Download - Mass Spectrometry and Proteomics - Application of Proteomics Research for New Drug Targets Study
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Mass Spectrometry and Proteomics - Application of Proteomics Research for
New Drug Targets Study
Yan-Hui Liu
Structural Chemistry/Mass Spectrometry
Schering-Plough Research Institute
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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)
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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
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Integration and Automation of Protein ID of 2D GelElectrophoresis Separated Microbial Proteomes by MS
in-gel digestion/peptide extraction
MALDI plate generation
MALDI TOF MS (PMF approach)
high-throughput ID using PS 1
acquire spectra
calibrate spectra
Protein ID
Significant ID?
YGenerate report
generate .pks files
Web-based dbsearch (Mascot)
extracted peptides (96-well plates)
Capillary LC/QTOF2 (sequence tag)
acquire spectra (DDA)
generate .pkl files
Web-based db search(Mascot)
N
Protein ID
Significant ID?
Y
2D gel spots (96-well plates)
MassPREP Station (Micromass)
MassLynx
or
MALDI PSD(sequence tag)
Protein ID
(spot ID, MS ID, protein ID)
N
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• 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
Peptide Mass Fingerprinting
Courtesy of Micromass, Ltd. (UK)
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MS Identification of Proteins Separated by 1D GelElectrophoresis/Chromatography or Proteins of Incompletely
Sequenced Genome
acquire spectra
calibrate spectra
generate .pks files
Web-based dbsearch (Mascot)
MALDI TOF MS (PMF approach)
Protein Separation Pattern
MassPREP Station (Micromass)
Generate report
acquire spectra (DDA)
protein digestion/peptide extraction
MALDI plate generation
high-throughput ID using PS 1
Protein ID
Significant ID?
Y
extracted peptides (96-well plates)
Capillary LC/QTOF2 (sequence tag)
generate .pkl files
Web-based db search(Mascot)
N
Protein ID
Significant ID?
Y
MassLynx
or
MALDI PSD(sequence tag)
Protein ID
(spot ID, MS ID, protein ID)
N
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• 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.
MS/MS and dB searching
Courtesy of Micromass, Ltd. (UK)
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2-D Spot Identification by PMFSYPRO Ruby stained E.coli gelZoomed view after
automated spot picking
pI 4.0 linear IPG pI 7.0
MW (kDa)
14.4
20.1
30.0
45.0
66.0
97.01
2
499.0 1999.4 3499.8 5000.2 6500.6 8001.0Mass (m/z)
0
7988.7
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Spot #1: unknown protein from gene y
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Protein identification of RP-HPLC peak 2: non-significant identification of rAd pX by peptide mass fingerprint
MS-Fit Search
30000 819.4595 #CHCA
Co
un
ts
Mass (m/z)
0
5000
10000
15000
20000
25000
500 1000 1500 2000 2500
3037.78
3053.953069.08
443.
2141
#
459.
2046
#
563.
2941
628.
2894
923.
4686
1020
.509
810
88.6
475
#
1123
.579
5
1671
.829
3
1993
.985
4
Try
psi
n
2547
.234
6
2273
.150
9
Rank MOWSE Protein Species SwissProt Protein NameScore MW (Da)/pI Accession #
1 77.6 37832.2/8.23 CHVP1 Q84424 MRNA Capping Enzyme2 57.2 48778.4/8.39 HAEIN P44856 NADH Dehydrogenase3 48.9 59428.2/7.07 ORENI P70091 Cytochrome P450 19A1333# 48.7 8845.7/12.88 ADE02 P14269 Late L2 MU Core Protein Precursor
(11 KD Core Protein) (Protein X)4 45.4 23123.2/9.06 BRUCA Q45110 25KD Outer-Membrane
Immunogenic Protein Precursor
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Obtain sequence tag of tryptic peptide (m/z 819.5) by MALDI/PSDObtain sequence tag of tryptic peptide (m/z 819.5) by MALDI/PSD
y4-N
H3 [4
59.7
8]
Mass (m/z)
Co
un
ts
0
1000
2000
3000
4000
5000
100 200 300 400 500 600 700 800
MH+
y4 [4
76.9
4]
y6 [6
72.8
3]
728.
43
777.
73 y7-N
H3 [8
01.5
4]
y5 [5
75.3
1]
y5-N
H3 [5
58.4
5]
618.
29
y6-N
H3 [6
55.7
8]
y3-N
H3 [3
62.2
3]
b3
[3
44.4
2]
y2 [3
22.5
3]
y2-N
H3 [3
05.6
2]
PG
F [3
02
.61
]
b2
[2
45.2
5]
a2
[217.4
8]
GF
[20
5.5
3]
y1 [1
74.9
7]
y1-N
H3 [
158
.13
]
R
[119.8
3]
R [1
11.9
7]
P/R
[7
0.0
0] V [7
2.0
1]
819.4555 : PSD
F P V P G F R
y7 y6 y5 y4 y3 y2 y1
b2 b3
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MS-Tag search against SwissProt identify the rAd pXMS-Tag search against SwissProt identify the rAd pX
* Substrate specificity of Ad protease: (M,I,L)XGG X or (M,I,L)XGX G
-------------------------------propeptide----------------------------------------------Late L2 MU Core Protein--
MALTCRLRFP VPGFRGRMHR RRGMAGH GLT GG MRRAHHRR RRASHRRMRG
-----------------------------propeptide------------------------
G ILPLLIPLI AAAIGAVPGI ASVALQAQRH
NovelNovel Adenovirus Protease Cleavage Site of pX (?)Adenovirus Protease Cleavage Site of pX (?)
ox Ad protease (?)
Rank # Sequence MH+ MH+ Protein Species SwissProt Protein Unmatched Caclc. Error MW (Da) Accession Name Ions (Da) (Da) /pI #
1 16/30 (R)FPVPGFR(G) 819.4517 0.0038 8845.7/12.88 ADE02 P14269 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 Peroxidase/Catalase
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ImmunoprcipitationImmunoprcipitation/SDS-PAGE for Membrane Protein/SDS-PAGE for Membrane ProteinIdentification (Identification (RasRas and A-Factor Converting Enzyme) 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 andpeptide sequence tags
Rank MOWSE Protein Species SwissProt.3.30.00 Protein IDScore MW (Da)/pI Accession #
1* 2.03e+004 35746.8/6.46 YEAST P00359 Glyceraldehyde 3-Phosphate Dehydrogenase 3 (GAPDH3)
2 777 35847.0/6.46 YEAST P00358 Glyceraldehyde 3-Phosphate Dehydrogenase 2 (GAPDH2)
* No RCE protein was identified by database search of the MALDI peptide mass map
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Peptide signal of under represented components of a protein mixture can be suppressed
in a MALDI MS spectrum using PMF approach
600 1280 1960 2640 3320 4000
Mass (m/z)
0
1.3E+4
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1753.7878
2591.33813264.7351
3569.8634
2207.1000
1470.8068
1420.7323833.4428
1185.6723
2303.1408667.3840
GAPDH 3
GAPDH 3GAPDH 3GAPDH 3
GAPDH 3
GAPDH 3
GAPDH 3 RCE or Keratin ?
RCE
RCE orKeratin ?
RCE
1162.0 1220.4 1278.8 1337.2 1395.6 1454.0
Mass (m/z)
0
1352.8
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1420.7323
RCE or Keratin ?
1185.6723
RCE
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5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00Time0
100
%
rcenewlc011: TOF MS Survey ES+
TIC
3.53e4
40.9739.21
30.29
27.94
24.86
23.03
0.5119.95
13.48
32.84
36.02
43.26
52.4949.02
57.66
1 µl injection of RCE in-gel digest
Z > 1
Exclude List Match
Set Collision Energy ~ m
MS-MS Spectrum
Charge State (z) and Mass (m)Determination
MS Survey Spectrum
Y
Y
N
N
Q-TOF/Capillary LC: Data dependent MS to MS-MS switchingfor automatic protein characterization
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Three precursor ions were selected for MS/MS fromthe survey scan of the peak eluted at 38.86-39.21 min
1l injection of RCE newrcenewlc01
rcenewlc01
rcenewlc01
rcenewlc01
4: TOF MSMS ES+TIC
3.06e3
3: TOF MSMS ES+TIC
2.22e4
2: TOF MSMS ES+TIC
4.93e4
1: TOF MS Survey ES+TIC
3.44e4
38.85 39.90 38.95 39.00 39.05 39.10 39.15 39.20 39.25 39.30 39.35Time
t = 39.09
t = 39.14
t = 39.04
m/z 816.41
m/z 722.33
m/z 1082.49
mass spectra
MS Survey Scan
MS/MS of 1082.49
MS/MS of 722.33
MS/MS of 816.41
1l injection of RCE newrcenewlc01 122 (39.040) Cm (122:123)
CE = 24.04: TOF MSMS 816.41ES+
38
rcenewlc01 122 (39.142) Cm (120:121)
rcenewlc01 163 (39.092)
rcenewlc01 884 (38.869)
3: TOF MSMS 722.33ES+125
2: TOF MSMS 1082.49ES+190
1: TOF MS Survey ES+23
100
%
0
100
%
0
100
%
0
100
%
0100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
86.09
129.10
229.11229.12
246.18451.22
451.75
599.35664.36 729.39
773.40
901.51
902.50901.46
818.41
773.90 972.55
973.51
1102.08 1220.55
1119.62 1220.64
1328.74 1418.79 1546.83
72.06
102.05234.14
305.19
392.22
855.41
505.29
428.22605.28
652.36
739.32
756.36 856.43 1080.52
1023.49
954.50
1512.671308.62
1254.59
1211.56
1210.58
1209.56
1191.551083.50
229.111209.53
1081.54
392.22
512.201521.75
1634.871524.78
1523.80
1409.70
1407.71
1311.66
1309.70
1309.67
1308.62
86.09
183.11
234.14
343.17
305.181063.51
1211.57
1210.57
1209.56
1130.54
1081.59
1081.54
1080.52
625.29
530.23
597.29
739.32 838.38909.44415.19
505.30
429.08
453.34
453.36
532.98 626.26
722.33816.41710.32
710.28852.66
729.97837.71
1025.91
1065.49
1595.32
1492.071447.53
1243.431177.24
1136.57
1083.99
1083.53
183.49
1083.01RCE or Keratin?
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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)
300D T L Q T L V G T P G Y R312
(RCE)
2: TOF MSMS 710.85ES+
DT L Q T L V G T PGYR bMaxR YG P T G V L T Q L TD yMax
100
%
0100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
86.09141.11
169.10
183.15
282.19312.16
650.32y6 749.39
y7
1421.66
1420..341402.72
924.43
686.65
367.27
395.26y3
458.22b4
492.26y4
559.28b5
593.31y5 771.42
b7
862.47y8
867.86
1263.771185.66
1091.57y10
1029.97
1073.56
981.46
963.52y9
924.97
y1y12
b1 b12
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Database search using both peptide mass fingerprint and peptide sequence tags identified the presence of RCE protein
MOWSE Likelihood Protein ID Protein Species No. of Matched Sequence Coverage Score MW (Da)/pI Peptides (%)
2.43e+031 1.23e+052 Glyceraldehyde 3- 35615/6.9 YEAST 36 70.39Phosphate Dehydrogenase 3 (GAPDH 3)
5.54e+019 6.58e+047 Glyceraldehyde 3- 35715/6.9 YEAST 27 46.22 Phosphate Dehydrogenase 2
(GAPDH 2)2.33e+008 1.27e+018 Glyceraldehyde 3- 35618/8.6 YEAST 13 29.31
Phosphate Dehydrogenase 1
(GAPDH 1)3.77e+002 4.14e+005 Hypothetical 11.5 KD 11518/6.6 YEAST 5 9.62
Protein in HTB2-NTH2Intergenic Region
5.40e+001 2.72e+005 CAAX Prenyl Protease 35911/9.1 YEAST 2 7.622 (RACE)
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The Matched Sequence Covered the Loop Region of RCE
Serine/Threonine HydrophobicHistidineGlutamic Acid
Proline
GlycineCysteine
Glutamine Asparagine
ArginineAlanine
M L Q F S T F L V L L Y I S I S Y V L P L Y A T S Q P E G S K R D N P R T I K S R M Q K L T I M L I S N L F L V P F L Q S Q L S S T T S H I S F K D A F L G L G I I P G Y Y A A L P N P W Q F S Q F V K D L T K C V A M L L T L Y C G P V L D F V L Y H L L N P K S S I L E D F Y H E F L N I W S F R N F I F A P I T E E I F Y
T S M L L T T Y L N L I P H S Q L S Y Q Q L F W Q P S L F F G L A H A H H A Y E Q L Q E G S M T T V S I L L T T C F Q I L Y T T L F G G L T K F V F V R T G G N L W C C I I L H A L C N I M G F P G P S R L N L H F T V V D K K A G R I S K L V 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
Cytosol
ER Lumen
24
6
45
63 75
93 106
128
229
207 260
242 297
279
MS/MS
MS/MS
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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
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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
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Growth Curves
unk4 OD 540 (Jan25/02) Before Dilution
0
0.1
0.2
0.3
0.4
0.5
0.6
T=0' T=1' T=2'
Time (Hours)
A 5
40
1 +
1 -
2 +
2 -
3 +
3 -
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Growth Curves
Unk4 OD 540 (Jan 25/02)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
T=0 T=1 T=2 T=3 T=4 T=5
Time (Hours)
A 5
40
1 +
1 -
2 +
2 -
3 +
3 -
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Proteomic T=0 Results
T=0, 3 (+IPTG)Cy3 image
T=0, 3 (-IPTG)Cy5 image
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Response Effect of Unk4 Knockout(proteomic T=3 results)
T=3, 2 (+IPTG)Cy5 image
T=3,2 (-IPTG)Cy3 image
down-regulated
up-regulated
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Review dB Search Results
assigned sequence of the peptide based on the MS/MS data
Protein ID
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Unk4 shutoff Protein IDs
Identified on Average Spot "normal" and Response to Volume Change
gene Identified Proteins shutoff gels unk4 shutoff (8 gels)
dihydroxy-acid dehydratase no increases 12.57acetolactate synthase (large subunit) no increases 14.611-pyrroline-5-carboxylate dehydrogenase yes decreases 3.161)formyltetrahydrofolate synthetase yes decreases 3.57
2)no hit-hypothetical protein no decreases 3.57chorismate mutase homolog no increases 4.381)threonine dehydratase/deaminase no increases 7.59
2)conserved hypothetical protein no increases 7.59ketol-acid reductoisomerase (2 spots-8 gels) no increases 10.52/10.46 nucleoside diphospate kinase yes decreases 3.771)xx no decreases 9.252A)copper transporting ATPase no decreases 9.25
2B)hypothetical protein no decreases 9.25phosphoribosylformylglycinamidine synthase I no decreases
ilvDilvB
ilvC
ilvA
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Expression Level Clustering After Unk4 Shutoff
Absolute DifferentialExpression (3h)
K-means clustering
-20
-15
-10
-5
0
5
10
15
AAB v AAC AAB v AAD AAB v AAE AAB v AAF AAB v AAG AAB v AAH
ilvD*
leuB
leuC
ilvB*
leuA
-23.7-21.9-20.6-18.4-18.4
ilvC*ilvA* -11.0
-15.9
xunk4 +7.7
nc + - + - + -1h 2h 3h
Incr
easi
ngE
xpre
ssio
n
* consistent with expression proteomic results
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Threonine deaminase
Acetolactate synthase
Ketol-acid reductoisomerase
Ketol-acid reductoisomerase
Dihydroxy-acid dehydratase
Ketol-acid reductoisomerase
Ketol-acid reductoisomerase
Acetolactate synthase
Valine, Leucine and Isoleucine Biosynthesis
Dihydroxy-acid dehydratase
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x = unknown proteiny = unknown proteinz = unknown proteinu = 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)
S. aureus Organization Valine, Leucine and Isoluecine Biosynthesis Regions
x y z u v ilvD ilvB ilvN ilvC leuA leuB leuC leuD ilvA
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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.
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Protocol for Quantification by
ICAT/MDLC/MS Approach
Trypsin digest
Reduce
Control
ICAT (1H)
Trypsin digest
Reduce
Experimental
ICAT (2H)
RPC or CE
Affinity select
Mix samples
MALDI or ESI
Bioinformatics
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The ICAT Approach
R. Aebersold Anal. Chem. January 2000.
control
CH-CH2-S-CH2-CO-NH Linker arm (1H8) biotin
experimental
CH-CH2-S-CH2-CO-NH Linker arm (2H8) biotin
Note the deuterium label
Trypsin digestion
m/zi n
ten s
i ty
1H8-ICAT labeled peptide x
2H8-ICAT labeled peptide x
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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
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Functional Study of Novel Target-SP1999 byFunctional Study of Novel Target-SP1999 byIdentification and Characterization of Its Natural LigandIdentification 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.
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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
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MS Identification of Novel Ligand for SP1999MS Identification of Novel Ligand for SP1999
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800m/z0
100
%
LC/ESI MS of Purified Ligand of SP1999
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z0
100
%
134
79
159
426328273 408
LC/ESI-MS-MS of m/z 426
227.0[(TFA)2-H]-
249.0[(TFA)2-2H+Na]-
346.1(AMP-H)-
426.1(ADP-H)-
(HPO3-H)-
(M-H3PO4-H)-
(M-H2O-H)-
(M-H)-
5’-ADP
O
OHOH
N
N
N
N
NH2
O
P
OH
O
O
P
OH
HO O
134
-HPO3
346-H2O
408
-HPO3
328
-Base
291-H2O
273
15979
(C10H13O9N5P2: high resolution MS)
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IL-10 Folding Study Using Chemistry Cross-linking/Mass SpectrometryIL-10 Folding Study Using Chemistry Cross-linking/Mass Spectrometry
Bifunctional Cross-Linkers
Linker name Linker structureSpacer arm
length
DMP(Dimethyl pimelimidate2HCl)
M.W. 259.18C CH2 CH2 CH2 CH2 CH2
-Cl+H2N
H3COC
NH2+Cl-
OCH3
9.2 Å
DMS
(Dimethyl suberimidate2HCl)
M.W. 273.2
C CH2 CH2 CH2 CH2 CH2
-Cl+H2N
H3COCH2 C
NH2+Cl-
OCH3
11 Å
L y s - N H 2
monomer 1 or 2monomer 1
Cross-Linking Reaction with Imidoesters
H 2 N - L y s
C ( C H 2 ) n
-C l + H 2 N
H 3 C OC
N H 2+ C l -
O C H 3
C ( C H 2 ) n
-C l + H 2 NC
N H 2+ C l -
H N - L y sL y s - N H
monomer 1 or 2monomer 1
CH 3 OH
SCHEM E 1.
2
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LC/ESI MS Total Ion Chromatogram (TIC) of Tryptic Digestion of DMP and DMS Modified hIL-102.66e6 cpsTIC of +Q1: from ES0397E0504ss, smoothed 2 times
% % MultiView -- © 1996, SCIEX, a division of MDS Health Group. % % Original concept: % Dr. Ron Bonner % Dr. Lyle Burton % % Development: % Dr. Lyle Burton % Yves Legault % Shengping Ma % % With the help of: % Dr. Victoria Barclay % Scott Champ % Rob McDermid %
20 30 40 50 60 70 80 90 100 110Time, min
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6In
tens
ity, c
ps
1
T22,23
T19
2
T12,13
T2T17
3
T18
4
T20
T3
56
a
T9
T10,11 b
c7d
8
fe9
T5
T1-T14 g
T7
T6
10
h11
i12
j
lk m
13
no
p
q
T8-T15
r
s14
t u vw15
T21
zy
x
3 .7 9 e4 cp sBio Sp ec Reco n stru ct fo r + Q1 : 8 2 .3 5 min (6 scan s) fro m ES0 3 9 7 E0 5 0 4 LL, su b tracted (scan s 1 8 6 to 2 5 2 )
% % Mu ltiView -- © 1 9 9 6 , SCIEX, a d iv isio n o f MDS Health Gro u p . % % Orig in al co n cep t: % Dr. Ro n Bo n n er % Dr. Ly le Bu rto n % % Dev elo p men t: % Dr. Ly le Bu rto n % Yv es Leg au lt % S h en g p in g Ma % % With th e h elp o f: % Dr. Victo ria Barclay % S co tt Ch amp % Ro b McDermid %
1 6 0 0 1 8 0 0 2 0 0 0 2 2 0 0 2 4 0 0 2 6 0 0 2 8 0 0Mass, amu
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
3 0 0 0 0
3 5 0 0 0
Inten
sity, cps
peak n 2205.2
6.66e6 cpsTIC of +Q1: from ES0597E0690s, smoothed 2 times
% % MultiView -- © 1996, SCIEX, a division of MDS Health Group. % % Original concept: % Dr. Ron Bonner % Dr. Lyle Burton % % Development: % Dr. Lyle Burton % Yves Legault % Shengping Ma % % With the help of: % Dr. Victoria Barclay % Scott Champ % Rob McDermid %
20 30 40 50 60 70 80 90 100 110Time, min
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
Inte
nsity
, cps
2 .9 8 e4 cp sBio S p ec Reco n stru ct fo r + Q1 : 8 3 .2 3 min (11 scan s) fro m E S 0 5 9 7 E0 6 9 0 s, su b tracted (scan s 1 6 4 to 2 2 0 )
% % Mu ltiView -- © 1 9 9 6 , S CIEX, a d iv isio n o f MDS Health Gro u p . % % Orig in al co n cep t: % Dr. Ro n Bo n n er % Dr. Ly le Bu rto n % % Dev elo p men t: % Dr. Ly le Bu rto n % Yv es L eg au lt % S h en g p in g Ma % % With th e h elp o f: % Dr. Victo ria Barclay % S co tt Ch amp % Ro b McDermid %
1 6 0 0 1 8 0 0 2 0 0 0 2 2 0 0 2 4 0 0 2 6 0 0 2 8 0 0Mass, amu
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
1 2 0 0 0
1 4 0 0 0
1 6 0 0 0
1 8 0 0 0
2 0 0 0 0
2 2 0 0 0
2 4 0 0 0
2 6 0 0 0
2 8 0 0 0
Inten
sity, cps
peak n 2218.9(b) DMS
T22,23
1
T19
2
T2
T17
3
4
T18
T20
T3
5 6 7 9 10
T9
T10,11
ac
d
e
T5
T1-T14
f
g
T7
T6
a’h
1 1, 1
2
jk/l m
13 n
o
p
qb’
rs
tc’
T8-T15
14 v u15
w
xy
z
T21
(a) DMP
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Table . DMS (11 Å spacer arm) Modified Lysine Residues Based on LC/ESI MS Results
Peak Tryptic Fragment Modified Lysine Residue MWcalcd. (Da) MWexpt. (Da) Lysine Accessibility orNitrogen Interatomic Distance (Å)
a XL-T16,17 Lys119 1058.2 1057.6 Exposed
c XL-T19,20 Lys134 1148.4 1147.8 Exposed
d XL*-T9,10 Lys99 1707.9 1707.2 Exposed
e T18-XL-T19,20
(on same monomer)Lys130-XL-Lys134(on same monomer)
1690.9 1690.2 13.69
f XL-T18,19 Lys130 1261.4 1261.1 Exposed
g XL-T9,10 Lys99 1721.9 1722.2 Exposed
h 2XL-T18,19,20 Lys130, Lys134 1893.2 1892.1 Both Exposed
j T’6,7-XL-T18,19 Lys50’-XL-Lys131 3262.5 3262.2 12.89
k XL*-T6,7 Lys49 2189.5 2188.8 Exposed
l T’5,6-XL-T19,20 Lys41’-XL-Lys135 2970.6 2970.0 16.47
m XL*-T5,6 Lys40 2010.4 2009.6 Exposed
n T4-XL-T5,6
(on same monomer)Lys35-XL-Lys41(on same monomer)
2219.7 2218.9 10.85
o XL-T6,7 Lys49 2203.5 2203.7 Exposed
p XL-T5,6 Lys40 2024.4 2024.1 Exposed
q T15(-ss-T8)-XL-T16,17
(on same monomer)Lys118-XL-Lys120(on same monomer)
5417.3 5416.6 15.42
r XL-T15(-ss-T8),16 Lys117 4794.5 4794.0 Exposed
s T8(-ss-T15)-XL-T9,10
(on same monomer)Lys89-XL-Lys100(on same monomer)
6081.0 6080.0 14.54
t XL*-T7,8(-ss-T15) Lys57 5527.3 5526.3 Exposed
u XL-T7,8(-ss-T15) Lys57 5541.3 5541.5 Exposed
v XL*-T21,22,23 Lys157 2857.4 2856.4 Exposed
w XL*-T21,22 Lys157 2743.3 2742.3 Exposed
x T’7,8(-ss-T15)-XL-T21,22 Lys58’-XL-Lys158 8096.4 8095.5 13.46
y XL-T21,22,23 Lys157 2871.4 2871.0 Exposed
z XL-T21,22 Lys157 2757.3 2757.0 Exposed
a’ XL-T4,5 Lys34 1198.5 1197.9 Exposed
b’ XL-T4,5,6 Lys34 or Lys40 2251.7 2250.7 Both Exposed
c’ 2XL-T4,5,6 Lys34, Lys40 2421.9 2420.1 Both Exposed
Note: XL-T denotes the cross-linker modified tryptic peptide of IL-10.
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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).
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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 Cross-linking/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 protein-protein interaction studies.
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AcknowledgementLeigh 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