurus]
gi|119922764PREDICTED:similarto MSTP006
gil153791208immunoglobulinsuperfamilycontaining leucine-richrepeat[Bos taurus]
gil28461189 lectin, galactoside-binding,soluble, 1 (galectin 1)[Bostaurus]
gi]29135329 glutathione S-transferase pi [Bs taurus]
gi|115497078splicing factor,arginine/serine-rich1 (splicing factor 2, alternate splicing factor) [Bostaurus]
gi|119924055 PREDICTED:similarto p167 isoform4 {Bostaurus]
gi|119879765 PREDICTEDsimilarto eukaryotictranslationinitiationfactor 4 gamma, 1 [Bos taurus]
gi|116004039 peptidylprolylisomerase C (cyclophilinC) [Bostaurus]
gil27806783 thioredoxin[Bos taurus]
gi]78369458 proteasome (prosome, macropain) subunit,alpha type, 1 {Bostaurus]
gil119908208 PREDICTED:similarto Calpain 2, (m/l1)largesubunit isoform 3 [Bos taurus]
gi|77736071 heterogeneous nuclearribonucleoproteinK (Bostaurus]
gi|84000347 interleukinenhancer bindingfactor 2 [Bos taurus]
gi|41386760 CD14antigen [Bos taurus]
gi|115495555 endoplasmicreticulumprotein 29 [Bos taurus]
gi|78369232 phosphorylase,glycogen, brain [Bos taurus]
gi|27806081 peroxiredoxin1 [Bostaurus]
gi|77736371 actin related protein203complex subunit 2 [Bos taurus]
gi|41386798 poly(A)bindingprotein, cytoplasmic1 [Bos taurus]
gi}61888866 tropomyosin1 alpha chain [Bos taurus]
gi|78042498 phosphoglyceratedehydrogenase [Bos taurus]
gil27806751 alpha-2-HS-glycoprotein[Bos taurus]
gir77736343 ErbB3-bindingprotein 1 [Bostaurus]
gi|27807377 glutamic-oxaloacetctransaminase 2, mitochondrial(aspartate aminotransferase 2) [Bos taurus]
gi]62177164 tumorprotein, translationally-controlled1 [Bos taurus]
group box 1 [Bostaurus]
gij41386729 high-mobility
gi(164420709retinolbindingprotein 4, plasma [Bostaurus]
gi]27806487 serine (or cysteine) proteinase inhibitor,clade F (alpha-2 antiplasmin, pigment epitheliumderivedfactor), member 1(Bos taurus]
gi|119924945PREDICTED:similarto agrin [Bos taurus]
gi]82524405 US01 homolog,vesicledocking protein[Bos taurus]
gi|76651183 PREDICTEDsimilarto histone H3 [Bos taurus)
gi|27806881 epididymalsecretory proteinE1 [Bos taurus]
31.13773 1.3796260.184951 0.015124221 3.408354 0.185321
23.21678 0.885651 0.5675610.447981718 2.157923 0.404807
0.125 2.345824 0.527841
19.72318 1.3324070.367264
53.76344 1.0250670.347011 0.845657197 2.129681 0.617309
27.58242 0.900734 0.198308 0.321008378 1.61418 0.719711
45.58823 1.392308 0.437747 0.027607744 3.050149 0.610149
69.65517 1.44846 0.234906 0.00699086 1.621779 1.029319
29.80392 0.682914 0.537326 0.084294821 1.290988 0.835646
48.77049 0.593049 0.500713 0.000548737 0.906695 0.377041
15.02836 1.245575 0.282183 0.065427349 2.665289 0.731677
40.72165 1.0459 0.218217 0.537002948 2.966651 0.609538
21.82857 1.221701 0.56517 0.467953282 3.302064 0.412142
23.35456 0.380074 D.254298 0.001622704 0.51614 0.273737
17.25543 0.996475 0.287665 0.992062477 2.37895 0.319461
21.68022 1.354057 0.380122 0.005344143 2.752202 0.538342
14.28571 1.117697 0.491219 D.502761117 3.589575 0.37128
0.1329 0.113432742 3.22781 0.844503
19.98002 1.106651
0.0343738 3.120213 0.498958
27.72074 0.877814 0.101158
25.42373 1.217784 0.38359 0.073445335 3.689365 0.478908
14.19878 0.900802 0.149951 0.089461939 0.956216 0.202782
53.84616 1.229495 0.386822 0.005714112 2.449846 0.497627
19.18819 1.360266 0.280167 0.023770178 3.010397 0.521807
20,20725 1.294087 0.252831 0.02996345 3.20688 0.406586
35.21595 1.082165 0.223792 0.314590585 2.006861 0.278274
34.23423 1.148276 0.284911 0.253928781 0.978402 0.619482
15.80611 0.876277 0.448436 0.540422033 0.929385 0.487573
30.83832 0.912492 0.447437 0.467843256 1.111225 0.269885
28.8835 1.089541 0.614847 0.712006947 2.065618 0.225223
19.96616 3.268542 0.853115 0.000759791 0.712782 0.425171
37.15596 1.17602 0.536588 0.290628484 2.803965 0.552609
23.13433 0.970701 0.110027 0.685964868 2.694891 0.528697
29.22374 0.81151 0.749682 0.481703528 1.977168 1.606058
17.61194 0.785806 0.30958 0.006301553 1.755499 0.247458
41.73913 1.274264 0.549719 0.074405983 4.509896 0.602457
19.87041 1.110245 0.176154 0.125980369 0.890141 0.292637
10.37966 0.906627 0.530205 0.478444551 0.970058 0.35082
22.56097 0.599339 0.62022 0.056459368 1.117795 0.400638
17.04805 0.974997 0.137881 0.699598479 2.415233 0.419959
1.84E-07 1.024216 0.341271
31.81818 0.626649 0.33961
41.68798 0.839077 0365111 0.197549662 2.282233 0.454349
19.88848 0.920482 0.522844 0.570787529 1.698239 0.686754
41.30435 1.103425 0.160067 0.116314216 4.245659 0.469967
16.42772 0.992998 0.340681 0.942123233 1.07211 0.349671
23.96007 0.794447 0.476544 0.14970632 0.626444 0.281415
39.41606 0.824377 0.407782 0.008142552 0.920387 0.454298
12.65109 0.823522 0.296224 0.069126059 1.038216 0.202334
28.26087 1.445789 0.458957 0.102862623 2.514228 0.577943
15.42857 0.802455 0.309292 0.218395134 1.249937 0.244271
44.73684 1.253298 0.301906 0.072725986 3.625316 0.623463
8.27E-09 1.087048 0.277356
48.14815 0.495177 0.226909
19.82841 1.663807 0.752503 0.232522007 3.749804 0.937328
0.296875 0.862362 0.499196
16.35514 0.802223 0.41405
35.55556 1.610276 0.374908 0.006413745 2.624683 0.359533
32.38095 1.122915 0.244218 0.118041828 2.950799 0.292574
50 1.234643 0.305858 0.058368077 2.890116 0.370763
23.32849 1.314471 0.187168 0.067196595 2.246554 0.525588
22.98739 0.852552 0.354759 0.287069522 1.537511 0.545863
2.47E-09 1.062364 0.299366
38.20755 0.684331 0.373857
5.D9E-062.949452 0.495424
51.42857 1.40006 0.110957
38.78327 1.12676 0.505423 0.399398202 2.98806 0.524133
16.09589 1.294738 0.547471 0.511199959 2.865098 0.986682
28.23276 1.162442 0.27411 0.209559792 2.827074 0.658521
28.71795 118807 0.413414 0.248192181 2.591724 0.911163
29.49062 D.718284 0.426758 0.067077633 0.447034 0.82842
32.17054 1.063647 0.500749 0.650900277 3.895887 0.816752
1957295 1.124417 0.46718 0.592429287 2.372325 0.962257
3969849 1.013692 0.364535 0.894837687 2.996943 0.608624
24 0.846992 0.409937 0.419687408 1.586726 0.414133
0.80341858 2.595454 0.010263
22.64151 1.04511 0.544222
49.29577 1.269786 0.233070 0.123991012 2.19207 0.041845
17.82364 0.927041 0.397486 0.623947485 2.364541 0.493862
21.72702 0.322124 0.472372 0.006198951 0.51348 0.379411
0.064191 0.890964497 2.208606 0.707947
15.98985 1.004709
26.97674 1.118437 0.227507 0.341451802 2.947352 0.228243
3662791 1.140074 0.510119 0361989591 2.504788 0.731534
29.30233 0.975179 0.471119 0.875466602 3.528455 0.59746
33.33333 0.420745 0.338166 0.023614332 0.636046 0.096835
30.04808 0.687616 0.308951 0.039742456 1.631605 0.34124
12.58035 0.882582 0.100124 0.037338157 D.694748 0.256876
14.77627 0818211 0.58442 0.612123221 1.900213 0.742402
52.20588 0.753026 0.441014 0.072713355 1.281729 0.001102
6.75E-05 2.180407 0.310342
30.87248 0.356994 0.193381
0.0003249
1.35E-05
0.0068653
0.0404264
0.1593815
0.0004622
0.2152592
0.4044338
0.1851973
0.0130567
0.0047413
0.0189709
0.0057
5.21E-05
0.0019531
0.0003907
0.0288445
0.0074
1.14E-05
0.4014582
1.31E-08
0.0025454
0.0625
0.0013815
0.8774691
0.6980291
0.2441812
0.0027932
0.0193949
0.0004158
0.0438219
0.3165338
9.24E-07
1.28E-06
0.2235103
0.6201094
0.4776424
0.0026333
0.8632156
0.0045123
0.0286247
0.0006011
0.7421875
0.0017773
0.1532154
0.4375
0.0201016
0.1660525
0.0007949
0.2377548
0.0733604
0.2610433
0.0002363
1.47E-06
0.0001726
0.0633805
0.1152917
0.2160807
4.97E-05
0.0002231
0.1192677
0.007377
0.0293373
0.0332025
0.0002715
0.1580827
0.0014924
0.114149
0.0076688
0.0004718
0.0052285
0.0139899
0.267018
0.0072522
0.0032679
0.0625
0.0068255
0.0276504
0.0238678
0.3344105
0.2272909
0.0013022
3.44296 0277661 0.000986481
2.696082 0.643891
2.12E-05
0.03125
2.89181 0.609906
1.770043 0442193 D.031622411
0.859 0.138907486
1.790095
2.868464 06141 0.000564349
1.633981 0842019 0.064122109
1.204605 0977631 0.544925809
0.413569 0656242 0.000976563
0447800 0.001499399
2.888502
2.913725 071626 0.008160725
3.197819 0422648 0.020047615
0.329036 0200248 0.0000591469
6.02E-05
2.130793 029614
2.730784 0.044717 0.001953125
4.16E-05
3.841086 0.308882
3.030507 0.843197 0.031103752
2.781147 0.000376 0.012530222
1.36E-05
3.455985 0.477728
0.952238 0.103006 0.503113248
1.856561 0444849 0.000420791
2.471896 0408662 0.002949993
2.984815 0479848 0.001737378
2.073149 0.408887 0.004217119
1.515731 0.010189 0.095180167
0.684979 0.320106 0.104607426
1.025236 0.440829 0.749919944
1.139716 0.280633 0.239601093
0.672148 0.791011 0.099381319
7.26E-05
2.658348 0.393801
0.753 0.121873938
2.155718
1.818467 1.701232 0.375548192
0.543177 0.640833 0.002266239
5.25E-07
4264045 0.032238
0.987084 0.162306 0.984072538
0.935079 0.200966 0.425633123
1.292816 0.418288 0.105518239
3.058982 0.319684 0.000334903
1.230284 0.827382 0.162334404
2.470278 0.488788 0.004058435
0.02492977
1.550913 008181
3.497581 0.0319 0.001311643
0.90306 0.378389 0.37475027
0.73261 0.482631 0.081605855
6.98E-05
0.609768 0.567618
0.723608 0.233962 0.005463519
2.818763 0.008003 0.009451117
D.325786 1.488483 0.204295759
3.338367 0.702696 0.002273803
1.64E-07
0.411653 0.378272
3.638376 0.221916 0.00459497
0.977635 0.618003 0.959597689
2.579216 0.603623 0.002451033
7.23E-07
3.035265 0.277031
2.536704 0.49682 0.001104549
1.833652 0.80870 0.212801969
1.263803 1.216474 D.597450382
0.916782 0.310311 0.064609318
9.37E-06
2.918305 0390989
2.769028 0031731 0.000309952
2.405706 0.79040 0.106135047
2.580231 0407060 0.002457434
2.416142 0.893766 0.031827392
0.487415 0.716581 0.033797768
3.665525 0.67863 0.000492021
1.868395 0.848406 0.200478112
3.170503 0.670037 0.001640537
1.658406 0.7137240.210314559
2.809743 0.724413 0.012307
2.20171 0.611100 0.081511689
2.892111 0013386 0.004805503
0311114 0940334 0.03899761
1.713963 0.8866600.424095043
2.502367 0.367001 0.02398561
0.00158855
2.915557 07084
0.0625
4.561559 0.60021
0.244006 0.090417 0.027453193
1.053292 0.297749 0.567994518
0.660421 0.106879 0.001688625
1.486134 0.797999 0.484810234
2.63243 0.898001 0.004206016
0.83957 0.22989 0.086816037
gi|119912531 PREDICTED:similarto mannose receptor,C type 2 {Bostaurus]
gi|114051730 cell growthregulatorwith EF-hand domain1 [Bos taurus]
gi|77735425 proteasome alpha 7 subunit [Bos taurus]
gi|119905546 PREDICTED:similarto Cu/Zn superoxide dismutase [Bos taurus]
gil27807147 C-type lectindomain family3 member A [Bostaurus]
gil119892706 PREDICTEDsimilarto chondroitn 4-0-sulfotransferase [Bos taurus]
gil119906216 PREDICTED:similarto Eukaryotictranslation elongationfactor 1 delta (guanine nucleotide exchange protein) isoform 1 [Bos taurus]
gi|148232266 fibulin1 [Bos taurus]
gi|166706929 spectrin, alpha, non-erythrocytic1 (alpha-fodrin)[Bos taurus]
gil1 15497888nuclear distributiongene C homolog [Bos taurus]
gi]62460514 angiopoietin-like7 [Bos taurus]
gi]84370163 SET translocation (myeloidleukemia-associated) [Bos taurus]
gi|62751847 ribosomalprotein LlOa [Bos taurus]
gi|119919151PREDICTED similarto AHNAKnucleoproteinisoform 1 [Bostaurus]
gil14052350 WD repeat domain 1 [Bos taurus]
gi|125630701collagen triplehelixrepeat containing 1(Bos taurus]
gi|76613946 PREDICTED:similarto Histone H1.5[Bostaurus]
gill 19904280PREDICTED:similarto collagen type 5 alpha 1 [Bos taurus]
gill 16003917far upstream element-bindingprotein [Bos taurus]
gil148228060 stem cell growthfactor [Bos taurus]
gil28461187 myosin,lightchain 6, alkali, smooth muscle and non-muscle [Bos taurus]
gil134085825 carboxypeptidase Z[Bos taurus]
protein[Bos taurus]
gil119893536
7
3 PREDICTED:similarto KIAA0755
giJ77 3582 ribosomalprotein S20 [Bos taurus]
gil1 19920777PREDICTED:similarto collagen,type IX, alpha 2 [Bos taurus]
gi|157785567calnexin [Bostaurus]
gi|125991882phospholipase C, delta 1 [Bostaurus]
gill 14326274crystallin,zeta (quinone reductase) [Bos taurus]
gi77735583 S-adenosylhomocysteinehydrolase [Bostaurus]
gil119923009PREDICTED similarto put. HMG-17protein [Bostaurus]
gi|119907521PREDICTED:similarto NuMAprotein [Bostaurus]
gil158937293fibroblastgrowthfactor receptor 1 [Bos taurus]
gil114052384heterogeneous nuclear ribonucleoproteinAl [Bostaurus]
gi]77736141 extracellularmatrixprotein 2, female organ and adipocyte specific [Bos taurus]
gi|27807389 bucentaur [Bos taurus]
gil27807481 ubiquitin-likeprotein SMT3B[Bos taurus]
taurus]
gir27807075
226 92 55 9 S100 calcium-bindingprotein A4 [Bos
oncostatin Mreceptor [Bos taurus]
gill
gi]91680535 proteasome activatorsubunit 1 [Bostaurus]
gil76633778 PREDICTED:similarto syndecan 4 [Bostaurus]
gi]45430017 apolipoproteinA- bindingprotein [Bos taurus]
gi|164448628carboxypeptidase X [Bos taurus]
gi|115496724heat shock protein, alpha-crystallin-related,B6 [Bos taurus]
gil27807275 matrixGta protein[Bos taurus]
gi|155372143 arginine-rich.mutated in earlystage tumors [Bos taurus]
gi|164420791transforming growthfactor, beta 2 [Bos taurus]
gil28603812 epsilon subunit of coatomer protein complex [Bostaurus]
gi|119917542 PREDICTED:similarto tolloid-like2 proteinisoform 2 [Bos taurus]
gi|27806419 seryl-tRNAsynthetase [Bos taurus]
gi|78042520 eukaryotictranslationinitiationfactor 48 [Bos taurus]
gi|61097917 cystatin E/M[Bos taurus]
[Bos taurus]
gil27806743 alpha-1-microglobuhn/bikunin
kinase 1, cytosolic[Bos taurus]
gill 14051347 cytidinemonophosphate (UMP-CMP)
gil27806279 caldesmon 1 [Bos taurus]
gi|29135295 aspartate aminotransferase 1 [Bos taurus]
gi|155371881 alanyt-tRNAsynthetase [Bos taurus]
gill 19908206 PREDICTED:hypotheticalprotein [Bos taurus]
gi|115496906 acidic([eucine-rich)nuclear phosphoprotein 32 family,member E [Bos taurus]
gill 19901449 PREDICTED:similarto PDNP1 [Bos taurus]
transplantation antigen P35B [Bos taurus]
gil114051291
6 4 tissue specific
CNDPdipeptidase 2 [Bos taurus]
gi|780425
gil116004251 granulin [Bostaurus]
gil76614948 PREDICTED similarto pepbdyl-Pro cis trans isomerase isoform 1 [Bos taurus]
gi]77736257 protein phosphatase 1, catalyticsubunit,beta isoform [Bostaurus]
gi|l 19909677PREDICTED:similarto MGC84382protein [Bos taurus]
gi|164448698 chaperonin containingTCP1, subunit 3 [Bos taurus]
gi|165905571 leucine aminopeptidase 3 [Bos taurus]
gi|156120555 glyoxalase domaincontaining 4 [Bos taurus]
gi|123858766 prefoldinsubunit 2 [Bos taurus]
gij99028963
7 9 5' nucleotidase,ecto [Bos taurus]
gil77735 6 complement component 1, r subcomponent [Bos taurus]
gil148233312 dermatan 4 sulfotransferase 1 [Bostaurus]
giJ77736509 S-phase kinase-associated protein 1 [Bos taurus]
gil115495701 pentraxin 3 [Bos taurus]
gi|116812902hemoglobinalpha chain [Bos taurus]
gil156120327thrombospondin 3 [Bos taurus
gi|27806825 peptidylglycinealpha-amidating monooxygenase [Bos taurus]
gi|154152039scrapie responsive protein 1 [Bos taurus]
gil119904972 PREDICTED:similarto Collagenalpha-2(iv) chain [Bostaurus]
gi|77735597 FK506-bindingprotein4 [Bos taurus]
gil 19914266 PREDICTED:similarto cartilage-associated protein(CASP)[Bos taurus]
g0122692537 damage-specific DNAbindingprotein 1, 127kDa [Bos taurus]
14.20839 1.017853 0.309243
37.40458 1.220882 0.639517
39.51613 1.034125 0.284267
39.14894 1.184763 0.734416
26.39594 0.483096 0.281244
30.04622 0.885377 0.267538
18.8383 0.803381 0.427767
14.58924 0.972503 0.354609
23.94822 0.939537 0.537312
24.6988 1.61261 0.33356
16.56977 1.117718 0.442446
13.71841 1.242814 0.444574
0.213483
42.39631 1.063042
38.43826 1.183134 0.141004
14.35644 0.775361 0.032555
22.63374 0.668077 0.351419
54.42478 0.830506 0.365389
24.63235 0.976096 0.373942
20.52877 D.903883 0.95509
28.7037 0.612661 0.22486
33.77483 1.249014 0.265085
13.35404 0.58929 0.278876
1
10.46622 -0.99926
31.93277 0.795183 0.275014
39.27227 0.779581 0.414449
12.98482 0.865918 0.226559
17.32804 0.849466 0.162996
26.66667 1.236331 0.482611
29.86111 0.875234 0.171927
66.66667 0.833021 0.303443
20.08487 0.739806 0.47084
12.31707 1.26736 0.31132
45.9375 1.06482 0.652055
20.58824 0.874987 0.217415
18.24324 1.310154 0.236449
47.36842 1.26D646
37.62376 1.433824 0.428222
18.82716 1.216908 0.41015
25.70281 1.269151 0.308154
19.59799 0.534016 0.478089
18.75 1.454892 0.430942
15.47945 0.606999 0.088434
21.34146 1.035913 0.613646
24.27184 0.913055 0.251079
23.46369 0.946534 0.167793
26.08696 0.802539 0.131196
35.06494 1.116745 0.06763
1803922 0.465451 0.239334
18.67704 1.579072 0.659546
25.40984 1.490223 0.17479
51.00671 1.57135 1.391916
13.06818 1.087494 0.33666
26.31579 1.139562 0.222354
24.85981 1.420384 0.346329
26.87651 1.147065 0.296698
22.52066 0.958442 0.235416
16.47727 1.231063 0.120557
11.78707 0.672156 0.366952
13.84462 1.300379 0.384962
21.80665 1.303009 0.469639
16.84211 0.74381 0.091638
14.65077 0.808832 0.513145
37.19512 0.497807 0.459888
30.88685 1.007481 0.422626
30.8 0.795847 0.531392
21.65138 0.826817 0.391769
36.41618 1.197426 0.605365
22.81879 1.175848 0.129091
27.92208 1.080303 0.153165
18.4669 0.939144 0.602384
13.19797 0.371729 0.255753
30.05319 0.643613 0.144348
0.669089 0.127617
22.69939
21.98953 1.513781 0.30821
39.43662 1.943351 0.46772
12.76151 0.748922 0.148359
13.06584 0.685843 0.618738
50 0.985846 0.423782
17.6681 0.923236 0.251127
1
19.17211 -0.99926
18.70324 0.983074
19.38597 0988155 0.074826
0.922926019 0.828111 0.340358
0.318717364 1.176168 0.550588
0.750738059 2.413955 0.230209
0.1484375 2.958899 0.768915
0.004993909 0.597662 0.235224
0.218332299 0.79729 0.238971
0.330534308 1.414743 0.412788
0.817151886 1.165717 0.360374
0.801387909 1.476593 0.107719
0.069751071 2.734684 0.214804
0.69642184 0.417986 0.0939
0.7417
0.251830874 3.8849
0.352668292 3.533629 0.263291
0.247244968 3.471307 0.304387
0.00262712 2.19931 0.270689
0.004703672 0.813613 0.270199
0.33330418 1.270872 0.533668
0.7421875 0.939595 0.336983
0.817537461 1.770837 0.953737
0.015625 0.920192 0.111661
0.093465616 3.086608 0.230374
0.041876022 1.020271 0.15032
-1 2.150729 0.20013
0.096156675 1.667681 0.361371
0.0078125 0.917289 0.262812
0.165656654 2.926706 0.8834
0.291038812 1.955129 0.245251
D.292766628 2.943605 0.583895
0.112621037 2.778236 0.556254
0.182038529 3.169982 0.864344
0.416795077 1.398908 0.748488
0.026835321 0.6248 0.249036
0.76892942 1.86465 0.820084
0.266463338 1.600423 0.362142
0.257355307 1.678449 0.069102
0.125911276 3047138 0.350288
0093076211 2.753903 0.491145
0.116358817 0.710875 0.427083
0.192091906 2.101795 0.621285
0.032408073 0.415467 0.598041
0.161253804 2.919583 0.152417
0.00050569 1.335219 0.315531
0.896815108 2.426789 1.124426
0.074444217 0.785242 0.412548
0.356197163 3.697732 0.617168
0.016999756 1.643629 0.367663
0.184486441 2.278645 0.134782
0.015355155 0.770373 0.053673
0.1390788 4.16785 0.93634
0.029291852 5.005955 0.732354
0.417271146 3.610196 0.825578
0.462935933 1.002925 0.259727
0.18674671 2.471173 0.39826
0.126510497 2.308451 0.428326
0.516777071 2.780204 0.085909
0.701296239 3.613826 0.280184
0.049539842 2.732936 0.643642
0.271090308 2.641361 0.649759
0.060269605 2.584342 0.728107
0.201449286 2.485554 1.304786
0.096115311 1.5355320.276254
0.320042318 1.213373 0.46849
0.022140592 1.146593 0.566521
0.965747982 3.146577 0.696114
0.396532407 1.931222 0.562766
0.350435816 1.537338 0.629118
0.533044454 2.139903 0.580549
0.087720645 3.282099 0.12729
0.331178713 2.247358 0.716627
0.820956401 1.255821 0.354889
0.010543925 0.445584 0.224777
0.101515473 0.693874 0.487356
0.098484838 1.605843
0.831632
0.078004648 1.861369 0.236121
0.010124121 1.317848 0.288852
0.01120273 0.935365 0.315655
0.267882545 1.056921 0.400266
0.943757758 0.439114 0.347235
0.636275225 1.109448 0.339989
-1 1.461249 0.842797
0.527972721 2.405346 0.348523
0.811770675 2.488301 0.320618
0.388534
0.053878
0.184089
0.3855219
0.001667
0.0078125
0.0074339
0.0625
0.1815515
0.280848
0.0129203
0.007428
0.0330213
0.0042834
2.46E-07
0.0768193
0.003729
0.0207383
0.4016913
0.3852008
0.2817003
0.046875
00007995
0.9308931
0.0826136
0.0285578
0.1626
0.0406038
0.0216417
0.0133519
0.0136238
0.0319157
0.3954241
0.0003068
0.0747428
0.0878642
0.0030006
0.643004 0.8993780.263761812
0.4375
0.864672 0403676
2.38417 0.368222 0.006189962
1.918399 1011161 0.031271826
0.660605 0.314855 0.034987006
0.776221 0.380156 0.112181895
1.70436 0.454989 0.095161529
1.207589 0.3572710.141190722
1.626208 0.983398 0.33146398
3.269898 0.363047 0.014371437
0.153246 1.0067D6 0.164939633
0.00074897
3.798151 0468633
3.520818 0.237968 1.07E-07
2.609466 0275736 0.087842949
2657109 0427772 0.006822752
0.721987 0409756 0.041342839
4.620782 0450048 0.013324411
0.954205 0.272693 0.616667519
1.737182 0.991753 0.289734467
0.762631 0.273554 0.011575853
2.771789 0.180657 0.00048544
0.98617 0.253463 0.999005344
2.183436 0.366483 0.141469321
1.855457 0.333426 0.012005374
0.754799 0.336487 0.014238535
2.265914 0.661015 0.035943935
1.547054 0.383386 0.099083505
2.743991 0.591988 0.015489679
2.111045 0.59152 0.033963277
3.391513 1.0386290.041581134
1.169949 0.2780150.264062386
0.983398 0.2254930.962779865
0.6758480.028937035
1.983742
0.378310561
1.15332 0.34888
2.018276 0.348796 0.036133204
2.973906 0.56251 0.003186068
3.182135 0.573603 0.009751096
0.713548 0.247116 0.002462396
2.040854 0.920073 0.186858663
0.435597 0.465745 0.014886286
2.524393 0.47828 0.039043115
0.558246 0.492702 0.04466754
2.486681 1.12189 0.175536569
0.651858 0.573383 0.00012207
0.00330936
3.852789 0701618
2.280779 0.323806 0.004963112
1.701141 0378442 0.208708142
0.813512 0.133918 0.069635347
3.146452 0.975216 0.041443138
4.290955 0.754442 0.039660058
1.308161 0.734717 0.34790038
0.827057 0.227758 0.087467767
0.008248
2.346831 0.40005
2.519663 0.386029 0.026058352
2.384269 0.018312 0006471147
3.364019 0202327 0.004324917
2.462334 0822684 0.108715161
2.137621 0.529138 0.067409869
2.987183 0951432 0.009201833
2.090313 1.226407 0.175255314
1.152352 0.232898 0.236409376
0.588216 0459439 0.047694216
4.95E-07
3.541769 0.290595
2.366375 0.76722 0.104143124
1.717631 0.502826 0.128879012
1.614271 0.320710 0.061593675
1.724988 0.6974980.183003339
3.123256 0.180034 0.00388226
2.620447 0622145 0.059184844
1.839502 0.426399 0.06740268
0.307881 0407087 0023818009
0.805619 0287647 0.387807689
1.694182 0.9747480.299648314
1.546302 0.1621990.020184636
1.21806 0.282345 0.073192769
0.869454 0.310559 0.326842981
0.710438 0.250022 0.08188384
0.146196 0.83028 0.017017848
0.838427 0.54257 0.676592696
1.978104 1.585442 0.53689562
2.328281 0.27950 0.003021512
2543983 0.145043 0.047660548
0.0005344
0.0098709
0.0191566
0.0993864
0.0230515
0.0033118
0.0866305
0.1921033
0.005232
0.0024956
0.0322574
0.0518435
0.006043
0.0223702
0.0320889
0.0099289
0.8955853
0.0075525
0.0408018
0.0265684
0.0076978
0.0613781
0.066527
0.0061718
0.1417659
0.0642906
0.3502469
0.5234901
0.0555497
0.1036383
0,2403691
0.0847148
0.0018764
0.109088
0.2714627
0.0117492
0.3575317
0.3014508
0.0233769
0.0430657
0.5127564
0.8211274
0.0264629
0.6856706
0.5391877
0.0056359
01103968
gi|115497736 olfactomedin-like3 [Bos taurus]
gi|119888815 PREDICTED:similarto GALEprotein isoform2 [Bos taurus]
9184370199 6-phosphogluconolactonase [Bostaurus]
gi82697343 cystatin B (stefin B)[Bos taurus]
gi|29135293 farnesyl diphosphatesynthase [Bos taurus]
gi|77736061 NSFLI (p97)cofactor (p47)[Bos taurus]
gi[115495441hexosaminidaseA[Bostaurus
gi[115497820parvalbumin[Bos taurus]
gil119915943 PREDICTED:similarto serine (or cysteine) proteinaseinhibitor,clade B (ovalbumin),member 1 isoform 1 [Bostaurus]
gil149642605 drebrin-like[Bostaurus]
gil119925682 PREDICTEDsimilarto adlican [Bos taurus]
gill 54152127 olfactomedin-tike21 [Bos taurus]
gi83035095 high-mobilitygroup box 2 [Bos taurus]
gi|27806555 legumain [Bostaurus]
gi|122692375 hypotheticalprotein LOC783871[Bostaurus]
gi|27805927 heat shock lOkDaprotein 1 [Bos taurus]
gi|119901068 PREDICTED:similarto Collagen, type IX,alpha 1. partial [Bos taurus]
gi|27805981 ribosomalprotein SA [Bostaurus]
gi77735725 cathepsin H [Bos taurus]
gi|84000203 esophageal cancer related gene 4 protein [Bos taurus]
gil1 19895711PREDICTEDsimilarto cytokine-likeproteinZCYTD7isoform 1 [Bos taurus]
gil119914936 PREDICTEDsimilarto histone Hlx [Bostaurus]
gil27806677 connective tissue growthfactor [Bos taurus]
gil119901084 PREDICTED:similarto CD109 [Bos taurus]
gi|119914223 PREDICTED:similarto KIAA2019protein [Bos taurus]
gi|115497124 asparagine synthetase [Bos taurus]
gi|77735669 lysyl-tRNAsynthetase [Bos taurus]
gil119907290 PREDICTED:hypotheticalprotein [Bos taurus]
gil119896208 PREDICTED:similarto PRDX3 protein [Bos taurus]
gil119919315 PREDICTED:similarto latent transforming growthfactor beta binding protein3 [Bos taurus]
gi[78045531 aspartyl-tRNAsynthetase [Bos taurus]
gi|114051185programmed cell death 5 [Bos taurus]
mannose-binding,1 [Bos taurus]
gij149773586
gi]77736560 stathmin l/oncoprotein 18 [Bos taurus]
gi[77736489 high-mobilitygroup nucleosome binding domain1 [Bos taurus]
gi|84000165 complementfactor I[8os taurus]
gil119919951 PREDICTED:similarto glucose-6-phosphate dehydrogenase G6PD [Bostaurus]
gil27805805 fatty acid bindingprotein 5 [Bostaurus]
gi[77735927 3-hydroxybutyratedehydrogenase, type 2 [Bos taurus]
gi1149642873hyaluronan and proteoglycanlinkprotein 3 [Bos taurus]
gil115432055tyrosine 3-monooxygenaseltryptophan5-monooxygenase activation protein, beta polypeptide [Bos taurus]
gil119906919 PREDICTED:similarto ribosomalprotein L6-tike[Bostaurus]
gi|155371891ADAMmetallopeptidasewiththrombospondin type I motif,1 [Bos taurus]
gi|76642911 PREDICTED:similarto IL-104nduciblechemokine [Bos taurus]
associated 8 [Bos taurus]
gill 15496496 matrix-remodelling
gil27806001 matrixmetalloproteinase14 [Bos taurus]
gil114052322 alpha-N-acetytgalactosaminidase[Bos taurus]
gil157279907parathymosin [Bostaurus]
gi]41386699 heat shock 70kDa protein2 [Bos taurus]
gil115496734 proteasome (prosome, macropain) 260 subunit,non-ATPaose,5 [Bostaurus]
gi]62461611 ribosomalprotein012 [Bos taurus)
gil119904908 PREDICTED:similarto CDEP[Bos taurus]
gil27806703 CD44antigen [Bos taurus]
synthetase [Bos taurus]
gill 14051578 histidyl-tRNA
4)-monophosphatase 1 [Bos taurus]
gil27805945
07 inositol(myo)-l(or
synthetase [Bos taurus]
gi|147902 9 glpcyl-tRNA
gi|62460568 eukaryotictranslation elongationfactor 1 beta 2 [Bostaurus]
gi|169658384 non-metastaticcells 1, protein (NM23A)expressed in [Bostaurus]
gi|155372073 cullin-associatedand neddylation-dissociated 1 [Bos taurus]
gi|28461173 elastin [Bostaurus]
gi|157074012 EF-hand domainfamily,member D2 [Bos taurus)
gi|62751650 poly(rC)binding protein1 [Bos taurus]
gij47564074 hypotheticalprotein LOC407769[Bos taurus]
gill25991938 cadherin 11,type 2 [Bos taurus]
gil119909757 PREDICTED:similarto E-selectinligand-1 [Bos taurus]
gil119909718 PREDICTED:similarto KIAA0015[Bos taurus]
gil134085807 capping protein (actinfilament) muscle Z-line,alpha 1 [Bos taurus]
gil114051892 UDP-N-acteylglucosaminepyrophosphorylase 1 [Bos taurus]
gi|70778754 adenine phosphoribosyltransferase[Bos taurus]
gill 19890624 PREDICTED:similarto insulin responsive sequence DNA bindingprotein-1 [Bostaurus]
gil62460548 sushi-repeat-containingprotein. X-linked2 [Bos taurus]
gil27806265 leukocytecell derived chemotaxin 1 precursor [Bostaurus]
gil27806093 endothelialproteinC receptor [Bos taurus]
gil 14051423ubiquitincarboxyl-terminalesterase L1 [Bos taurus]
gi|87196507 unactiveprogesterone receptor, 23 kD[Bos taurus]
gi[76641864 PREDICTED:hypothetical proteinisoform 1 [Bostaurus
gi[27807173 S100 calcium bindingprotein A10 [Bos taurus]
gill 14053237GDP-mannose pyrophosphorylaseB [Bos taurus]
factor-like 3 [Bos taurus]
gi)64370167 ADP-ribosylation
gi176691395 PREDICTED:similarto mammaryserum amyloidA3.2 [Bos taurus]
A4 hydrolase [Bos taurus]
gi[77735515
gi[76615127 PREDICTEDsimilarto Dihydrolipoamidedehydrogenase isoform2 [Bos taurus]
lectin,
leukotriene
16.25616 0.557809 0.109217
20.64394 0.986212 0.602951
32.55814 1.08729 0.010834
51.02041 1.190769 0.389515
16.71388 0.97314 0.518484
29.18919 1.296683 0.801161
15.12287 0.999343 0.237049
40.90909 0.99826 0.482785
21.17647 1.1133760.319955
23.64066 1.187723 0.140487
13.23736 1.086712 1.000353
11.46245 0.814567 0.347086
0.5384
29.1866 0.905687
24.94226 1.28063 0.311188
35.46512 0.421234 0.665214
42.15686 1.352487 0.680836
37.22628 0.589893 0.184696
28.81356 0.874403 0.276509
14.32836 0.753775 0.331318
17.68707 0.905053 0.397999
21.88841 0.636762 0.502956
43.45794 0.922147 0.373228
12.03438 0.504088 0.520528
26.02041 0.80244 0.306074
28.78556 1.307125 0.774543
16.22103 0.730472 0.983032
19.10112 1.413725 0.478522
16.90722 1.546651 0.400086
38.7234 1.626509 0.450798
15.59829 0.624843 0.001418
18.16367 1.29131 0.109172
48 1.10207 0.263788
12.35521 1.530055 0.083158
57.64698 1.413739 0.619969
33.66337 0.749563 0.504437
12.13592 1.154973
15.13513 1.008522 0.522136
17.03764 1.228877 0.078394
17.55102 1.032032 0.198887
33.33333 0.975716 0.37877
45.93496 1.067162 0.281426
27.52613
0.758 0.14045
15.6701 1.07832 0.340438
40 0.774014 0.398277
21.81426 0.609769 0.30D607
11.85567 0.908796 0.955072
16.54501 0.831156 0.063374
33.33333 1.188807 0.59957
25.1981 0.904301 0.552239
21.47117 1.075324 0.911732
54.54546 0.819442 0.214485
1
19.40727 -0.99926
13.6612 1.645602 0.128848
22.59332 1.122038 0.220312
23.10469 1.079881 0.611607
23.81597 1.606374 0.0542D6
30.22222 1.222485 0.401154
55.92105 1.213016 0.389997
12.27642 0.910426 0.466138
13.25301 0.46035 0.302444
28.92562 1.479102 0.407377
15.44944 0.951504 0.060815
31.03448 1.760139 0.026391
9.798995 1.029332 0.216288
11.81435 0.968227 0.130439
18.33031 1.054522 0.1086
27.27273 1.106632 1.132295
9.386973 1.639564 0.121794
22.22222 1.071031 0.77651
15.90361 0.751705 0.061726
10.75269 0.778538 0.412148
18.50746 0.873375 0.345613
22.82158 0.902405
22.22222 1.641466 0.274248
32.5 -0.99926
1
20.33898 0.804241 0.024704
37.1134 0.836268 0.428373
9.722222 1.080336 0.475313
1
19.78022 -0.99926
76.33588 0.288428 0.456861
17.18494 1.394948 0.087069
12.37721 1.717126 1.088033
0.05829103 1.515813 0.038220.0296611 0.729204 0.199797
0.967447781 3.903813 0.381253 0.0125738 2.981569 0.208893
0.039985804 1.630192 0.384234 0.2405187 1.707107 0.290343
0.172527176 2.16493 0.309373 8.46E-05 1.8108 0.315601
0.0625 1.993744 0.479779
0.877111348 2.074039 0.496513
0.417270753 2.111991 1.432306 0.236042 3.13167 0.59002
0.999520984 1.681304 0.472008 0.2732886 1.00883 0.481833
0.0625 2.21725 0.735683
0.995001014 2.075344 0.571627
0.401371937 2.752236 0.26127 0.0015985 2.825008 0.253982
0.241424229 3.737837 0.158998 00379909 3.369437 0.205367
0.89210617 0.928769 0.237273 0.5873691 0.876498 0.048336
0.441956589 0.920045 0.215988 0.5261371 0.596049 0.438993
0692930167 1.715068 0.347504 0.0634901 2.901278 0.334401
0.350910137 1.305949 0.188029 0.2225851 0.989315 0.947051
0.229761274 1.151609 0.049037 0.119084 1.221621 0.251098
0.289690003 3.832442 0.82003 0.0184602 3.778411 0.686969
0.003749994 0.787361 0.425489 0.1844166 0.624327 0.487785
3.470368 0.498863
0.351077261 3.869427 0.407906 0.0145405
0.091258679 2.119895 0.421949 0.0150507 1.40763 0.189489
0.312978304 2.037898 0.923839 0.0113757 1.524122 1.032493
0.154553615 0.932442 0.397235 0.64954111 1.128668 0.230758
0.644629952 1.853161 0.283439 0.0336409 2.989851 0.319115
0.0321576 0.747283 0.5165610. 1864789 0.13033 0.991603
0.03393584 0.742776 0.361113 0.0165521 0.805539 0.294339
0.608019143 3.38677 1.199377 0.2887992 2.90209 0.92197
0.632936793 1.49994 0.490593 0.3508227 1.346408 0.543093
0.378409805 3.158358 0.731462 0.1946331 2.786114 0.882606
0.268612245 0.261942 0.578954 0.1315333 0.111001 1.49149
0.27106167 4.012125 0.423475 0.0148501 4.140285 0756945
0.000942204 1.436095 0.219236 0.1907879 0.819444 0.760568
0.130976267 1.720934 0.282647 0.1631461 1.881404 0.139305
0.587298948 1.787634 0.644411 0.3236882 1.535001 0.633431
0.60717466 1.704893 0.262832 0.0386577 1.71455 0.266353
0.367858 0.313355 1.667756 0.552267
0.296846802 1.234679
0.198595925 2.461898 0.432901 0.0096732 3.105833 0.402257
0.393053895 0.893997 0.199503 0.4195872
0.977048396 2.595589 0.545915 0.1766078 2.907788 0.443163
0.11692543 4.054718 0.075811 0.0170667 2.344424 0.482357
0.67154207 2.979959 0.207751 0.0006472 1.967751 0.358372
0.649016468 0.921851 0.139075 0.084606 0.888852 0.190702
0.617276161 2.581333 0.370805 0.0243184 2.088619 0.081218
0. 155436407 2.262101 0.56771 0.2125714 1.437656 0.813034
0.510480475 1.607625 0.208313 0.002043 1.636837 0.455804
0.161353081 0.983356 0.298251 0.7840996 0.94777 0.377036
0.184549596 0.671404 0.651627 0.4193901 0.472828 0.554075
0.87267598 1.541102 0.826353 0.4897151 1.205964 1.212126
0.018435872 1.521106 0.031548 0.0009691 0.887839 0.160644
1.976041 0.82029
0.311175001 2.415955 0.851656
0.515898683 2.14605 0.332899 1.03E-05 2.719939 0.384057
0.896403774 2.130784 1.273523 0.4451057 1.9598641.61162
0.075765251 2.19407 0.797832 0.6683928 2.034711 0.447271
-1 2.688501 0.80084 0.0932213 1.690321 0.385162
0.384738395 0.489597 0.367648 0.010623 0.591614 0.323209
0.477515858 2.002624 0.115951 0.0530599 2.129274 0.645424
0.838749351 2.202377 0.247301 0.0159043 2.870485 0.965361
0.000135907 4.327654 0.211279 0.0002809 3.225096 0.367893
0.079414648 2.816158 0.49834 1.79E-07 2.871123 0.488319
0.340100794 3.35112 0.806727 0.5658469 3.685255 0.630437
0.753663294 2.446792 0.225866 0.0796139 2.721135 0.165432
0.512771 0.180011
0.005116389 0.50723 0.122546
0.0691202 2.330451 0.438356 0.0119723 2.591688 0.797542
0.181876504 2.491774 0.406024 0.031161 2.44942 0.659596
0.014542702 6.579779 0.508267 0.0843201 4.937296 0.418019
0.826355089 1.269545 0.29725 0.3664875 1.191328 0.456152
0.708197466 1.711253 0.62285 0.3363318 1.469913 0.734501
0.111768
0.496408999 1.355215 0.146074 0.1541969 1.377506
-1 2.25468 0.090837
0.884212225 -1.01428
1
0.076378038 2.416769 0.233459 0.0834175 2.558425 0.299703
0.844289155 3.287405 0.451077 0.0227638 3.177302 0.330875
0.748 0.248511
0.007446011 0.877509 0.164043 0.1581004
0.222237
0.433162454 1.022105 0.381825 0.9738584
0.21868
0.0625
0.271256636 1.728857 0.13948
0.020129491 1.03466 0.29098 0.9117492 0.93193 0.241818
1.468517 0.759323
0.69276435 1.565913 0.352563
-1 2.612931 0.782133 0.0942004 1.88813 0.26381
0.035467011 2.201792 0.360675 0.1427529 1.627975 0.02169
0.408045367 2.869626 0.661961 0.002605 4.900832 0.220089
0.721457043 2.67214 0.42058 0.028887 2.71291 0.503566
-1 3.079884 0.177022 0.0496288 2.920123 0.200703
5.79E-07 0.321994 0.462015 1.25E-06 0.321651 0.470007
0.010734163 3.895395 0.414711 0.0148731 3.50586 0.562383
0.495190248 2.184036 0.52265 0.2052057 2.318915 0.371718
0.209789
0.056579
0.199785382
0.005659271
0.161500826
0.000324255
0.0625
0.01096698
0.938567978
0.023727802
0.001
25798
0.052031586
0.126394813
0.257159665
0.015030236
0.993323057
0.331938612
0.01101608
0.073576372
0.024258935
0.012317972
0.10535932
0.28099731
0.012995765
0.0097754
0.039489852
0.252382552
0.450155858
0.251223404
0.205612544
0.041796827
0.70648233
0.067028078
0.389420646
0.03517643
0.140513744
0.003755483
0.677325398
0.126183586
0.169642131
0.011459212
0.154086587
0.00186993
0.517568894
0.023020513
0.781783441
0.225327496
0.791974892
0.244335628
0.017803395
3.09E-06
0.544310075
0.018504045
0.214474083
0.019768668
0.248204185
0.023359158
0.002636469
8.67E-08
0.03472345
0.050742599
0.941672
0.169139
0.0080776
0.0005009
0.761549
0.777775
0.0379748
0.000303379
0.039588963
0.074831083
0.080537143
0.55299561
0.467101739
0.103388318
0.034236561
0.097314623
0.012565068
0.108900501
0.254028191
0.024898749
0.717119088
0.227343469
0.125071838
0.013495405
1.65E-06
0.037364144
0.05752439
1.77E-06
0.030065774
0.133631271
gil157073982von Willebrandfactor A domaincontaining1 [Bos taurus]
gil77735861 vacuolar proteinsorting29 [Bostaurus]
gi77735805 leucinezipper transcriptionfactor-like 1 [Bos taurus]
gil27806849 melanomainhibitoryactivity[Bos taurus]
gil27806297 biliverdinreductase B (flavinreductase (NADPH))[Bostaurus]
gil145279649tumor necrosisfactor alpha[Bostaurus]
hypotheticalprotein[Bostaurus]
gi|119888578 PREDICTED:
gi|115497946 LanClantibioticsynthetase componentC-like 1 [Bos taurus]
giJ84000233 hypotheticalproteinLOC517857[Bos taurus]
gi|77735537 ephrin-A1[Bostaurus]
gi|119884774 PREDICTEDsimilarto profilin1 [Bostaurus]
1 [Bostaurus]
gi|27806161
3 84 tissue inhibitorof metalloproteinase
protein1 [Bostaurus]
PREDICTEDsimilarto Oxysterol-binding
gi|119921
4
hydrolasedomaincontaining2 [Bostaurus]
giJ780 2510 haloaciddehalogenase-like
gi]77735687 proteasome(prosome, macropain) subunit,betatype 6 [Bostaurus]
gi|41386683 beta-2-microglobulin[Bos taurus]
gi|164414726 thymosin beta 4 [Bos taurus]
gi|27806365 UDP-glucosedehydrogenase [Bos taurus]
gi|77735683 S100 calcium bindingproteinA2 [Bostaurus]
gi|75812956 GTP bindingproteinRabla [Bostaurus]
gi|77735697 threonyl-tRNAsynthetase [Bostaurus]
gi|114053301 receptoraccessoryprotein5 [Bostaurus]
gil126722829 BCL2-associatedathanogene 3 [Bos taurus]
gi|148235020 cold shock domainproteinA [Bos taurus]
gil114053087 glutathioneS-transferase M3[Bos taurus]
gi|149642641 D-dopachrometautomerase [Bostaurus]
gi]77404290 ribosomalproteinS3 [Bos taurus]
gil123959772 leucine rich repeat neuronal 1 [Bos taurus]
gil77735465 D component of complement (adipsin) [Bostaurus]
protein isoform1 [Bos taurus]
giJ76608584 PREDICTED:similarto KIAA0653
gi[27806273 chemokine(C-C motif)ligand 20 [Bos taurus]
gil119927320PREDICTED:similarto lamininB2,partial [Bostaurus]
gij78042550 glutaredoxin3 [Bos taurus]
gil119879173PREDICTED:similarto putativeMAPKactivating proteinisoform 2 [Bos taurus]
similarto ChainA, CrystalStructureOf HumanRangap1-Ubc9 isoform2 [Bostaurus]
gil76652407 PREDICTED:
U [Bos taurus]
gil125991756
6 3 3 heterogeneous nuclear ribonucleoprotein
synthetase [Bostaurus]
gil2780 5 tryptophanyl-tRNA
gi[76635398 PREDICTED:similarto DDX6proteinisoform 1 [Bos taurus]
gil29135323 sialyltransferase 1 [Bos taurus]
similarto chondroitin6-sulfotransferase [Bostaurus]
gi[76631783 PREDICTED:
gill 14052973glypican 1 [Bos taurus]
gil122692293chloride intracellularchannel4 [Bos taurus]
gi77735939 proliferatingcell nuclear antigen [Bos taurus]
gi|119910239 PREDICTED:similarto gamma-adaptin[Bos taurus)
gi|47564098 cell divisioncycle 10 [Bostaurus]
gi|84000371 H2Ahistone family,member V [Bos taurus]
gi|134085671 histone cluster 1, Hic [Bos taurus]
gi78042544 galactosidase, beta 1 [Bos taurus]
[Bostaurus]
gi[84000197
7 FK506bindingprotein 3,25kDa
gil2780584 crystallin,lambda 1 [Bostaurus]
gil115497422 glucosamire (N-acetyl)-6-sufatase [Bos taurus]
gi|77736031 chaperonincontaining TCP1,subunit 6A [Bostaurus]
box polypeptide 36[Bos taurus}
gil122692521 DEAH(Asp-Glu-Ala-His)
similarto OAFhomolog(Drosophila)[Bostaurus]
gi|76635454 PREDICTED:
gi|77736207 growthfactor receptor-bound protein 2 [Bos taurus]
gi|77735815 rrbosomalprotein L30[Bos taurus]
giJ62751458 endoplasmicreticulumthioredoxinsuperfamilymember. 18 kDa [Bos taurus]
gi45430019 ribosomalprotein L12[Bos taurus]
3 [Bos taurus)
gil27806083 peroxiredoxin
gi62751982 proteasome (prosome, macropain) subunit, alphatype.5 [Bostaurus]
gi|155371989 methionineadenosyltransferase 11,alpha[Bostaurus]
gi|1 14052601hypotheticalprotein LOC530784[Bos taurus]
gi|149642987 S100 calcium binding proteinAl1 (calgizzarin)[Bos taurus]
gil76669880 PREDICTED:similarto LRTS841 [Bos taurus]
gi|77736139 proteasome (prosome, macropain) subunit, beta type,3 [Bos taurus]
gi|119901198PREDICTED:similarto Man9-mannosidase[Bostaurus]
gil122692459ABI gene family,member 3 (NESH)binding protein[Bos taurus]
gil114051602KH domaincontaining,RNA binding,signal transduction associated 1 [Bostaurus]
12.83293 0.931581 0.253872
27.41936 1.130883 0.10817
14.71572 1.322494 0.505015
23.07692 0.896063 0.274897
25.24272 1.19426 0.338181
8.974359 10.26339 1.608471
33.33333 1.255328 0.529528
8.270676 1.213854 0.158229
23.751.143714 0.520116
0.810966 0.52612
14.14634
26.42857 0.88314 0.23018
28.98551 0.970092 0.035717
14.10891 1.432519 1.117848
16.98842 0.785426 0.23742
22.17573 0.902672 0.145471
19.49152 1.609752 0.326146
72.72727 0.937717 0.310371
0.944 0.546123
16.39676
27.835050.876233 0.48295
34.10405 0.943646 0.094966
14.79945 0.859343 0.104989
19.57672 1.081227 0.165518
14.52992 0.840595 0.256045
22.07447 1.009469 0.221745
49.77778 1.017097 0.070453
32.20339 1.07376 0.895094
0.4677
27.98354 0.588744
10.19553 0.864345 0.2882
9.65251 0.563551 0.595564
5.688623 1.004959 0.25053
18.75 0.175335 0.04058
1390729 1.077214 0.122751
20.65868 0.99249 0.344049
13.49353 0.846562 0.390174
34.17721 1.264753 0.169236
18.30303 1.130716 0.155434
15.57895 1.018586 0.414788
17.59834 0.821274 0.587027
18.76543 0.784499 0.47048
17.53653 1.314842 0.086855
20.75134 1.186429 0.907828
20.1581 1.138384 0.095079
25.28736 0.847545 0.3874
11.0303 1.533588 1.213802
12.91866 0.901104 0.578761
45.3125 0.767522 0.154416
65.25822 0.817959 0.208153
14.24196 0.725925 0.431444
25.44643 0.867531 0.398158
12.14953 1.169827 0.457059
18.21429 0.760507 0.335275
19.77401 0.772838 0.47251
16.83168 0.732151 0.151561
25.17731 1.175187 0.27629
24.42396 1.372247 0.069782
38.26087 1.181253 0.764009
22.67442 1.089121 0.718946
38.78788 0.945167 0.038057
22.56809 0.768652 0.942605
1
31.95021 -0.99926
15.6962 1.392466 0.503745
10.79365 1.074493 0.419049
9.708738 0.927106 0.459521
20.49608 0.790905 0.611498
20.4878 1.336283 0.347604
19.42337 0.704295 0.233639
11.89802 0.943457 0.538466
21.21896 0.582443 0.70544
0.561370934 1.071063 0.199089
0.10369116 2.25123 0.848307
1.432508
0.335599871 0.738496 0.309989
0.225598036 1.94962 0.625445
0.009506114 1.043652 0.411732
0.302118766 4.705827 0.879107
0.091615507 2.901684 0.109101
0.583065838 2.722416 1.070718
0.613421 0.5701
0.0390625 2.49032 0.336657
1.308818 0.050452
0.629770703 2.411433 0.707693
0.126766471 2.651395 0.412343
0.222907663 2.427291 0.229025
0.024347998 1.118331 0.36153
0.596139681 6.453733 0.278919
0.86668958 2.401798 0.403245
0.566780294 2.049911 0.558286
0.434522862 3.356788 0.413392
0.209293288 1.913765 0.256355
0.509091202 4.331859 0.133176
0.233579823 2.130448 0.660564
0.919238372 3.504716 0.471729
0.69849654 3.077448 0.239657
0.896568411 1.611407 0.189353
0.260310714 2.273715 0.805692
0.335496087 1.181397 0.324037
0.138162472 0.585334 0.091417
0939136611 0.334446 0.30689
0.007275412 0.45052 0.189858
0.429894005 1.023182 0.00042
1.734352 0.33765
2.685494 0.369555
0.215488391 3.871949 0.129965
0.35165662 2.985644 0.513389
0.9823
0.940178567 1.71781
0.619061102 1.253473 0.658952
0.48468537 1.041339 0.128786
0.015662464 1.009076 0.024297
1
0.765556137 -1.01428
0.218574049 2.682452 0.191887
0.242201857 1.473169 0.450713
0.60272586 1.583943 0.255512
0.777492668 2.627683 0.394435
0.050718956 1.377428 0.139293
0.138142118 1.410168 0.20276
0.372150115 1.863763 0.202559
0.46858875 2.704596 0.166508
0.609700183 2.80394 0.195191
0.34492051 1.45D658 0.603864
0.466993694 2.348802 0.322914
0.149252811 1.229275 0.289036
0.442918937 2.368498 1.027467
0.075
0.068380505 3.667679
0.732484837 3.522881 0.52881
1
0.847410241 -1.01428
0.205892189 3.691938 0.193913
0.558666155 1.50584 0.286954
-1 1.824061 1.281265
0.407245916 2.110537 0.796037
0.783715444 1.884966 0.307009
0.723099805 2.052029 0.715259
0.578211681 0.682088 0.77761
0.337542728 3.710544 0.482928
0.201401163 1.149164 0.097311
0.863524662 0.422662 0.257946
0.36285151 1.349311 0.519302
3.320728
0.460558359
0.335263055
0.339822332
0.974364258
0.545225704
0.5647226 1.443218 1.0218470.449906825
0.1433237 1.947455 0.7378770.147658919
0.17445 2.910279 1.505746 0.214312616
0.0598521 0.776968 0.220637
0.0570018 1.785884 0.940802 0.075396343
0.8339538 10.8504 1.518189 0.007001955
0.0150849 3.128766 1.0002520.044667797
0.0017209 2.61229 0.1785690.005336706
0.1477175 2.464121 0997691 0.148761828
0.0842976 0.703241 0456227 0.122131253
9.02E-06
1.22E-05 3.027809 0401923
0.0615547 0.379411 0511780 0.163511214
0.0923627 1.983863 0.0763760.184405477
0.0282469 2.304034 0434077 0.039430197
0.010853 2.109202 0.1350490.005036076
0.4925552 1.63145 0484437 0.057707358
0.0003113 7.332573 0.235170.000146953
0.1434794 2.50919 0.089830.029986705
0.0877555 2.606138 0.4373 0.030923861
0.1567652 3.584035 0.4171960.100036468
1.9765 003727 0.016719328
0.124386
0.028603 3.904594 068587 0.015547378
0.1069098 2.142663 0.897016 0.108541195
0.0224042 3.865134 0.400497 0.019243706
0.5671233 2.483742 0627781 0.204676729
0.1259579 3.455764 0031901 0.007936662
0.2243478 2.492973 0.258773 0.086514746
0.3605628 1.418432 0.292037 0.089630587
0.0043993 0.568377 0.537349 0.124535298
0.03125
5.23E-05 0.5641 0.214774
0.09457953
0.0726815 0.310432 0352916
0.0149939 1.082923 0.216119 0.538829433
0.1904942 1.983858 0086821 0.038702029
0.1170882 3.068018 0.063427 0.017431026
0.0302497 3.81154 0.351494 0.080679787
0.1457719 3.458487 0.495D49 0.121591531
0.436910788
0.4713422 1.674737 0.88536
0.6305649 1.01386 0.358876 0.944212072
0.7484005 1.24845 0.16535 0.210607741
0.6498587 1.105178 0.097416 0.099640677
-1 1.082899 0.063705 0.204752902
0.0613723 2.119185 0.583501 0.227727609
0.0625 1.205623 0.31191 0.106078454
0.1743667 1.446848 0.20476 0.162961893
0.1275546 2.591611 0.731529 0.226181408
0.0316113 1.895719 0.379887 0.050063854
0.0556207 3.931079 0.189636 0.002966252
0.1029734 1.569368 0.584936 0.351888895
0.0045842 2.703866 0.315668 0.015323242
0.0597197 3.040205 0.392492 0.107723999
0.4399357 0.834813 0.550225 0.647913246
0.1185965 2.643298 0.322171 0.101084937
0.4042864 1.127218 0.1983 0.399893253
0.3411324 2.815247 0.667527 0.192449106
0.0784065
0.0182012 3.574233 0.325252
0.1306551 3.805666 0.110959 0.025621412
-1 2.15683 0.14969 0.059500004
0.25454044
0.0467477 2.827037 0908164
0.0750611 1.171559 0.3561150.349964129
0.5215266 1.576041 1.554514 0.64873546
0.311478 2.028654 0.5566810.230181937
0.1516395 1.661418 051494 0.089996198
0.1328952 2.407481 0.101143
0.3303916 0.577906 0.4494980.097124639
0.1149155 3.478751 0.8899 0.152654038
0.2322929 1.044494 0.056572 0.035564114
0.629888 0480153 0.307839705
0.0913148 1411991
1.011494 0600944594
0.4636148
0.052742037
0.002049768