Introduction to 2D LC-MS/MS

(Yuanming Luo)Institute of Microbiology

Chinese Academy of Sciences

Fully integrated 2D-LC/ion trap MS

Hardware Improvement ---- New Orthogonal Ion

Source

Square Quadrupole

New Inter-Octapole Lens

New Endcap Electrodes

Entrance Lens

Attomole Sensitivity !!!

1D-strong cation exchange column (Biobasic SCX)

Pressure cell

Xcalibur-control the instrument

Bioworks 3.1-database search software package containing SEQUEST

Application of 2D LC-MS/MS Molecular weight determination 2D gel spots (especially the spots that can’t be identified

by PMF analysis) Protein complex (after primary factionation) Proteome separation and identification Multi-dimensional liquid chromatography MS-based

differential proteomics Quantitative proteomics (including ICAT or stable isotope

labeling-based differential proteome analysis) Analysis of posttranslational modification (data

dependent)

Molecular weight determination of myoglobin by BIOMASS

Calculation

Mr:16951.38+/-0.33

High throughput gel

spot analysis

Tandem RP ColumnsTandem RP Columns

Automated Protein Identification of 2-D gel Automated Protein Identification of 2-D gel spotsspots

??

Sensitivity and Throughput !!!Sensitivity and Throughput !!!

SEQUESTCross-Correlation

ComparisonProtein identified

Digest

High throughput gel spot analysis

1. Protein mixture is separated by 2D gel electrophoresis

2. Excise target gel spot3. Perform in-gel digestion with trypsin. 4. Extract peptides from gel spot.5. Run peptide mixture with ProteomeX in

1D High Throughput mode.6. Data search by TurboSEQUEST software

Analysis of 2D Gel Spots Using ProteomeX ProteomeX High Throughput Method

RT: 0.00 - 102.10

0 2 4 6 8 10 12 14 16 18 20Time (min)

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1000

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10069.67

74.3851.4222.49 75.4066.1142.2728.29 82.7513.98 97.5610.92

61.66

51.90 65.73 70.4921.95 45.20 82.5928.6419.53 41.2213.77 83.90 100.31

22.68

51.85 79.6772.3570.5823.16 35.66 40.14 82.2922.0517.203.68 95.10

61.76

21.8351.89 65.77 70.2936.77 43.0734.16 80.86 82.4420.55 100.618.820.48

63.9323.06

29.58 39.32 51.7279.8765.8835.12 42.52 59.2017.44 82.2715.18 97.304.34

61.60

21.90 51.85 70.3748.8119.62 37.34 72.64 81.2627.7712.701.26 98.7485.70

22.74 51.8379.8663.79 70.37

54.5648.0029.56 81.1944.4721.2811.13 84.87 92.898.54

NL: 2.39E7Base Peak F: + c Full ms [ 300.00-2000.00] MS

GelSpot_tPA1_C1

NL: 9.02E6Base Peak F: + c Full ms [ 300.00-2000.00] MS

gelspot_tpa2_c2

NL: 1.16E7Base Peak F: + c Full ms [ 300.00-2000.00] MS

gelspot_tpa3_c1

NL: 1.41E7Base Peak F: + c Full ms [ 300.00-2000.00] MS

gelspot_tpa4_c2

NL: 2.11E7Base Peak F: + c Full ms [ 300.00-2000.00] MS

gelspot_tpa5_c1

NL: 1.15E7Base Peak F: + c Full ms [ 300.00-2000.00] MS

gelspot_tpa6_c2

NL: 8.00E6Base Peak F: + c Full ms [ 300.00-2000.00] MS

gelspot_tpa7_c1

Spot 1

2

3

4

5

6

7

Found t-PA

Found t-PA

Found t-PA

Global Protein Identification

Global Protein Identification

Protein mixture Protein digests

SCX column fractionationReverse column

separation

Auto MS/MS detection

Tandem MS spectraBioWorks data base search

Results

Plumbing Diagrams for Proteome X.

1D-SCX column

2D-RP1 column

2D-RP2 column

Global Protein Identification1. Extract proteins from cell lysates2. Reduce proteins to peptide

fragments by tryptic digestion.3. Analyze peptide mixture by 2D LC-

MS/MS with ProteomeX.4. Peptide and proteins identified by

TurboSEQUEST software.

Protease Digestion of Proteins

1D LC-MS/MS of proteins from A431 cell lysates

RT:0.00 - 600.00

0 50 100 150 200 250 300 350 400 450 500 550 600Time (min)

0

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10033.95

431.84

35.121163.80

26.66652.24

42.921138.32

76.43667.61

68.711160.5350.66

486.92 117.86563.10 138.63

703.32148.78371.00

14.27344.05 84.72

1154.55200.37563.16

17.79388.10

118.32431.94 258.97

776.40140.051839.77 228.84

619.48 269.22444.83

520.911912.57

432.24675.178.40

439.73 362.07675.25

341.64675.16

382.40675.26 524.05

1511.36465.75488.12294.93

576.29171.601154.56

123.05926.00

268.841285.77226.50

794.7812.29

390.90113.04897.74

575.63444.75

NL:2.28E9Base Peak MS A431_30minG_1029

NL:2.65E9Base Peak MS a431_60ming_1029

NL:1.28E9Base Peak MS a431_120ming_1029

NL:1.49E9Base Peak MS a431_240ming_1029

NL:4.60E8Base Peak MS a431_1213_8hrg

30 min gradient

60 min

120 min

240 min

480 min

2D LC-MS/MS of proteins from A431 cell lysates

Analysis of proteins from A431 cell lysates

56240min

105480min

44120min

2260min

1630min

# of Proteins Identified

Gradient1D

49120 hr.

33710 hr.

1445 hr.

# of Proteins Identified

Total Run Time2D

Yeast Protein separationYeast Protein separationRT: 0.00 - 147.01

0 20 40 60 80 100 120 140Time (min)

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10043.98

43.1025.6313.19

45.4948.383.85

130.2253.42 79.4862.96 121.87111.6097.29

27.67 43.08

23.30

19.68 39.1629.34

18.10 51.05 132.9053.67 128.66116.9114.07 104.0761.26 94.5975.82 135.2528.23

24.11

33.8623.21 36.15

44.14 57.3116.5014.65

62.00 122.3775.8410.12 130.00102.9097.14

57.2146.5038.4029.91

25.02

19.7915.27132.44124.4758.331.30 101.2175.95 98.84

NL:1.37E9Base Peak MS Yeast_120minG_01

NL:4.29E9Base Peak MS yeast_120ming_02

NL:2.67E9Base Peak MS yeast_120ming_03

NL:4.13E9Base Peak MS yeast_120ming_04

20 mM Ammonium chloride,

40 mM Ammonium chloride,

70 mM Ammonium chloride,

100 mM Ammonium chloride,

Yeast Protein SeparationYeast Protein SeparationRT: 0.00 - 147.01

0 20 40 60 80 100 120 140Time (min)

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10052.6632.77

18.84 46.16 57.1444.10

12.57

1.57 75.8658.03 87.56 129.57121.87 137.2296.2841.06

26.8522.22

13.3432.88

50.9312.54 128.3954.17 132.4157.88 75.96 87.63 124.3989.932.55

18.5217.01

42.0727.731.34

1.89 30.91 75.8650.18 57.70 76.9914.82 87.6770.04 134.87129.1497.96 113.09

NL:4.05E9Base Peak MS yeast_120ming_05

NL:3.24E9Base Peak MS yeast_120ming_06

NL:1.02E9Base Peak MS yeast_120ming_07

140 mM Ammonium chloride,

180 mM Ammonium chloride,

220 mM Ammonium chloride,

Yeast proteinsYeast proteinsReference Score Hits Entriesgi|129922|sp|P14828|PGK_KLULA PHOSPHOGLYCERATE KINASEgi|66895|pir||KIVKGL 65809.5 18 4 4 3 0 91 95 96 101 255 260 686 689 824 873 888 902 913 925 926 948 960 1050 : 864 994 1630 1765 : 1333 1507 1682 1749 : 886 910 1760 : gi|6319594|ref|NP_009676.1| translational elongation factor EF-1 alpha; Te 56564.0 12 1 1 0 0 76 215 239 336 1020 1024 1159 1163 1338 1362 1518 1526 : 113 : 538 : : gi|119145|sp|P16017|EF1A_CANAL ELONGATION FACTOR 1-ALPHA (EF-1-ALPHA)gi|1 46500.4 0 11 1 1 0 : 76 215 239 1020 1024 1159 1163 1338 1362 1518 1526 : 113 : 1706 : gi|1172457|sp|P41757|PGK_CANMA PHOSPHOGLYCERATE KINASEgi|83923|pir||JT095 46303.5 12 1 0 0 0 255 260 824 860 873 888 913 925 926 948 960 1050 : 994 : : : gi|6324637|ref|NP_014706.1| Ribosomal protein L3 (rp1) (YL1); Rpl3p [Sacch 42592.4 10 0 1 1 0 133 138 143 383 387 468 471 482 484 1531 : : 386 : 1305 : gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae]gi|1 41268.3 10 2 1 0 0 38 56 58 69 77 262 269 270 302 313 : 180 1413 : 163 : : gi|6319279|ref|NP_009362.1| Pyruvate kinase; Cdc19p [Saccharomyces cerevis 38052.7 23 3 0 0 0 29 38 59 70 73 92 132 136 166 288 291 421 474 483 900 1121 1198 1246 1613 1619 1632 1638 1714 : 163 480 526 : : : gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae]gi|1 36687.9 13 2 1 0 1 8 11 39 42 129 377 396 486 488 590 802 1751 1814 : 1014 1621 : 234 : : 458 gi|6321968|ref|NP_012044.1| enolase; Eno2p [Saccharomyces cerevisiae]gi|1 34389.6 13 2 1 0 1 19 50 329 349 441 442 445 820 1028 1037 1434 1522 1548 : 346 2014 : 160 : : 1674 gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 34030.4 13 7 0 0 0 49 113 116 212 215 336 567 608 804 1082 1275 1317 1320 : 121 125 615 647 797 1253 1272 : : : gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase; Pgk1p [Saccharomyc 26362.2 7 4 1 0 0 185 187 307 313 404 1743 1785 : 109 373 1746 1804 : 1811 : : gi|6322790|ref|NP_012863.1| aldolase; Fba1p [Saccharomyces cerevisiae]gi| 26245.4 2 0 0 0 0 266 269 : : : : gi|6319279|ref|NP_009362.1| Pyruvate kinase; Cdc19p [Saccharomyces cerevis 25546.9 9 0 0 0 0 61 131 218 276 309 310 1005 1399 1404 : : : : gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 23954.0 7 1 0 0 1 97 143 204 294 356 365 492 : 297 : : : 264 gi|6325112|ref|NP_015180.1| sequence similar to Hes1p; Kes1p [Saccharomyce 23937.7 3 0 2 2 0 611 736 1234 : : 76 1362 : 215 1547 : gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase; Pgk1p [Saccharomyc 23524.5 3 2 1 0 1 171 173 298 : 175 1060 : 1226 : : 378 gi|6321609|ref|NP_011686.1| phosphatidylserine decarboxylase located in va 23254.1 2 0 1 3 4 475 1103 : : 404 : 397 451 1007 : 139 185 400 1607 gi|6322526|ref|NP_012600.1| phosphatidylinositol kinase homolog; Tor1p [Sa 21082.5 0 3 2 1 2 : 7 80 770 : 1518 1690 : 1024 : 75 1020 gi|14318479|ref|NP_116614.1| Actin; Act1p [Saccharomyces cerevisiae]gi|11 21011.4 2 0 0 0 0 372 384 : : : : gi|12230852|sp|O74343|YH2X_SCHPO HYPOTHETICAL 76.4 KDA PROTEIN C1A4.09 IN 20063.6 0 1 1 1 1 : 1603 : 847 : 1607 : 1590 gi|119336|sp|P00924|ENO1_YEAST ENOLASE 1 (2-PHOSPHOGLYCERATE DEHYDRATASE) 19580.1 4 0 0 0 0 177 260 302 313 : : : : gi|6324313|ref|NP_014383.1| Pbi2p [Saccharomyces cerevisiae]gi|124818|sp| 19318.9 5 0 0 0 0 62 70 73 1878 1882 : : : : gi|6323004|ref|NP_013076.1| member of 70 kDa heat shock protein family; Ss 19151.9 7 0 0 0 1 208 211 213 215 1467 1609 1610 : : : : 1288 gi|6325331|ref|NP_015399.1| Transketolase 1; Tkl1p [Saccharomyces cerevisi 18932.7 5 1 0 0 0 217 218 223 224 1328 : 420 : : : gi|6321631|ref|NP_011708.1| Glyceraldehyde-3-phosphate dehydrogenase 3; Td 18650.3 3 0 0 0 0 1590 1603 1607 : : : : gi|6321977|ref|NP_012053.1| 6-phosphogluconate dehydrogenase; probable GND 18647.0 4 0 0 1 0 647 1529 1530 1534 : : : 695 : gi|7492246|pir||T40586 nucleolar protein involved in pre-rRNA processing - 18417.0 6 8 12 10 8 1075 1243 1276 1414 1508 1540 : 1009 1076 1247 1482 1585 1587 1715 1739 : 277 880 895 967 1148 1180 1213 1216 1296 1386 1625 1773 : 137 587 962 1149 1287 1312 1451 1538 1556 1763 : 140 911 1042 1355 1356 1450 1681 1713 gi|7491534|pir||T40003 hypothetical protein SPBC25H2.08c - fission yeast 18270.9 0 1 2 0 0 : 143 : 133 138 : : gi|6325016|ref|NP_015084.1| 82 kDa heat shock protein; homolog of mammalia 18036.9 6 2 0 0 0 5 31 132 800 1229 1235 : 37 1237 : : :

Yeast proteinsYeast proteinsgi|6323376|ref|NP_013448.1| Ribosomal protein L26A (L33A) (YL33); Rpl26ap 4748.4 2 1 1 0 0 1624 1627 : 1625 : 652 : : gi|6321007|ref|NP_011086.1| Transcriptional regulator which functions in m 4747.8 0 0 0 1 0 : : : 33 : gi|6323292|ref|NP_013364.1| Ylr262c-ap [Saccharomyces cerevisiae]gi|21318 4746.0 0 1 0 0 1 : 876 : : : 732 gi|6322314|ref|NP_012388.1| Yjl147cp [Saccharomyces cerevisiae]gi|1353019 4744.7 0 0 0 1 0 : : : 460 : gi|6324034|ref|NP_014104.1| Ynl295wp [Saccharomyces cerevisiae]gi|1353106 4744.4 0 1 0 0 0 : 396 : : : gi|6324151|ref|NP_014221.1| Ribosomal protein S3 (rp13) (YS3); Rps3p [Sacc 4743.3 2 0 0 0 0 279 332 : : : : gi|6323691|ref|NP_013762.1| 4741.6 0 0 3 0 3 : : 1877 1949 2145 : : 981 1314 1853 gi|6322806|ref|NP_012879.1| p58 polypeptide of DNA primase; Pri2p [Sacchar 4738.8 0 0 1 0 1 : : 971 : : 558 gi|1171671|sp|Q09711|NCS1_SCHPO HYPOTHETICAL CALCIUM-BINDING PROTEIN C18B1 4737.8 0 0 0 1 0 : : : 397 : gi|7496455|pir||T19442 hypothetical protein C25A1.7a - Caenorhabditis eleg 4737.0 0 0 1 2 1 : : 1597 : 185 1645 : 1521 gi|83218|pir||S19440 hypothetical protein YCR029c - yeast (Saccharomyces c 4736.0 0 0 1 0 0 : : 50 : : gi|6323487|ref|NP_013559.1| Ylr454wp [Saccharomyces cerevisiae]gi|1363732 4735.9 1 1 2 2 0 724 : 6 : 638 1305 : 359 1200 : gi|6319642|ref|NP_009724.1| 4735.1 0 0 0 0 1 : : : : 224 gi|6319850|ref|NP_009931.1| non-mitochondrial citrate synthase; Cit2p [Sac 4729.1 1 1 0 0 1 646 : 1020 : : : 1619 gi|6324552|ref|NP_014621.1| 3'-5' exoribonuclease complex subunit; Dis3p [ 4728.2 0 0 1 1 0 : : 1637 : 329 : gi|7490292|pir||T38695 conserved hypothetical protein SPAC3C7.09 - fission 4726.5 0 0 1 1 0 : : 766 : 1441 : gi|6320126|ref|NP_010206.1| 4724.8 0 0 0 2 3 : : : 342 712 : 598 637 1671 gi|1077218|pir||S49776 hypothetical protein YDR179w-a - yeast (Saccharomyc 4723.5 0 0 1 0 0 : : 50 : : gi|1352297|sp|P48996|DP27_CAEEL CHROMOSOME CONDENSATION PROTEIN DPY-27gi| 4723.3 1 0 0 1 2 1196 : : : 482 : 122 896 gi|6323875|ref|NP_013946.1| involved in silencing; Esc1p [Saccharomyces ce 4722.7 1 0 1 0 1 1103 : : 1722 : : 792 gi|1175464|sp|Q09796|YAA2_SCHPO HYPOTHETICAL 111.5 KD PROTEIN C22G7.02 IN 4719.0 1 0 1 0 1 1954 : : 294 : : 214 gi|6323762|ref|NP_013833.1| Ymr115wp [Saccharomyces cerevisiae]gi|2497155 4717.5 0 0 0 1 0 : : : 243 : gi|6322171|ref|NP_012246.1| Ribosomal protein L2B (L5B) (rp8) (YL6); Rpl2b 4715.9 1 1 0 0 0 42 : 25 : : : gi|2495228|sp|Q12578|HIS7_CANGA IMIDAZOLEGLYCEROL-PHOSPHATE DEHYDRATASE (I 4715.9 0 1 0 0 0 : 610 : : : gi|15213983|sp|O94489|EF3_SCHPO ELONGATION FACTOR 3 (EF-3)gi|7492556|pir| 4715.5 1 0 1 0 0 24 : : 326 : : gi|6321594|ref|NP_011671.1| Cystathionine beta-synthase; Cys4p [Saccharomy 4712.9 2 0 0 0 0 284 652 : : : : gi|6323722|ref|NP_013793.1| (putative) involved in sister chromosome cohes 4712.4 1 0 0 0 1 561 : : : : 198 gi|113719|sp|P12807|AMO_PICAN PEROXISOMAL COPPER AMINE OXIDASE (METHYLAMIN 4712.2 4 6 4 3 5 1168 1335 1362 1468 : 1169 1228 1471 1496 1499 1613 : 1217 1394 1436 1745 : 1249 1530 1550 : 1151 1324 1337 1462 1742 gi|6320232|ref|NP_010312.1| 4707.7 0 0 0 0 1 : : : : 202 gi|6322088|ref|NP_012163.1| 4707.5 0 0 0 2 0 : : : 116 1740 :

Protein # 1708

2D LC-MS/MS of Yeast proteins2D LC-MS/MS of Yeast proteins

• Time: 15 hours

• Gradient: 5 – 65% Acetonitrile in 2 hrs in each step

• Proteins searched by Bioworks 3.1

• Proteins identified: 1708

• Throughput: 113.8 proteins/hr

Viewing Results

Synclein alpha

TIC

Filters for SEQUEST Results

Xcorr ： +1>1.5, +2>2.0, +3>2.5 ∆CN: >0.1 When three or fewer peptides for an

individual protein passed the criteria (1) the spectrum quality (S/N, match rate) (2) some continuity must be present among

the b or y fragments (3) if proline is predicted to be present, then

the corresponding y fragment should give an intense peak.

(4) unidentified intense peaks should be verified as being either doubly charged.

Filters for SEQUEST Results

On-Line Phosphopeptide Enrichment (IMAC capture)

Flow Path of an Automated 2D (IMAC + RP)-MS/MS System for the Analysis of Phosphopeptides

SCX

Col

umn

11

6

2

3

54

Pump 1

injector

1

6

2

3

54

Sample loop

Sample valve

To Waste

IMA

C

RP

11

6

2

3

54

Pump 1

injector

1

6

2

3

54

Pump 1

injector

1

6

2

3

54

InjectorSample loop

LCQ Deca XP Plus- mass spectrometer

RP2

10 –port valve in mass spectrometer

Analytical Pump

Sample Pump

1D-IMAC Column

Procedure Used for Automated 2D LC(IMAC+RP)- MS/MS Analysis of Phosphopeptides

Step 1: Load IMAC column

Step 2: Load peptides on IMAC column. Flow-through peptides captured by RP2 column.

Step 3: Wash IMAC column. The bound peptides are then eluted by phosphate buffer on to RP1, while the flow-through peptides trapped on RP2 are being analyzed by LC/MS.

Step 4: The bound phosphopeptides on RP1 are analyzed by LC/MS/MS.

Capture of FQ*SEEQQQTEDELQDK Phosphopeptide of -Casein Digest in the 2D LC(IMAC+RP)-MS/MS System

Non-phosphorylated peptidesflow through IMAC column and captured by and eluted from RP2

Phosphorylated peptide (m/z=1031.7, FQ*SEEQQQTEDELQDK) captured by IMAC column, bound to RP1, and eluted.

NL: 1.34E9

NL: 1.80E8

position for m/z=1031.7 on C2 column

RP2 column

RP1 column

Neutral Loss Scanning Confirmed the Major Ion at m/z=1031.6 as a P-peptide

Neutral lossfragment (-49)

M+2H+-49MS/MS of 1031.6

Phosphorylated peptide (m/z=1031.7,

FQ*SEEQQQTEDELQDK)

Bioworks 3.1 Search Identified the P-peptide with m/z=1031.6 as FQ*SEEQQQTEDELQDK

1+ 2+(M+2H)-49 (M+2H)-49

Proteins - Differential Expression (EGF treated and untreated cells)----Alternative method for differential analysis

of protein expression compared to 2DE strategy

Protein differential expression1. Divide A431 cell sample in two:

a) Half stimulated by EGFb) Half control

2. Lyse cells3. Extract proteins from lysates4. Digest with trypsin5. Run 2D LC-MS/MS of digests with

ProteomeX6. Proteins identified by TurboSEQUEST

software7. Compare “stimulated” vs. “control”

Automated 2D LC-MS/MS Analysis of Human A431 Cell Proteins

    

RT: 0.00 - 87.01

0 10 20 30 40 50 60 70 80Time (min)

0

50

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0

50

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0

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Rel

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Abu

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0

50

100

36.47974.2

11.84652.2

20.801154.5

8.17513.5 27.76

866.214.06981.2 34.75

814.46.03

652.945.66955.3

37.28974.1 74.00

1010.451.00614.2

53.93804.3

59.861179.0

66.46971.0

77.031024.2

20.34896.2

34.70970.7

37.53974.231.70

925.6 38.011335.0

26.50784.4

50.68496.2

13.92581.510.95

566.942.02322.0

52.10522.3

1.21712.6

54.90716.3

65.23369.3

73.06494.5

85.51371.0

78.78371.0

23.831154.7

50.28991.6

22.25820.7

37.57974.6

31.161113.3

51.75522.2

20.32896.1

46.23955.2

1.36712.6

13.92464.4

74.101010.4

85.00370.9

54.11760.2

68.691418.3

18.07652.3

23.73839.4

28.851079.1

52.371043.736.47

814.414.08655.0

1.06712.6

45.61955.3

12.34729.2

56.98315.9

72.50390.9

84.35370.9

81.03370.9

62.62563.3

NL:4.02E9Base Peak MS A431_040402_01

NL:7.25E8Base Peak MS a431_040402_02

NL:6.96E8Base Peak MS a431_040402_03

NL:1.26E9Base Peak MS a431_040402_04

RT: 0.00 - 87.01

0 10 20 30 40 50 60 70 80Time (min)

0

50

100

0

50

100

0

50

100

Rel

ative Abu

ndan

ce

0

50

100

35.16947.6

50.21991.830.76

832.018.73893.3 19.50

654.614.64653.5 51.82

522.227.91653.8

8.92578.0 35.81

1286.341.74322.0

53.03522.6

72.10390.8

83.25370.9

80.53370.9

59.38842.3

64.81371.0

23.93797.2

31.93866.3

26.10878.616.37

600.150.62496.2

12.80495.8

1.22712.5

38.721103.9

42.18322.0

73.18508.6

56.97315.9

85.04370.9

69.94508.7

65.29369.3

78.99370.9

33.02932.8

27.32889.5

51.72522.3

24.01697.0 50.30

496.211.49417.31.35

712.5 36.50900.1

38.62894.59.30

630.8 52.55522.4

72.16390.9

59.49842.4

83.45370.9

80.72371.0

69.191269.7

22.56682.7

30.16822.8

17.43480.8

15.47478.7

23.90668.8

50.63496.3

52.17522.232.76

932.60.82

712.4 38.09894.7

42.17321.9

73.04508.7

72.03508.5

56.86315.9

86.81370.9

80.31370.9

NL:9.00E8Base Peak MS a431_040402_05

NL:1.10E9Base Peak MS a431_040402_06

NL:3.95E8Base Peak MS a431_040402_07

NL:1.04E9Base Peak MS a431_040402_08

120 mM

10 mM

20 mM

40 mM

60 mMNH4Cl 0 mM

80 mM

160 mM

Automated 2D-LC-LC/MS-MS Analysis of Human A431 Cell Proteins (continued)

200mM

300mM

500mM

900mM

RT: 0.00 - 87.02

0 10 20 30 40 50 60 70 80Time (min)

0

50

100

0

50

100

0

50

100

Rel

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0

50

100

15.05506.3 51.86

522.450.26496.3

1.20712.5

41.68322.1

13.02503.1

31.88569.4

15.60473.0

9.31452.8

29.42677.9

59.03886.5

82.29370.8

72.08391.0

62.99347.9

1.14712.6

52.16522.250.64

496.232.45569.6

42.15321.912.85

603.41.91

712.5 49.37496.5

19.02857.9

32.95932.5

85.51370.9

79.64370.9

30.87832.1

63.71347.9

77.53370.9

53.47508.3

50.36991.7

1.09712.4

51.731043.5

32.17569.7

2.00712.5

41.92321.99.27

446.415.78824.2

81.51370.9

25.96769.1

78.98370.9

63.24348.0

72.13390.9

59.23886.4

1.04712.5

50.64496.31.69

712.432.37569.6

52.17522.3

42.27321.95.69

712.5 19.62712.5

70.87508.7

37.95331.9

63.72348.0

46.88399.1

83.46370.9

72.54390.9

29.70347.9

54.66371.1

NL:4.11E8Base Peak MS A431_040402_09

NL:2.46E8Base Peak MS a431_040402_10

NL:2.95E8Base Peak MS a431_040402_11

NL:2.70E8Base Peak MS a431_040402_12

Total Proteins Identified= 709, using Bioworks 3.1 with TurboSequest(Xcorr = 1.5, 2.0, and 3 for charge states +1, +2, and +3, respectively)

Proteins Differentially Expressed in Control and EGF-Stimulated A431 Cells

Proteins Differentially Expressed in Control and EGF-Stimulated A431 Cells (continued)

*Only those proteins with two or more peptides identified were compared

Proteins Identified in Both Control and EGF-Treated A431 Cells

Proteins Common to Control and EGF-treated A431 Cells (continued)

Proteins Common to Control and EGF-treated A431 Cells (continued)

Differential Protein quantitation

-quantitative proteomics

Stable isotope labeling (SIL) for quanlitative proteomics

Metabolic labeling (13C, 15N)Post-biosynthetic labeling (ICAT reagent)Post-digest isotope Labeling of tryptic

peptides(18O)

Metabolic labeling with [13C6]Arg in the elucidation of EGF signaling

Cells were grown in medium containing either normal or [13C6] arginine.

8 h of serum starvation, the labeled cells were stimulated with 150ng/ml EGF for 10 min, whereas the unlabeled cells were left untreated.

Cells were lysed and combined in a 1:1 ratio followed by incubating at 4°C with Grb2 fusion protein bound to GSH-sepharose beads for 4h.

Wash with lysis buffer, boiled in sample buffer, and resolved on a 4-12% gel.

Bands of interest were excised and subjected to in gel digestion.

Mass spectrometric analysis

Strategy to study activated EGFR complex

SH2 domain of Gb2 binds tyrosine-phosphorylated proteins including EGFR, Shc etc.,

Quantification of protein ratios from peptide doublets. Top panels show mass spectra of peptides of different identified proteins, bottom panels show mass spectra of peptides from EGF-stimulated cells upon detection of autophosphorylation

Metabolic labelingAdvantages: (1) all sample-to-sample

variability induced by subsequent biochemical experiments can be eliminated. (2) metabolism-related dynamic labeling involved in a specific physiological process.

Drawbacks: (1)only works in cell culture systems that tolerate isotope-substituted media (which is actually often not the case), which may not be compatible with a particular biological investigation. (2)Total isotope substitution is required for reliable for MS-based quantification, which renders the approach rather expensive. (3)Difficulty in establishing an enrichment method

Differential Quantitation with isotope-coded Differential Quantitation with isotope-coded affinity tags (ICAT)affinity tags (ICAT)

1. Divide the previous sample (hGH in plasma) into two identical pools.

2. Reduce and alkylate (D0 ICAT for one plasma pool and D8 ICAT for the other), separately. Mix the two pools and digest the whole mixture with trypsin.

3. Sample clean-upa) ion exchange to remove excess ICAT reagentb) avidin affinity to capture the ICAT-labeled peptides

4. Collect the ICAT-peptide fractions and run LC-MS/MS.

5. Data analysis by Bioworks 3.1a) TurboSEQUEST for protein identificationb) XPRESS for relative quantitation

ProteomeX (2D)

Collect the flow through Frxn ProteomeX(2D)

ProteomeX (1D)

The structure of ICAT reagent

Data Dependent Mass Tag Setting for ICAT

1+2+3+

TurboSEQUEST Search ParametersTurboSEQUEST Search Parameters

Turbosequest parameters are set as usual except the amino acid modification and differential mass need to be set as in above

Bioworks 3.1 (SEQUEST and XPRESS) Bioworks 3.1 (SEQUEST and XPRESS) Search ResultsSearch Results200mM NH4Cl

Differential Quantitation by Bioworks (XPRESS) Differential Quantitation by Bioworks (XPRESS) SoftwareSoftware

NYGLLYCFR(T16 peptide of human growth hormone)

*

After finishing the TurboSEQUEST search, click the XPRESS function to locate the correct cysteine-containing peptide sequence (identified from its MS/MS spectrum) with the ratio of D0 and D8 ion intensities (integrated from its parent ion spectrum) as shown in above.

RT: 35.09 - 35.37

35.10 35.15 35.20 35.25 35.30 35.35Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

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35.29

35.25

35.20 35.33

35.16

35.16

35.33

35.29

35.20

NL:9.68E5

Base Peak

m/z=

798.6-799.6

MS

NL:1.09E6Base Peak

m/z=

794.4-795.4 MS

M/Z = 799.1 Signal = 0.986

M/Z = 794.9 Signal =1.09

D0/D8 = 1.1

Zoom In MS spectra

(+2) Charge D8

(+2) Charge D0

Using the highest MS intensity

Advantage: (1)Largely reduce the complexity of peptide mixture; (2)Easy to enrich.

Drawbacks: (1) 14% protein sequences do not contain cysteine-containing tryptic peptides (800-2500Da),19% contains just a single such peptide (alternatively, cleavable ICAT reagents). (2) requirement of protein over 100 g.

Post-digestion isotope labeling (18O)

Artifacts (i.e. side reactions) inherent to chemical labeling can be avoided.

All peptides can be used for identification and quantification

Available for gel-separated proteins

Samples of interest are first digested with trypsin.Aliquots are subsequently incubated with either

16O water or18O water in the presence of trypsin. Labeling efficiencies of individual peptides of the H2

18O-treated sample are determined by MALDI-TOFMS of a small portion of the sample. Mixtures of 16O- and 18O-labeled samples are then applied on the MALDI plate, and relative abundances are derived from relative isotopomer abundances.

General scheme of post-digest 18O labeling procedure

Time course of trypsin-catalyzed post-digest labeling of 1 pmol BSA tryptic digest. The exchange rate of C-terminal oxygen atoms is dependent on the peptide sequence. Fast exchanging peptides show complete labeling after <10 min (a). However, for some peptides close to quantitative labeling could only be achieved after incubation for 2 h (c).

Practical considerations for stable isotope labeling in quantitative proteomics

Predictable mass difference between labeled and unlabeled samples

Easy to enrich

An example of Data dependent MS/MS mode-reject high abundant proteins(GDH-2)

Glutamate dehydrogenase 2

1193.29

1759.92

Data dependent setup for rejecting high abundant GDH-2 Just ion of

interest

Post-Translational Modifications

Modifications

Modifications (continued)

Modifications (continued)

Phosphorylation

Protein identification: Phosphorylation

Data Dependent (with Dynamic Exclusion)

MS/MS spectrum of m/z 980-982%

Rel

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400 600 800 1000 1200 1400 1600 18000

10

20

30

40

50

60

70

80

90

100

931.8

1311.5

922.7 1099.5764.6551.1452.5 1591.51410.6 1689.71083.1366.4 665.3 1786.8

Y’’12

+1

(MH2 - H3PO4)2+

Y’’10

+1

Arg-Leu-Ser-Leu-Val-Pro-Asp-Ser-Glu-Gln-Gly-Glu-Ala-Ile-Leu-Pro-Arg

Y”12

+1 Y”10

+1

Serine Not Phosphorylated

SerinePhosphorylated

Glycosylations

Glycosylation

Glycosylation

Glycosylation

Identifying Glycosylation – MS full scanRT: 0.00 - 140.00

0 10 20 30 40 50 60 70 80 90 100 110 120 130Time (min)

010

2030405060708090

100R

elat

ive

Abu

ndan

ce46.78

31.08 34.5529.80 44.6626.55

5.3256.23

6.06 97.6149.19 96.92 121.72113.398.59 57.42 98.2595.9321.91 110.9821.55 88.2461.02 117.3564.9113.23 86.7570.50 75.92 122.46 138.88

NL:2.79E10TIC MS

#583 RT: 15.57 AV: 1 NL: 7.23E8T: + c Full ms [ 200.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

5

10

15

20

Rel

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527.5

217.0 575.8

1064.0234.1 445.0 634.8286.8 1095.7762.3 1060.7807.9 1267.6 1595.2 1780.21514.8 1987.9

Glycopeptide region MS from

glycopeptide ionMS scan

+3+2

Asn

FuN

(select to do MS/MS)

Glycopeptide region

12

34

Other region perform only MS and MS/MS

Identifying Glycosylation – MS/MS 584 RT: 15.59 :

T: + c Full ms2 1064.00@65.00 [ 280.00-2000.00]

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

5

10

15

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35

40

45

50

55

60

65

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75

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Rel

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1267.6

1185.81413.1

1449.6

1369.41450.3

366.0657.0 1003.5

1478.91085.1739.5453.8 966.8 1845.7 1933.3923.0

AsnFu

AsnFu

AsnFu

AsnN Fu

N

AsnFu

AsnFu

(select to do MS to 3)

+2

+2

+2+2

+2+1+1

Identifying Glycosylation – MS3

TPA_iontree_2_010524173638 #585 RT: 15.61 AV: 1 NL: 4.09E5T: + c Full ms3 1064.00@65.00 1267.64@65.00 [ 335.00-2000.00]

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

5

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15

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Rel

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1333.2

1011.31478.3666.6

1479.2993.5

1537.61085.21334.61185.7

740.3892.4 1987.5

528.0

551.2 768.61697.2

1460.4586.1930.3 1315.8 1859.1

Asn

AsnFu

Asn

Fu+2

AsnFu

Asn

Fu

+2 Asn

AsnFu

oxidized

Asn

Fu

(select to do MS to 4)

Fu

AsnFu

Identifying Glycosylation – MS4

SCH2COOH

TPA_iontree_2_010524173638 #586 RT: 15.63 AV: 1 NL: 8.63E4T: + c Full ms4 1064.00@65.00 1267.64@65.00 1333.22@65.00 [ 355.00-2000.00]

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

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728.4 1213.4

1130.7

506.2

833.01053.4

636.2 823.4 1240.5618.3 798.0

492.2

C-T-S-Q-H-L-L-N-R

C-T-S-Q-H-L-L-N-RDehydro-alanine form

Peptide onlyC-T-S-Q-H-L-L-N-R

B6 B6

Y7

Y7-H2O

y2

Y2 / Y6(+2)

B5

B5

Dehydro-alanine formFurther CNH2 loss on N-terminal

B7-H2O

B7(select to do MS to 5)

Summary of one glycopeptide fragmentation pathway (a biantennary

glycopeptide)

AsnFu

MS MS/MS

AsnFuN

MS to 3

Asn

AsnFu

AsnFu

AsnFu

AsnFu

211(1064.4 +3)

210(1450.6 +2)

MS to 4 or 5

Peptide only (1131 +1)

LCQ-deca (nanospray)

LCQdecaXP (microspray)

(1333 +1)

(1186 +2)

(1085 +2)

(1105 +2)

(1004 +2)

(1333 +1)

AsnFu

(1267 +2)

De Novo Peptide Sequencing

Why De Novo Peptide Sequencing ?

Determination and/or confirmation of peptide sequences derived from proteins that are:

not in the databases (including DNA sequence)

with amino acid modifications

De novo sequencing software (PARSER II)

Ref: Zhang ZQ, McElvain JS. De Novo peptide sequencing by two-dimensional fragment correlation mass spectrometry. Anal Chem, 2000, 72 (11): 2337-2350

MS, MS2 and MS3 spectra collected with peak parking

16 18 20 22 24Time (min)

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Base Peak

400 600 800 1000 1200m/z

496.1

990.6

Full Scan MS

200 400 600 800 1000m/z

0

20

40

60

80

100730.4

389.2261.1

233.1 616.4

732.4502.4

Full Scan MS2

***

*

*

100 200 300 400m/z

120.2

86.1121.2

400 800 1200m/z

713.4

714.5389.2502.3

Full Scan MS3@233.1

Full Scan MS3@730.4

100 200 300 400 500m/z

0

233.1

234.1200 400 600

m/z

261.2226.1

243.0

129.1

372.2

354.1

200 400 600 800 1000m/z

581.2

599.3

Full Scan MS3@261.1

Full Scan MS3@389.2

Full Scan MS3@616.4

Determination of Peptide Sequence by MS3 De Novo Sequencing Software --- Biowork 3.1

Peptide = FINNIGANK

Sequencing Tryptic Peptide (m/z 585.1) by MS3 De Novo Sequencing Software

Peptide = TGPNLHGLFGR

Thank You!