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 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

• 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)

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

• 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)

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

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

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

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

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

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

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

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?

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

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)

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

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

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 -

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 -

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)

T=3, 2 (+IPTG)Cy5 image

T=3,2 (-IPTG)Cy3 image

down-regulated

up-regulated

Review dB Search Results

assigned sequence of the peptide based on the MS/MS data

Protein ID

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

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

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

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

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

Trypsin digest

Reduce

Control

ICAT (1H)

Trypsin digest

Reduce

Experimental

ICAT (2H)

RPC or CE

Affinity select

Mix samples

MALDI or ESI

Bioinformatics

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

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 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.

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 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)

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

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

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.

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 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.

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