Mass Spectrometry and Proteomics Application of Proteomics Research for
New Drug Targets Study
Yan-Hui Liu
Structural Chemistry/Mass Spectrometry
Schering-Plough Research Institute
Protein ID by MS in Combination with Various
Protein Fractionation/Separation Methods
• 2DE-MS
– ability to observe global changes in the total cellular protein
complement and post-translational modifications associated with
differential gene expression or compound treatments.
– specific classes of proteins may be absent or under-represented:
very acidic or basic proteins, extremely small or large proteins,
membrane proteins, and low abundance proteins.
• Multi-Dimensional Liquid Chromatography (MDLC)-MS:
– SCX/RP-HPLC of enzymatically digested total cell lysate
– signature peptide approach: Isotope-Coded Affinity Labeling
– protein pre-fractionation prior to 1, 2-DE or MDLC
• Immunoprecipitation or Affinity Pre-enrichment
Combined with 1, 2-DE, or MDLC-MS
– identify biomarkers and binding partners of target protein(s)
Functional Study of Drug Targets Using
Proteomics and Mass Spectrometric Tools
• Analysis of Protein Expression -- Expression
Proteomics
– strategies
– identification of the function of antibacterial target Unk4
(S. aureus) through 2-D gel electrophoresis followed by
mass spectrometric protein identification
• Analysis of Protein Function -- Functional
Proteomics
– functional study of novel drug target by identification
and characterization of its natural ligand
• Analysis of Protein Structure/Protein Complexes - Chemical Cross-linking/Mass Spectrometry
Integration and Automation of Protein ID of 2D Gel
Electrophoresis Separated Microbial Proteomes by MS
2D gel spots (96-well plates)
MassPREP Station (Micromass)
in-gel digestion/peptide extraction
MALDI plate generation
extracted peptides (96-well plates)
MALDI TOF MS (PMF approach)
Capillary LC/QTOF2 (sequence tag)
or
acquire spectra
calibrate spectra
high-throughput
ID using PS 1
generate .pks files
Web-based db
search (Mascot)
MALDI PSD
(sequence tag)
acquire spectra (DDA)
MassLynx
generate .pkl files
Web-based db search
(Mascot)
Protein ID
Protein ID
Protein ID
N
Significant ID?
N
Significant ID?
Y
Y
Generate report
(spot ID, MS ID, protein ID)
Peptide Mass Fingerprinting
• MALDI-MS
•Comparing
experimental PMF with
theoretical PMF from
protein databank
• Facilitates rapid protein
identification from simple
mixtures (e.g. 2D PAGE)
by MALDI MS
Courtesy of Micromass, Ltd. (UK)
MS Identification of Proteins Separated by 1D Gel
Electrophoresis/Chromatography or Proteins of Incompletely
Sequenced Genome
Protein Separation Pattern
MassPREP Station (Micromass)
protein digestion/peptide extraction
MALDI plate generation
extracted peptides (96-well plates)
MALDI TOF MS (PMF approach)
acquire spectra
Capillary LC/QTOF2 (sequence tag)
or
calibrate spectra
high-throughput
ID using PS 1
generate .pks files
Web-based db
search (Mascot)
acquire spectra (DDA)
MassLynx
MALDI PSD
(sequence tag)
generate .pkl files
Web-based db search
(Mascot)
Protein ID
Protein ID
Protein ID
N
Significant ID?
N
Significant ID?
Y
Y
Generate report
(spot ID, MS ID, protein ID)
MS/MS and dB searching
• Using fragmentation
model to compare
experimental MS/MS
spectra with theoretical
MS/MS spectra generated
from the in silico digestion
of known proteins.
• Facilitates rapid protein
identification by matching
experimentally
characterised peptides to
databank proteins.
Courtesy of Micromass, Ltd. (UK)
2-D Spot Identification by PMF
Zoomed view after
automated spot picking
SYPRO Ruby stained E.coli gel
MW (kDa)
1
97.0
66.0
45.0
2
30.0
90
80
20.1
7988.7
100
Spot #1: unknown protein
from gene y
14.4
% Intensity
70
60
50
pI 4.0
40
30
20
10
0
499.0
1999.4
3499.8
5000.2
Mass (m/z)
6500.6
0
8001.0
linear IPG
pI 7.0
Protein identification of RP-HPLC peak 2: non-significant
identification of rAd pX by peptide mass fingerprint
3037.78
30000
CHCA
819.4595 #
3053.95
3069.08
5000
2547.2346
2273.1509
Trypsin
1993.9854
10000
1671.8293
15000
923.4686
1020.5098
1088.6475 #
1123.5795
Counts
20000
443.2141 #
459.2046 #
563.2941
628.2894
25000
0
500
1000
1500
2000
2500
Mass (m/z)
MS-Fit Search
Rank
MOWSE
Score
Protein
MW (Da)/pI
Species
SwissProt
Accession #
Protein Name
1
2
3
3#
77.6
57.2
48.9
48.7
37832.2/8.23
48778.4/8.39
59428.2/7.07
8845.7/12.88
CHVP1
HAEIN
ORENI
ADE02
Q84424
P44856
P70091
P14269
4
45.4
23123.2/9.06
BRUCA
Q45110
MRNA Capping Enzyme
NADH Dehydrogenase
Cytochrome P450 19A133
Late L2 MU Core Protein Precurso
(11 KD Core Protein) (Protein X)
25KD Outer-Membrane
Immunogenic Protein Precursor
1000
100
V [72.01]
200
300
400
Mass (m/z)
500
600
700
y1
y7-NH3 [801.54]
y6 y5 y4 y3 y2
777.73
728.43
y7
y6-NH3 [655.78]
y6 [672.83]
5000
y4-NH3 [459.78]
819.4555 : PSD
618.29
y5-NH3 [558.45]
y5 [575.31]
3000
y4 [476.94]
PGF [302.61]
y2-NH3 [305.62]
y2 [322.53]
b3 [344.42]
y3-NH3 [362.23]
y1 [174.97]
GF [205.53]
a2 [217.48]
b2 [245.25]
y1-NH3 [158.13]
R [111.97]
R [119.83]
2000
P/R [70.00]
Counts
Obtain sequence tag of tryptic peptide (m/z 819.5) by MALDI/PSD
MH+
F P V P G F R
b2 b3
4000
0
800
MS-Tag search against SwissProt identify the rAd pX
Rank
#
Sequence
Unmatched
Ions
MH+
Caclc.
(Da)
MH+
Error
(Da)
Protein
MW (Da)
/pI
Species SwissProt
Accession
#
Protein
Name
1
16/30
(R)FPVPGFR(G)
819.4517
0.0038
8845.7/12.88
ADE02
Late L2 MU
Core Protein
Precursor
(11 KD Core
Protein)
( Protein X)
2
20/30
(K)VPFFPGR(G)
819.4517
0.0038
82989.3/5.06
BACST P14412
P14269
Peroxidase/
Catalase
Novel Adenovirus Protease Cleavage Site of pX (?)
-------------------------------propeptide----------------------------------------------Late L2 MU Core Protein-MALTCRLRFP VPGFRGRMHR RRGMAGH GLT GG MRRAHHRR RRASHRRMRG
ox
Ad protease (?)
-----------------------------propeptide-----------------------G ILPLLIPLI AAAIGAVPGI
ASVALQAQRH
* Substrate specificity of
Ad protease: (M,I,L)XGG X or (M,I,L)XGX G
Immunoprcipitation/SDS-PAGE for Membrane Protein
Identification (Ras and A-Factor Converting Enzyme)





Express yeast RCE (tag with S-protein) and  Rce/ Afc (negative control); make lysate
Immunoprecipitate with S-protein agarose; separate by SDS-PAGE
Cut band of ~35kDa; digest with trypsin
Analyze with MALDI MS; search database using peptide mass fingerprint
Analyze with Q-TOF/capillary LC; search database using both peptide mass fingerprint and
peptide sequence tags
Rank MOWSE
Score
Protein
MW (Da)/pI
Species
SwissProt.3.30.00
Accession #
Protein ID
1*
2.03e+004
35746.8/6.46
YEAST
P00359
2
777
35847.0/6.46
YEAST
P00358
Glyceraldehyde 3-Phosphate
Dehydrogenase 3 (GAPDH3)
Glyceraldehyde 3-Phosphate
Dehydrogenase 2 (GAPDH2)
* No RCE protein was identified by database search of the MALDI peptide mass map
Peptide signal of under represented components
of a protein mixture can be suppressed
in a MALDI MS spectrum using PMF approach
1753.7878
100
1.3E+4
GAPDH 3
1352.8
100
RCE or
Keratin
90
90
80
?
70
% Intensity
80
70
1420.7323
60
50
RCE
40
1185.6723
% Intensity
30
20
60
10
0
1162.0
50
0
1220.4
1278.8
1337.2
1395.6
1454.0
Mass (m/z)
40
GAPDH 3
30
RCE
667.3840
20
RCE or
Keratin
833.4428
RCE or
Keratin
?
GAPDH 3
1420.7323
RCE
2207.1000
GAPDH 3
1185.6723
?
2303.1408
GAPDH 3
2591.3381
1470.8068
GAPDH 3
3264.7351
3569.8634
10
0
600
GAPDH 3
0
1280
1960
2640
Mass (m/z)
3320
4000
Q-TOF/Capillary LC: Data dependent MS to MS-MS switching
for automatic protein characterization
rcenewlc01
100
MS Survey Spectrum
TIC
1: TOF MS Survey ES+
40.97
39.21
1 µl injection of RCE in-gel digest
3.53e4
Charge State (z) and Mass (m)
Determination
43.26
30.29
N
32.84
Z>1
%
27.94
Y
52.49
49.02
Exclude List Match
24.86
Y
36.02
23.03
N
0.51
57.66
19.95
13.48
Set Collision Energy ~ m
0
5.00
MS-MS Spectrum
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
Time
Three precursor ions were selected for MS/MS from
the survey scan of the peak eluted at 38.86-39.21 min
4: TOF MSMS ES+
TIC
3.06e3
1l injection of RCE new
rcenewlc01
t = 39.04
CE = 24.0
4: TOF MSMS 816.41ES+
38
1l injection of RCE new
rcenewlc01 122 (39.040) Cm (122:123)
901.51
100
m/z 816.41
451.75
664.36 729.39
229.12
451.22
599.35
229.11
246.18
86.09
129.10
rcenewlc01
t = 39.14
m/z 722.33
mass
spectra
t = 39.09
2: TOF MSMS ES+
TIC
4.93e4
%
505.29
234.14
305.19
234.14
183.11
86.09
1083.50 1211.56
1308.62
1191.55
1254.59
1512.67
2: TOF MSMS 1082.49ES+
190
512.20
505.30
597.29
0
rcenewlc01 884 (38.869)
100
RCE or Keratin?
%
0
1080.52
625.29
415.19
305.18
530.23
343.17
453.34
Time
38.85 39.90 38.95 39.00 39.05 39.10 39.15 39.20 39.25 39.30 39.35
1023.49
605.28
1210.58
0
rcenewlc01 163 (39.092)
392.22
100
%
1: TOF MS Survey ES+
TIC
3.44e4
652.36
739.32
428.22
MS/MS of 722.33
1209.56
1209.53
856.43 1080.52
1081.54
954.50
756.36
453.36
429.08
1546.83
3: TOF MSMS 722.33ES+
125
855.41
72.06
m/z 1082.49
rcenewlc01
1119.62 1220.64
973.51
1328.74 1418.79
1102.08 1220.55
972.55
818.41
392.22
229.11
102.05
rcenewlc01
773.90
0
rcenewlc01 122 (39.142) Cm (120:121)
100
3: TOF MSMS ES+
TIC
2.22e4
MS/MS of 816.41
902.50
901.46
773.40
%
739.32
909.44
838.38
1063.51
1308.62
1209.56
MS/MS of 1082.49
1309.67
1521.75
1081.54 1210.57 1309.70
1407.71 1523.80
1081.59 1211.57
1311.66 1409.70 1524.78 1634.87
1130.54
1: TOF MS Survey ES+
23
1083.01
183.49
722.33
816.41
1083.53
710.32
710.28
532.98 626.26
729.97
837.71 1065.49
852.66
1025.91
MS Survey Scan
1083.99
1136.57
1177.24
1243.43
1492.07
1447.53
1595.32
m/z
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
Q-TOF LC-MS-MS of precursor ion at m/z 710.85 (2+)
Observed MW of the peptide: 1419.6812 Da (RCE or Keratin?)
(Eluted at 38.734 min)
2: TOF MSMS 710.85ES+
DT
R
L
YG
Q
T
P
100
L
T
G
650.32
y6
V
G
L
V
T
T
749.39
y7
492.26
y4
86.09
141.11
395.26
282.19
559.28 593.31
312.16 y3
y5
183.15
b5
458.22
367.27
b4
L
TD
924.97
963.52
y9
981.46
862.47
y8
169.10
Q
924.43
867.86
%
bMax
yMax
PGYR
1091.57
y10
1073.56
686.65
771.42
b7
1029.97
1420..34
1185.66 1263.77
1402.72
0
1421.66
m/z
100
200
300
400
500
600
700
800
900
1000
y12
1100
y1
D T L Q T L V G T P G Y R312
300
b1
b12
(RCE)
1200
1300
1400
Database search using both peptide mass fingerprint and
peptide sequence tags identified the presence of RCE protein
MOWSE
Score
Likelihood
2.43e+031
1.23e+052
5.54e+019
2.33e+008
3.77e+002
5.40e+001
Protein ID
Glyceraldehyde 3Phosphate
Dehydrogenase 3
(GAPDH 3)
6.58e+047 Glyceraldehyde 3Phosphate
Dehydrogenase 2
(GAPDH 2)
1.27e+018 Glyceraldehyde 3Phosphate
Dehydrogenase 1
(GAPDH 1)
4.14e+005 Hypothetical 11.5 KD
Protein in HTB2-NTH2
Intergenic Region
2.72e+005 CAAX Prenyl Protease
2 (RACE)
Protein
Species
MW (Da)/pI
No. of Matched Sequence Coverage
Peptides
(%)
35615/6.9
YEAST
36
70.39
35715/6.9
YEAST
27
46.22
35618/8.6
YEAST
13
29.31
11518/6.6
YEAST
5
9.62
35911/9.1
YEAST
2
7.62
The Matched Sequence Covered the Loop Region of RCE
MLQFSTFLVL LYISISYVLPLYATSQPEGSKRDNPRTIKS
RMQKLTIMLISNLFLVPFLQSQLSSTTSHISFKDAFLGLG
IIPGYYAALPNPWQFSQFVKDLTKCVAMLLTLYCGPVLDF
VLYHLLNPKSSILEDFYHEFLNIWSFRNFIFAPITEEIFY
TSMLLTTYLNLIPHSQLSYQQLFWQPSLFFGLAHAHHAYE
QLQEGSMTTVSILLTTCFQILYTTLFGGLTKFVFVRTGGN
LWCCIILHALCNIMGFPGPSRLNLHFTVVDKKAGRISKLV
S I W N K C Y F A L L V L G L I S L K D T L Q T L V G T P G Y R I T L (315)
* The underlined sequence were confirmed by MS/MS
MS/MS
MS/MS
Cytosol
6
63
75
128
207
260
279
24
45
93
106
229
242
297
ER Lumen
Serine/Threonine
Hydrophobic
Histidine
Glutamic Acid
Glycine
Cysteine
Proline
Glutamine
Arginine
Alanine
Asparagine
Identification of the Function of Unk4 for Antibacterial
Drug Discovery via Expression Proteomics
•
Proteins of unknown function
– 14% bacterial broad spectrum
targets
– 82% gram positive only
– 45% gram negative only
– 30% fungal targets
•
Access function by expression
proteomics
– identify global protein expression
patterns associated with conditional
gene expression or chemical
perturbations
– identify potential function/pathway
for unknown targets
– develop novel biochemical assays
based on identification of protein
function derived from pathway
analysis
Experimental Conditions
Overnight culture(+IPTG,+Cm)
Subculture (+IPTG,+Cm) and grow to OD540 0.8
Wash out IPTG
Inoculate 3 flasks to OD540 0.1(+IPTG,+Cm)
Inoculate 3 flasks to OD540 0.1(-IPTG,+Cm)
Grow 2hrs,dilute each 2-fold
(+IPTG,+Cm to +IPTG,+Cm)
(-IPTG,+Cm to -IPTG,+Cm)
Follow growth for 5 hours
Growth Curves
unk4 OD 540 (Jan25/02) Before Dilution
0.6
0.5
0.4
1+
A 540
12+
0.3
23+
3-
0.2
0.1
0
T=0'
T=1'
Time (Hours)
T=2'
Growth Curves
Unk4 OD 540 (Jan 25/02)
2
1.8
1.6
1.4
1+
1.2
A 540
12+
1
23+
0.8
3-
0.6
0.4
0.2
0
T=0
T=1
T=2
T=3
Time (Hours)
T=4
T=5
Proteomic T=0 Results
T=0, 3 (+IPTG)
Cy3 image
T=0, 3 (-IPTG)
Cy5 image
Response Effect of Unk4 Knockout
(proteomic T=3 results)
up-regulated
down-regulated
T=3, 2 (+IPTG)
Cy5 image
T=3,2 (-IPTG)
Cy3 image
Review dB Search Results
Protein ID
assigned sequence of the
peptide based on the MS/MS
data
Unk4 shutoff Protein IDs
gene
ilvD
ilvB
ilvA
ilvC
Identified on
Average Spot
"normal" and Response to Volume Change
Identified Proteins
shutoff gels unk4 shutoff
(8 gels)
dihydroxy-acid dehydratase
no
increases
12.57
acetolactate synthase (large subunit)
no
increases
14.61
1-pyrroline-5-carboxylate dehydrogenase
yes
decreases
3.16
1)formyltetrahydrofolate synthetase
yes
decreases
3.57
2)no hit-hypothetical protein
no
decreases
3.57
chorismate mutase homolog
no
increases
4.38
1)threonine dehydratase/deaminase
no
increases
7.59
2)conserved hypothetical protein
no
increases
7.59
ketol-acid reductoisomerase (2 spots-8 gels)
no
increases
10.52/10.46
nucleoside diphospate kinase
yes
decreases
3.77
1)xx
no
decreases
9.25
2A)copper transporting ATPase
no
decreases
9.25
2B)hypothetical protein
no
decreases
9.25
phosphoribosylformylglycinamidine synthase I
no
decreases
Expression Level Clustering After Unk4 Shutoff
Absolute Differential
Expression (3h)
ilvA*
ilvC*
-23.7
-21.9
-20.6
-18.4
-18.4
-11.0
-15.9
15
10
5
0
Increasing
Expression
ilvB*
ilvD*
leuA
leuB
leuC
K-means clustering
-5
-10
-15
-20
unk4
x
*
+7.7
nc
consistent with expression proteomic results
+
AAB v AAC
-
AAB v AAD
1h
+
AAB v AAE
-
AAB v AAF
2h
+
AAB v AAG
-
AAB v AAH
3h
Valine, Leucine and Isoleucine Biosynthesis
Threonine deaminase
Acetolactate synthase
Ketol-acid reductoisomerase
Ketol-acid reductoisomerase
Dihydroxy-acid dehydratase
Acetolactate synthase
Ketol-acid reductoisomerase
Ketol-acid reductoisomerase
Dihydroxy-acid dehydratase
S. aureus Organization Valine, Leucine and
Isoluecine Biosynthesis Regions
x
y
z
u
v
ilvD
ilvB
ilvN
ilvC
leuA
x = unknown protein
y = unknown protein
z = unknown protein
u = unknown protein
ilvD = dihydroxy-acid dehydratase (20566-22281)
ilvB = acetolactate synthase (large subunit) (22282-24078)
ilvN = acetolactate synthase (small subunit) (24063-24332)
ilvC = ketol-acid reductoisomerase (24394-25473)
leuA = isopropylmalate synthase (25479-27032)
leuB = 3-isopropylmalate dehydrogenase (27029-28081)
leuC = isopropylmalate dehydratase subunit (28095-29465)
leuD = isopropylmalate dehydratase subunit (29566-29937)
ilvA = theonine dehydratase/deaminase (30031-31335)
leuB
leuC
leuD
ilvA
Unk4 Hypothesis
• Because an Unk4 depletion causes an up-regulation of an
amino acid biosynthesis pathway -- it may affect a
transporter/transporter component, either by processing or
allowing secretion. This transporter could allow entry of
amino acid or peptide fragments into S. aureus.
• Testing the hypothesis
– confirm the 2-D DIGE expression proteomics results by
Isotope-Coded Affinity Tags (ICAT)/Multi-Dimensional Liquid
Chromatography (MDLC)/MS approach.
– identify Unk4 interacting partners through MS studies of
protein complexes.
Protocol for Quantification by
ICAT/MDLC/MS Approach
Control
Experimental
Reduce
Reduce
Trypsin digest
Trypsin digest
ICAT (1H)
ICAT (2H)
Mix samples
Affinity select
RPC or CE
MALDI or ESI
Bioinformatics
The ICAT Approach
control
CH-CH2-S-CH2-CO-NH
Linker arm (1H8)
biotin
Trypsin digestion
Note the deuterium label
experimental
CH-CH2-S-CH2-CO-NH
Linker arm (2H8)
biotin
1H8-ICAT
labeled peptide x
2H8-ICAT
m/z
R. Aebersold Anal. Chem. January 2000.
labeled peptide x
Frequency of Low Abundance Amino Acids
and Post-Translational Modification (E. coli)
Amino acid/PTM
Av. number per protein
tyrosine
3.5
cysteine
2.8
histidine
2.1
methionine
1.7
tryptophan
1.1
glycosylation
0-5
phosphorylation
0-5
Functional Study of Novel Target-SP1999 by
Identification and Characterization of Its Natural Ligand

SP1999 is highly expressed in brain, spinal cord, and blood
platelets.

SP1999 was identified as a GPCR by BLAST homology search.

Phylogenetic analysis of SP1999 showed that it shares 43%,
32%, and 28% sequence identity with classes of GPCRs of
KIAA0001, H963, and GPR34. It shares little sequence
homology with known P2Y receptors, another classes of
GPCRs. *This suggests that the natural ligand for SP1999
would be non-nucleotide.
Purification of SP1999 Ligand(s)
Rat spinal cord (100 ng)
RP-HPLC C18 column
Cation exchange column (SP/8HR)
Anion exchange column (MonoQ)
DEAE column
RP-HPLC C18 column
Fractions for MS structural determination
MS Identification of Novel Ligand for SP1999
227.0
[(TFA) 2-H]-
249.0
[(TFA) 2-2H+Na]-
346.1
(AMP-H) -
LC/ESI MS of Purified Ligand of SP1999
5’-ADP
426.1
(ADP-H)-
(C10H13O9N5P2: high resolution MS)
159
79
O
LC/ESI-MS-MS of m/z 426
NH2
N
O
HO P O P O
OH
N
O
N
N
134
OH
OH OH
-HPO3
-Base
(HPO3-H)
-
-H2O
(M-H)(M-H3PO4-H)(M-H2O-H)-
291
-H2O
408
-HPO3
273
328
346
IL-10 Folding Study Using Chemistry Cross-linking/Mass Spectrometry
Bifunctional Cross-Linkers
Linker nam e
Spacer arm
length
Linker structure
DM P
(Dim ethyl pim elim idate2HCl)
-
Cl + H 2N
+
NH 2 Cl
C
M .W . 259.18
CH 2
CH 2
CH 2
CH 2
CH 2
9.2 Å
-
C
H 3 CO
OCH 3
DM S
-
(Dim ethyl suberim idate2HCl)
+
+
Cl H 2N
NH 2 Cl
C
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
-
C
H 3 CO
OCH 3
M .W . 273.2
SCHEME 1.
Cross-Linking Reaction with Imidoesters
mon omer 1
L y s -N H 2
-C
l+ H
2
H
2
N -L y s
N H 2 + C l -
N
C
(C
H 2 )n
C
H 3 C O
O
C H 3
2
-C
l+ H
2
L y s -N H
CH
3
OH
N H 2 + C l -
N
C
mon omer 1
mon omer 1 or 2
(C
H 2 )n
C
H N -L y s
mon omer 1 or 2
11 Å
Peak
a
c
d
e
Table . DMS (11 Å spacer arm) Modified Lysine Residues Based on LC/ESI MS Results
Tryptic Fragment
Modified Lysine Residue MWcalcd. (Da) MWexpt. (Da) Lysine Accessibility or
Nitrogen Interatomic Distance (Å)
XL-T16,17
Lys119
1058.2
1057.6
Exposed
t
u
v
XL-T19,20
XL*-T9,10
T18-XL-T19,20
(on same monomer)
XL-T18,19
XL-T9,10
2XL-T18,19,20
T’6,7-XL-T18,19
*
XL -T6,7
T’5,6-XL-T19,20
*
XL -T5,6
T4-XL-T5,6
(on same monomer)
XL-T6,7
XL-T5,6
T15(-ss-T8)-XL-T16,17
(on same monomer)
XL-T15(-ss-T8),16
T8(-ss-T15)-XL-T9,10
(on same monomer)
*
XL -T7,8(-ss-T15)
XL-T7,8(-ss-T15)
XL*-T21,22,23
w
x
y
XL -T21,22
T’7,8(-ss-T15)-XL-T21,22
XL-T21,22,23
Lys157
Lys58’-XL-Lys158
Lys157
2743.3
8096.4
2871.4
2742.3
8095.5
2871.0
Exposed
13.46
Exposed
z
a’
b’
XL-T21,22
XL-T4,5
XL-T4,5,6
Lys157
Lys34
Lys34 or Lys40
2757.3
1198.5
2251.7
2757.0
1197.9
2250.7
Exposed
Exposed
Both Exposed
c’
2XL-T4,5,6
Lys34, Lys40
2421.9
2420.1
Both Exposed
f
g
h
j
k
l
m
n
o
p
q
r
s
*
Lys134
Lys99
Lys130-XL-Lys134
(on same monomer)
Lys130
Lys99
Lys130, Lys134
Lys50’-XL-Lys131
Lys49
Lys41’-XL-Lys135
Lys40
Lys35-XL-Lys41
(on same monomer)
Lys49
Lys40
Lys118-XL-Lys120
(on same monomer)
Lys117
Lys89-XL-Lys100
(on same monomer)
Lys57
Lys57
Lys157
1148.4
1707.9
1690.9
1147.8
1707.2
1690.2
Exposed
Exposed
13.69
1261.4
1721.9
1893.2
3262.5
2189.5
2970.6
2010.4
2219.7
1261.1
1722.2
1892.1
3262.2
2188.8
2970.0
2009.6
2218.9
Exposed
Exposed
Both Exposed
12.89
Exposed
16.47
Exposed
10.85
2203.5
2024.4
5417.3
2203.7
2024.1
5416.6
Exposed
Exposed
15.42
4794.5
6081.0
4794.0
6080.0
Exposed
14.54
5527.3
5541.3
2857.4
5526.3
5541.5
2856.4
Exposed
Exposed
Exposed
Note: XL-T denotes the cross-linker modified tryptic peptide of IL-10.
Nine inter-lysine distances of hIL-10 inferred (4 intramolecular, 5 intermolecular) from chemical
cross-linking/mass spectrometry studies using DMP and DMS. One of the cross-links, Lys50’XL-Lys120 (10.9 Å), could be placed at the lower end of DMP's spanning range (9.2 Å) given its
inability to be cross-linked by DMS (11 Å).
Summary
• Protein Identification
– Integrated MS techniques (MALDI MS and ESI Q-TOF MS/MS)
with 1-DE, 2-DE, chromatographic separation methods, and
bioinformatics tools for identification of proteins for drug targets
studies and target validation.
• Protein Functional Study
– Identified SP1999 as a novel P2Y (G-linked) receptor for ADP.
This result allowed the functional hypothesis to be generated and
confirmed, which led to the validation of SP1999 as a viable target
for anti-throbotic therapy.
• Protein Structural Study
– The information of inter-lysine distances and solvent accessibility
of lysine residues of hIL-10 derived from Chemistry Crosslinking/Mass Spectrometry study matched very well with those
obtained from crystallography study.
– This method will provide valuable distance-constraint and amino
acid solvent accessibility information for protein structural modeling
using bioinformatics tools. It can also be applied for proteinprotein interaction studies.
Acknowledgement
Leigh Ann Giebelhaus
Todd Black
Fang L. Zhang
Frederick J. Monsma, Jr.
Nicholas J. Murgolo
Wei Ding
Gary Vellekamp
David Wylie
Fred Poeter
Shihong Wang
Guodong Chen
James Pai
Birendra N. Pramanik
John Piwinski