Table 1: Comparison of different proteomics-based techniques
METHOD
DESCRIPTION
ADVANTAGES
DISADVANTAGES
SENSITIVITY
2D GEL ELECTROPHORESIS/MASS SPECTROMETRY
 Separation of complex
proteins via 2D gel
electrophoresis based
charge and size
 Major
protein
identification by MS
 Detects about 20002500 spots/gel
 Ability
to
identify
unknown proteins
 Detects
protein
modification
(phosphorylation and
methylation)
 Used
for
various
biological
samples,
including
tissue,
blood
and
other
biological fluids
 Proteins expressed at
low abundance may be
missed
 Unsuited for diagnostic
application
 Limited
reproducibility
and high rate of false
identification
 Limited
dynamic
range
 semi-quantitative
 Detection sensitivity
is in the nanogram
range (50 ng/spot
for Coomassie Blue;
1 ng/spot for silver
stain)
 Using
fluorescent
2D-differential
gel
electrophoresis (2DDIGE),
sensitivity
improves by 10 fold
(CyDye label)
LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY
 LC
to
separate
proteins in a sample,
with sequential LC for
improved separation
efficiency
 MS to systematically
identify the major
proteins
 Detects over 1000
proteins/run
 Individual
protein
immobilization on a
solid-support (glass or
membrane)
 Individual
proteins
identified by labeled
antibodies
 Detects over 1000
proteins/array
 Ability
to
identify
unknown proteins
 improved separation
efficiency compared
to 2D gel
 Used
for
various
biological
samples,
including
tissue,
blood
and
other
biological fluids
 Proteins expressed at
low abundance may be
missed
 Unsuited for diagnostic
application
 Limited
reproducibility
and high rate of false
identification
 Limited
dynamic
range
 semi-quantitative
PROTEIN ARRAY
 High sensitivity and  Limited
protein
specificity
availability from complex
 Good
quantitation
protein
production
range
process (expression and
 High
purification)
throughput/density
 Limited access to a
amenable
for
large number of affinity
automation
antibodies for detection.
 Economical and low
sample consumption
 Lots of data from
single experiment
 Software
and
hardware tools may
be shared with DNA
microarray
REVERSE PHASE PROTEIN ARRAY
 Detection sensitivity
is in the nanogram
range or ~20 cells
 1% false positive
rate
 Detection sensitivity
is in the ng/ml range
 Multiple whole-cell or
tissue
lysate
immobilization
on
individual spots on a
solid support (similar
to tissue microarray
format)
 Presence of specific
proteins are detected
by antibody
 Detects
<
100
proteins/array
 Highly
sensitive
detection of proteins
 High throughput, i.e.
a large number of
samples on one slide
 Minimal
sample
required
 Reduced number of
antibodies needed to
detect protein
 Capture
antibodies
are spotted and fixed
on a solid surface
 Proteins
(antigens)
are captured on the
array surface and
detected by a second
antibody specific for a
different epitopes than
capture
antibody
(sandwich format)
 Detects
<
100
proteins/array
 Highly specific from
dual
antibody
detection
 Highly sensitive
 High throughput and
amenable
for
automation
 Possible to detect
protein modifications
(phosphorylation,
methylation, etc) by
modification-specific
antibodies
 Suitable for clinical
applications
 Detection sensitivity may
be compromised from
loss
native
protein
conformation
when
surface spotted
 Limited sensitivity to
detect low abundance
proteins
 Specificity
may
be
compromised from nonspecific antibody binding
(i.e. potential for high
background)
 Limited
number
of
available
signaling
protein-specific
antibodies
 Detection sensitivity
is in the picogram
range
 Increased sensitivity
 Using laser capture
microdissection,
lysates
can
be
analyzed with as
few as 10 cells
ANTIBODY ARRAY
 Protein complexity and
denaturation may affect
antigen-antibody
interaction
 Need for high-affinity
and specific antibodies
for
capture
and
detection
 Limited
dynamic
ranges of 2 or 3 orders
of magnitude
 Detection sensitivity
is in the low pg/ml
range
PATHWAY ARRAY
 Complex proteins in a
sample
(cells
or
tissue) are separated
via gel electrophoresis
 Proteins
then
transfers
to
nitrocellular
membrane
 Proteins detected by
multichannel
immunoblot (similar to
Western Blot)
 Detects up to 300
proteins/run
 Highly sensitive with
detection
of
low
abundance proteins
 Highly specific (as
determined
by
immunoreactivity and
size)
 High accuracy and
reproducibility
 Minimal
antibody
required for each
sample
 Detects
protein
modifications
(phosphorylation,
methylation, etc)
 Limited availability of
signaling-related
antibodies
 Relative low throughput (one sample per gel
 Limited
dynamic
ranges of 2 or 3 orders
of magnitude
2
 Detection limit of 1
ng for each protein
with
chemiluminescence;
0.1
ng
with
fluorescence
 Linear
detection
range is 100 fold for
ECL and 1,000 for
fluorescence.
 Either
capture
antibody or proteins
are coated on beads
 Detection of proteins
by labeled antibodies
(similar to antibody
array or ELISA)
 Detects
50-100
proteins/run




BEAD-BASED ARRAY
Highly sensitive and  Protein complexity and
specific
denaturation
affecting
High throughput and
antigen-antibody
amenable
for
interaction
automation
 Need for high-affinity
Detecst
protein
and specific antibodies
modifications
for
capture
and
(phosphorylation,
detection
methylation, etc) by  Limited dynamic ranges
modification specific
of 2 or 3 orders of
antibodies
magnitude
Suitable for clinical
applications
3
 Detection limit is
sufficient to capture
low
abundance
protein
analytes
down to the pg/mL
range
Supplemental Table 1: Microarray technologies used in genomic and epigenetic analysis:
MICROARRAY
CHARACTERISTIC FEATURE
GENE EXPRESSION
ARRAY [1]
 Simultaneous monitoring of expression levels for >45,000
transcripts to study the effects of certain treatments, diseases, and
developmental stages on gene expression using high-density arrays
 Does not detect splicing variants as probes are designed to
interrogate the 3' end of the transcripts
 Requires as little as 2 ug of starting mRNA for reverse transcription
and labeling
ALTERNATIVE SPLICING
ARRAY [2]
 Used to assess the expression of alternative splice forms of
thousands of genes
 Exon arrays have a different design that employs probes designed to
detect each individual exon for known/predicted genes
 Used to detect different splicing isoforms
 Requires as little as 100 ng of starting mRNA
MICRORNA ARRAY [3]
 A high-throughput technique to assess cancer-specific expression
levels for hundreds of miRNAs in a large sample numbers
 500 human miRNAs have been recorded in mirbase
(http://microrna.sanger.ac.uk/sequences/)
 miRNAs are involved in gene expression regulation.
SNP ARRAY [4]
 Used to identify single nucleotide polymorphisms among alleles
within or between populations
 Evaluates germline mutations in individuals or somatic mutations in
cancers, assessing loss of heterozygosity, or genetic linkage
analysis
 Can measure more than 900,000 SNPs in the whole genome
COMPARATIVE GENOMIC
HYBRIDIZATION
ARRAY[5]
 Used to detect loss, gain and amplification of copy number at the
chromosomal level
 Can detect small gains and losses, e.g. Inter-marker distance of
~100-700 base pairs
 Combined with SNP array, can be used for genome wide association
studies
CHIP-ON-CHIP ARRAY [6]
 Combines chromatin immunoprecipitation (chip) with microarray
technology ("chip").
 High throughput (genome-wide) identification and analysis of DNA
fragments bound by specific proteins such as histones,
transcriptional factors.
 Used to investigate interaction between protein and DNA
 Identifies binding sites of DNA-binding proteins in a genome-wide
basis.
DNA METHYLATION
ARRAY [7]
 DNA methylation is an abnormal heritable epigenetic modification
process occurring in cancer cells whereby cpg dinucleotides are
methylated at the C5 position of cytosine
 The methylation of the 5’ regulatory regions of genes results in gene
silencing
 Methylated DNA are captured with 5-methlycytidine antibody or
methyl binding domain proteins and hybridized to a DNA array which
contains 385k to 2.1M probes
4
Supplemental Table 2: List of antibodies included in the immunoblot array (partial list)
_____________________________________________________________________________
Cell signaling: ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-AKT (Ser473), HGF, HGFR, pHGFR
(Y1234/Y1235), IGF, IGFR, TGF, TGFR, Notch 4, Notch 1, p38, p-p38 (Thr180/Tyr182), JNK, p-JNK
(Thr183/Tyr185), FGFR, p-FGFR (Tyr653/654), VEGFR, p-VEGFR (Tyr951), PKC, p-PKCalpha
(Ser657), p-PKCα/β(Thr638/641), PTEN, p-PTEN (Ser380), PI3K, Ras, Raf, EGFR, p-EGFR (Tyr1068),
p-EGFR (Tyr1148), p-EGFR (Tyr1173), Her2, p-Her2 (Tyr1221/1222), PDK1, p-PDK1 (Ser241), mTor,
p-mTor (Ser2448), HSP90, NF-kB, IKB, c-Kit, c-Kit (Tyr719), PDGFR, GSK3, beta-catenin, p-be,tacatenin (Ser33/37/Thr41), stat3, p-stat3 (Ser727), stat5, p-stat5 (Tyr694), smad, p-smad (Ser463/465),
CREB, p-CREB (Ser133), Frizzled receptor, APC
Cell Growth/Cell Proliferation: Rb, P21, P27, P15, P16, P18, P19, CHK1, CHK2, DP-1, MDM2,
BRCA1, BRCA2, GADD45, 14-3-3β
Cell cycle: CDK2, CDK4, CDK6, CDC2p34, CDC25A, CDC25B, CDC25C, Cyclin B, Cyclin D, Cyclin E,
Rb
Invasion/metastasis: VEGF, NF-kappaB, IKK, E-cadherin, N-cadherin, HSP90, TGF-beta, osteopontin,
KISS1, KAI1, uPA, uPAR, MMP9, ICAM-1, FAK, EphB2, EphB3
Transcription factor: p-c-Jun, ETS1, c-MYC, E2F-1, GATA, Stat1, p-Stat3, p-Stat5, p-Smad1, p-RB,
PR, ERa, ERb
Apoptosis/Autophagy: Bax, FAS, BAD, BCL2, BID, BAK, cleaved Caspase 3, cleaved Caspase 8,
cleaved Caspase 9, TRAF, p53, XIAP, NFKB, IKB, Bcl-xL, Smac, LC-3I, LC-3II, Cytochrome C, TNF,
AKT1, Survivin, RIP
Angionesis: VEGF, VEGFR, E-cadherin, PDGF, PDGFR, TGF-beta, TGF-beta Receptor, TNF alpha,
COX-2, FGF, FGFR, EPO, Ang, Endoglin, Neuropilin, MMP9
DNA repair: P53, ATM, Phospho-ATM (Ser1981), ATR, PCNA, BRCA1, Rad52, TDP1, ERCC1, RCA1,
BTG2, CCNH, DNMT1, GADD45A, PTTG1, XRCC5
Epithelial-to-mesenchymal transition/Adhesion: E-cadherin, catenin, Ep-CAM, HCAM, ICAM1,
VCAM1
_____________________________________________________________________________
5
Supplemental Table 3: Effect of Cdk6 and XIAP silencing on cell viability, cell cycle distribution and
necrosis
Cell
Viability
(%)
(G0/G1
phase)
(S Phase)
(G2+M
Phase)
Negative Control
100
66.37
15.9
17.76
2.14
CDK6 siRNA
8.26
83.81
4.6
10.89
14.46
XIAP siRNA
14.02
71.56
6.96
11.24
31.38
Cell Cycle Distribution (%)
6
Necrosis
(%)
Supplemental Table 4: The expression of signaling transduction proteins in HCCs from 4 patients
SIGNALING
PROTEINS
PATIENT SAMPLES
A
B
C
D
Akt
0
2651
1724
1438
BRCA1
0
1778
0
0
652
0
0
0
cPKCα
13879
462
269
2058
ERK1/2
0
2308
186
0
HIF-3α
0
885
0
0
9104
702
8470
9571
0
0
2036
10094
cdk6
p27
XIAP
7