proteomics standards research group sprg2006: a proteomics ... · proteomics standards research...

A BR F

Proteomics StandardsResearch Group

sPRG2006:A Proteomics Standard

www.abrf.org/sprg

Phil Andrews - University of Michigan

David Arnott - Genentech, Inc.

Mary Ann Gawinowicz - Columbia University

Jeffrey A. Kowalak - National Institutes of Health (Chair)

William S. Lane - Harvard University (EB Liaison)

Kathryn S. Lilley - University of Cambridge

Larry Martin - East West University

Stephen E. Stein - NIST

Proteomics Standards ResearchGroup (sPRG) Members

ABRF


ABRF


Mission Statement

• Promote and support the development and use of standards in Proteomics.

• Identify and implement technical standards that reflect the ABRF’s commitment to accuracy, clarity, and consistency.

• Standards include, but are not limited to, reference materials, data sets, conditions and procedures that give researchers independent criteria to evaluate their abilities to produce predictable, consistent results.

• Strongly support efforts for standardization of the recording and reporting of proteomics experiments.

sPRG Websitewww.abrf.org/sprg

ABRF


ABRF


Authors described a data set of peptide tandem mass spectra generated from a control mixture of18 purified proteins that can be employed to evaluate strategies for tandem mass spectral analysis.

Purvine, S., Picone, A.F., and E. Kolker, E. (2004) “Standard Mixtures for Proteome Studies” OMICS 8: 79-92

Proteomics Standardsin the Literature

Authors introduced a mixture of 23 peptides and 12 protein digests to serve as possible standard for proteome analyses.

Keller, A., Purvine, S., Nesvizhskii, A.I., Stolyar, S., Goodlett, D.R. and E. Kolker, E. (2002) “Experimental Protein Mixture for Validating Tandem Mass Spectral Analysis” OMICS 6: 207-212

• Human proteins, purified from their biological source or recombinantly expressed.

• Subjected to multiple analytical methodologies, e.g., 1D PAGE, IEF, and RP-HPLC, in order to assess required levels of purity (> 95%).

• Protein concentration was determined by amino acid analysis.

• Five picomole aliquots of each protein were combined and lyophilized in a 1 mL polypropylene tube.

ABRF


Materials

• Prior to distribution to participating labs, the prototype protein standard was distributed to the sPRG committee members and analyzed by 5 different member laboratories for validation.

• In three RG laboratories, the standard was analyzed using a shotgun approach by digestion with trypsin of the entire proteinmixture followed by on-line 1D LC-MS/MS, on-line 2D LC-MS/MS, off-line capillary RP-HPLC, TOF/TOF

• In two RG laboratories, the protein mixture was partially separated by 1D SDS-PAGE, followed by in-gel tryptic digestion and on-line 1D LC-MS/MS.

ABRF


Standard Validation

Results Submission

• Results were submitted anonymously through an on-line survey and included experimental details, instrument(s) used, and a complete list of all proteins identified with accession numbers.

• A web site (www.proteomecommons.org/dev/abrf/) was provided for the voluntary submission of raw mass spectral data to be made available for future studies.

ABRF


http://www.proteomecommons.org/dev/abrf/

129

78

sample requests data returned

ABRF


ABRF


60.5% Data Return

7537

10

22 1

AmericaEuropeCanadaAustraliaJapanArgentina

ABRF


sPRG2006 RequesterDemographics

46

32 ABRF Member

Non-member

sPRG2006 ABRF MembershipABRF


ABRF


53

7

11

7 Academic

Biotech/Pharma/Industrial

Government

Manufacturer/Vendor

sPRG2006 Laboratories

ABRF


52

11

85 1

Both core functions andnon-core lab research

Core only

Non-core research lab

Other

No Response

sPRG2006 Facility Type

7

71

yesno

ABRF


Did you receive more than 1 sample?

2

76

yesno

Did you have prior knowledge of another lab’s results?

ABRF


sPRG2006 Proteomics Standardwww.abrf.org/sprg

Accsn Entry Protein Accsn Entry ProteinP01008 ANT3_HUMAN Antithrombin-III precursor P01343 IGF1A_HUMAN Insulin-like growth factor IAP02768 ALBU_HUMAN Serum albumin P12081 SYH_HUMAN Histidyl-tRNA synthetaseP08758 ANXA5_HUMAN Annexin A5 P02788 TRFL_HUMAN LactotransferrinP55957 BID_HUMAN BH3 interacting domain death agonist P41159 LEP_HUMAN LeptinP00167 CYB5_HUMAN Cytochrome b5 P61626 LYSC_HUMAN Lysozyme CP99999 CYC_HUMAN Cytochrome c P00709 LALBA_HUMAN Alpha-lactalbuminP04040 CATA_HUMAN Catalase P61769 B2MG_HUMAN Beta-2-microglobulinP62937 PPIA_HUMAN Peptidyl-prolyl cis-trans isomerase A P02144 MYG_HUMAN MyoglobinP02741 CRP_HUMAN C-reactive protein Q15843 NEDD8_HUMAN NeddylinP00915 CAH1_HUMAN Carbonic anhydrase 1 P01127 PDGFB_HUMAN Platelet-derived growth factor B-chnP08311 CATG_HUMAN Cathepsin G Q06830 PRDX1_HUMAN Peroxiredoxin 1P01031 CO5_HUMAN Complement C5 P16083 NQO2_HUMAN NRH dehydrogenase [quinone] 2P00918 CAH2_HUMAN Carbonic anhydrase 2 P02753 RETBP_HUMAN Retinol-binding proteinP07339 CATD_HUMAN Cathepsin D P01112 RASH_HUMAN GTPase HRasP06732 KCRM_HUMAN Creatine kinase M-type P63165 SUMO1_HUMAN Small ubiquitin-related modifier 1P15559 NQO1_HUMAN Quinone reductase 1 P00441 SODC_HUMAN Superoxide dismutase [Cu-Zn]P01133 EGF_HUMAN Pro-epidermal growth factor P02787 TRFE_HUMAN SerotransferrinP05413 FABPH_HUMAN Fatty acid-binding protein P01375 TNFA_HUMAN Tumor necrosis factorP06396 GELS_HUMAN Gelsolin P10599 THIO_HUMAN ThioredoxinP08263 GSTA1_HUMAN Glutathione S-transferase A1 P10636 TAU_HUMAN Microtubule-associated protein tauP09211 GSTP1_HUMAN Glutathione S-transferase P P62988 UBIQ_HUMAN Ubiquitin

P69905 HBA_HUMAN Hemoglobin alpha chain P51965 UB2E1_HUMAN Ubiquitin-conjugating enz E2 E1P68871 HBB_HUMAN Hemoglobin beta chain P63279 UBE2I_HUMAN Ubiquitin carrier protein 9P10145 IL8_HUMAN Interleukin-8 O00762 UBE2C_HUMAN Ubiquitin-conjugating enzyme E2 CP01344 IGF2_HUMAN Insulin-like growth factor II

ABRF


ABRF


Source Entry Accession Total Unique

# ofLabs

7770

8v29

504v

7207

965

215

9816

658

941

2911

527

960v

1701

742

457

1287

446

013

0715

2608

192

616

2245

521

079

1008

537

266

9733

0v53

908

7752

674

643

6256

227

406

0014

422

069

9850

640

003

2551

906

511

1499

710

6200

011

641

5195

854

601

9174

172

791

9675

105

013

6908

926

402

1898

400

700

8165

8834

8723

258

4788

620

4763

1v53

017v

7078

829

105

2101

310

266v

7148

914

474

3850

158

696

F475

501

903

2862

910

812

3196

053

178

2134

7943

691

7418

720

014

1389

532

344

3956

339

440

1508

6691

919

3218

1R

0001

RR

0002

RR

0003

RR

0004

RR

0005

R

sPRG2006 Proteins: 48 48 47 46 46 46 46 45 45 45 45 44 44 44 44 43 43 43 43 42 42 41 41 40 40 40 38 37 37 37 37 36 36 36 36 36 35 35 34 34 33 32 32 32 31 30 30 30 30 29 29 27 27 26 26 25 24 24 24 24 23 21 19 19 18 17 16 15 9 6 5 4 1 0 49 47 49 48 48sPRG2006 Proteins Unique Peptides: 808 497 883 646 576 568 562 712 702 547 442 520 477 349 419 631 347 222 306 261 340 185 381 462 219 570 195 160 118 177 338 190 36 221 198 290 328 410 224 235 109 80 253 100 337 156 158 122 371 111 213 222 383 128 95 283 317 191 70 162 311 77 137 179 18 156 193 117 101 13 5 23 1 0 381 408 317 390 645

sPRG Observed Median Unique: 12 6 13 9 9 10 9 11 12 10 7 9 8 6 6 10 5 4 5 5 6 3 6 8 5 8 4 3 2 4 7 4 1 4 4 5 8 8 5 5 2 1 7 3 6 4 4 4 7 3 6 7 8 4 2 8 5 6 2 4 12 3 6 9 1 9 11 7 12 2 1 6 1 0 6 6 4 6 8Epithelial & Ig's Mean Unique 17 10 19 14 13 12 12 16 16 12 10 12 11 8 10 15 8 5 7 6 8 5 9 12 5 14 5 4 3 5 9 5 1 6 6 8 9 12 7 7 3 3 8 3 11 5 5 4 12 4 7 8 14 5 4 11 13 8 3 7 14 4 7 9 1 9 12 8 11 2 1 6 1 0 8 9 6 8 13

Incorrect (Only lab): 0 0 5 0 0 0 8 0 0 0 0 0 0 1 6 1 2 2 3 2 4 0 2 5 6 12 1 0 3 7 8 0 0 1 2 17 0 17 0 6 3 0 2 4 3 0 3 6 9 0 4 0 8 4 22 0 2 7 11 25 1 3 0 1 20 7 1 3 4 1 4 17 11 32 0 0 0 0 0

sPRG50 ALBU_HUMAN P02768 2102 76 55 53 70 55 50 49 46 67 60 42 48 39 38 36 42 69 51 26 20 26 31 28 39 36 14 40 17 20 10 14 41 15 1 14 15 13 20 39 24 36 12 7 21 8 46 8 25 8 48 11 24 10 63 8 12 13 34 25 7 20 24 12 11 19 1 8 10 12 12 6 8 26 28 27 26 53CATA_HUMAN P04040 1153 74 41 16 41 26 28 27 26 38 25 30 23 26 22 16 23 26 15 12 21 12 15 10 20 23 3 25 10 9 6 12 21 12 1 18 8 23 14 21 19 11 8 5 16 4 21 7 3 7 14 5 9 10 6 5 7 15 28 14 7 7 17 3 11 7 1 10 10 11 1 13 21 11 16 48ANT3_HUMAN P01008 1100 74 32 21 39 27 22 12 25 31 35 29 21 19 22 14 17 23 11 12 21 9 12 5 21 23 12 27 10 10 5 4 17 13 1 16 9 20 18 26 10 10 6 4 10 4 20 10 5 5 19 6 11 13 16 8 6 19 20 15 3 6 18 10 18 1 13 13 8 15 1 12 15 12 15 32GELS_HUMAN P06396 1190 73 42 24 40 36 25 34 39 34 31 28 26 26 28 19 18 42 10 10 17 13 14 7 17 28 8 23 6 5 6 3 14 12 1 14 15 20 25 22 7 1 5 3 14 6 8 10 7 5 18 3 9 11 22 9 5 23 22 9 8 20 11 11 15 12 11 8 13 1 14 21 15 15 36TRFE_HUMAN P02787 1898 71 64 46 75 56 45 46 45 51 37 47 35 56 43 25 41 50 25 18 22 23 33 20 30 38 46 16 8 6 12 30 13 1 9 27 26 27 35 24 19 4 11 28 8 34 15 32 11 4 19 26 23 20 4 39 48 26 4 15 27 8 11 1 10 26 7 20 27 26 25 29 40SYH_HUMAN P12081 1066 70 44 22 54 22 33 13 31 30 24 33 21 25 17 18 21 31 18 11 16 13 19 3 12 26 9 27 9 5 8 7 18 5 1 9 6 14 7 27 7 2 7 1 12 1 17 6 3 4 6 4 12 18 5 5 21 36 13 2 3 14 2 14 1 16 18 29 16 7 19 36TAU_HUMAN P10636 1059 70 28 22 29 29 28 38 27 24 35 18 18 28 17 16 19 36 15 11 14 11 9 7 22 20 5 22 6 6 4 6 15 12 1 12 11 16 18 18 10 13 4 2 7 5 34 4 5 6 35 4 12 11 32 7 4 35 13 7 11 15 3 11 1 11 4 8 13 21 14 24KCRM_HUMAN P06732 1224 68 35 22 40 25 33 25 30 49 33 23 17 32 24 15 18 33 25 11 13 13 21 8 16 24 18 23 13 11 8 6 12 1 11 15 19 22 20 14 17 8 6 16 8 17 11 11 19 8 16 24 21 10 22 24 19 21 11 16 1 12 24 7 17 14 19 9 14 24GSTP1_HUMAN P09211 426 68 14 6 16 9 7 9 8 24 13 10 12 8 9 6 9 12 5 2 9 3 6 4 6 10 3 10 3 1 2 3 6 4 1 6 2 5 10 6 5 6 1 2 7 2 12 3 2 4 4 2 1 7 4 6 7 2 1 11 2 6 6 9 1 6 9 6 6 7CYB5_HUMAN P00167 322 68 8 6 13 6 6 4 7 11 10 6 6 5 5 5 6 5 4 5 4 5 6 4 6 8 2 4 4 4 2 4 4 4 1 3 5 4 5 5 7 2 1 1 5 3 2 4 3 2 6 7 6 3 2 5 1 4 5 7 4 1 5 7 5 3 4 4 3 8TRFL_HUMAN P02788 1252 67 46 51 45 45 23 37 27 28 41 15 23 18 15 15 22 38 22 11 13 13 23 12 21 29 8 30 9 9 4 10 14 11 1 10 1 2 9 27 11 27 5 5 16 6 24 8 10 48 7 14 2 40 5 15 14 2 4 8 14 7 6 6 26 35 27 31 51PRDX1_HUMAN Q06830 549 67 21 11 23 13 16 13 13 14 18 16 9 11 12 7 7 14 6 4 4 7 11 5 9 13 9 14 4 3 4 6 3 1 5 3 13 11 10 6 4 3 1 10 4 4 6 2 8 4 6 5 10 6 6 3 5 2 12 15 1 12 5 9 8 7 5 7 10LEP_HUMAN P41159 353 66 8 4 13 7 8 8 8 7 16 7 7 8 7 8 4 15 6 4 5 5 4 3 7 3 5 5 3 2 3 4 6 6 1 4 6 5 7 5 4 8 3 1 1 14 4 4 5 3 3 3 4 2 2 4 2 1 5 4 4 5 7 7 5 4 3 7MYG_HUMAN P02144 439 65 17 7 22 12 19 14 10 14 8 6 11 9 6 5 8 9 4 8 4 6 3 10 9 3 7 2 3 4 4 8 3 1 4 4 5 8 5 2 4 3 10 3 1 2 10 2 3 14 9 2 1 10 6 2 9 3 5 1 14 7 7 6 3 6 12CAH2_HUMAN P00918 712 64 22 20 23 19 16 12 16 35 32 14 14 17 15 9 16 24 11 6 12 8 10 9 19 10 4 10 7 5 5 8 6 1 13 3 9 12 10 10 8 5 6 5 9 5 13 5 11 3 24 4 7 5 1 9 6 8 9 6 2 11 15 14 14 15CAH1_HUMAN P00915 554 64 14 12 18 13 11 12 13 23 24 15 15 13 9 9 12 14 12 5 5 8 8 6 12 10 4 12 6 5 3 10 4 1 7 3 5 8 8 10 9 2 1 10 5 2 4 14 4 6 7 13 4 7 10 1 9 1 6 7 5 7 20 4 4 13UBE2C_HUMAN O00762 428 64 17 3 17 9 11 14 13 12 11 12 9 6 12 9 8 4 6 7 6 8 8 3 11 8 6 7 6 2 4 9 3 1 8 8 5 10 3 3 5 1 4 12 2 5 2 5 3 2 5 4 8 1 1 3 9 10 1 8 1 9 5 5 6 12HBB_HUMAN P68871 425 64 14 6 13 8 12 10 11 16 15 10 7 10 11 10 12 6 3 8 6 6 4 9 12 4 4 2 8 7 4 1 6 5 8 8 4 7 8 1 4 7 3 1 2 2 4 8 4 3 7 2 1 8 5 3 7 4 4 8 1 8 8 7 7 11UBIQ_HUMAN P62988 493 63 10 5 10 10 3 6 7 19 24 10 5 9 4 12 1 4 3 4 4 3 6 90 4 3 1 2 5 1 3 6 4 6 7 3 12 3 3 7 2 3 4 2 5 6 17 1 2 1 5 47 8 5 5 5 16 3 2 5 5 7 4 7 7BID_HUMAN P55957 383 63 11 3 13 8 7 13 6 12 14 7 12 6 9 8 17 10 6 6 6 5 5 7 4 5 7 3 5 4 6 11 8 1 8 3 5 10 3 4 6 2 1 1 10 4 4 4 3 3 6 1 1 6 4 3 4 1 7 5 6 8 6 3 6HBA_HUMAN P69905 288 63 11 7 9 6 7 6 10 6 14 9 7 4 5 4 5 7 5 3 7 1 4 2 10 5 5 6 3 2 1 4 8 3 1 4 4 2 4 4 3 1 2 2 3 1 6 2 2 3 5 1 4 2 1 2 6 9 4 4 6 3 1 5 5CATD_HUMAN P07339 521 62 16 8 23 14 13 13 11 21 12 13 9 12 12 14 9 10 7 3 7 7 10 1 8 10 9 13 6 4 2 3 12 3 1 2 4 11 12 10 5 2 2 8 5 1 2 4 4 5 7 7 13 3 6 12 11 1 7 14 12 12 11 12CATG_HUMAN P08311 452 62 15 8 14 14 15 11 13 15 14 10 9 15 15 6 4 8 7 6 2 5 9 2 5 9 3 7 5 2 4 5 8 3 1 4 2 4 12 5 2 2 7 3 10 7 3 4 2 6 11 2 6 2 1 11 1 7 11 7 13 10 12 16PPIA_HUMAN P62937 378 61 13 9 12 14 14 11 9 17 11 11 9 11 7 7 10 15 6 2 6 4 5 2 5 5 12 7 2 2 3 10 1 6 7 5 7 10 2 2 4 1 9 1 2 2 3 6 2 2 6 3 1 6 1 4 4 1 6 5 4 7 7RASH_HUMAN P01112 422 60 16 10 19 14 12 12 11 11 12 11 11 11 13 7 9 12 10 4 8 6 6 4 9 8 4 12 5 1 4 4 4 1 2 2 6 7 12 4 5 2 2 6 3 5 4 3 3 3 1 8 9 4 1 9 1 11 6 4 8 10LYSC_HUMAN P61626 255 58 11 8 10 10 10 7 6 14 5 7 4 9 7 2 5 5 2 4 3 3 2 2 7 5 4 2 2 1 5 1 1 1 4 4 4 2 2 1 1 4 3 1 2 11 1 1 5 2 9 2 1 3 1 5 2 4 11 4GSTA1_HUMAN P08263 368 57 18 4 28 12 16 13 12 9 5 12 2 13 15 3 6 4 1 2 2 5 4 1 13 9 7 5 2 3 7 4 1 4 6 8 1 3 4 4 4 3 2 5 10 4 2 11 7 2 1 10 9 1 3 8 3 8 7SODC_HUMAN P00441 247 57 12 6 8 9 6 10 8 5 4 7 7 4 4 4 12 6 1 1 5 2 1 5 7 5 2 16 5 1 1 1 3 3 4 6 4 1 1 1 3 2 6 1 2 2 1 8 1 2 5 1 4 4 5 1 3 2 6NEDD8_HUMAN Q15843 181 57 6 6 6 5 2 6 6 4 2 5 2 2 5 3 3 8 3 3 3 2 3 1 3 5 3 1 2 1 2 6 1 1 2 4 3 3 2 1 2 3 3 1 6 1 2 7 2 1 2 3 6 1 2 2 4 3 4ANXA5_HUMAN P08758 438 55 21 5 26 7 12 5 9 11 10 20 6 9 9 3 17 3 5 1 3 2 4 2 4 9 7 16 3 6 2 1 2 4 7 8 11 1 6 5 1 7 5 12 2 10 13 2 19 1 10 12 11 12 7 11 21CO5_HUMAN P01031 159 52 10 4 4 8 3 4 5 6 5 4 3 3 2 11 4 1 1 3 2 2 2 4 2 3 2 1 1 3 1 1 1 2 1 11 1 1 1 2 1 5 2 1 7 1 1 1 4 1 2 1 4 3PDGFB_HUMAN P01127 259 50 12 4 11 7 9 10 2 6 13 9 5 6 6 5 4 8 5 4 3 9 5 5 4 6 2 3 1 2 8 2 5 3 3 2 1 6 1 4 1 1 13 8 1 4 3 4 3 6 8 6UBE2I_HUMAN P63279 241 49 11 4 12 10 9 2 8 6 11 10 5 8 7 5 5 3 3 5 7 1 3 3 6 4 8 4 2 2 3 4 2 3 7 4 1 1 2 5 4 4 7 2 2 3 3 5 4 5 6THIO_HUMAN P10599 169 49 6 2 7 8 6 8 4 4 7 6 5 4 4 4 2 3 2 1 5 2 2 4 4 1 1 6 1 1 2 1 3 6 1 1 3 1 1 2 1 2 1 5 1 4 5 3 5 3 8NQO2_HUMAN P16083 208 47 11 8 14 9 2 9 5 5 13 5 5 1 4 5 1 9 3 4 3 2 1 7 7 3 4 1 2 2 4 1 1 2 3 2 1 1 1 3 5 5 2 2 6 6 4 4 10CRP_HUMAN P02741 147 42 7 3 7 5 4 2 3 4 6 8 3 7 3 3 2 2 1 3 3 4 1 3 5 3 5 3 1 1 4 1 3 5 2 1 3 2 1 5 3 3 4 8SUMO1_HUMAN P63165 133 42 6 3 4 6 6 4 3 4 3 3 3 2 5 2 2 3 1 1 2 4 1 6 4 3 3 1 1 2 1 1 2 2 3 7 1 2 5 2 4 3 5 7NQO1_HUMAN P15559 184 39 10 8 15 7 5 6 9 9 1 4 1 4 4 4 1 1 3 2 2 4 8 4 7 4 3 1 3 1 5 4 3 4 1 1 6 8 2 4 15B2MG_HUMAN P61769 113 39 6 4 1 8 2 5 4 3 4 4 3 3 2 2 2 2 4 3 1 4 4 2 1 3 1 2 1 1 2 3 1 4 11 1 1 1 2 2 3IGF1A_HUMAN P01343 106 35 7 2 4 5 5 9 5 5 2 4 1 4 1 1 1 4 3 3 4 3 1 1 4 1 3 1 1 6 1 1 4 3 2 2 2RETBP_HUMAN P02753 122 34 9 4 6 9 5 4 5 5 2 5 5 6 2 3 2 2 2 2 3 1 2 2 1 2 2 2 3 4 1 1 7 3 4 6IL8_HUMAN P10145 85 34 3 4 4 6 3 4 3 3 6 3 1 2 1 2 1 1 1 3 2 1 4 1 1 1 2 3 1 2 6 1 2 1 4 2IGF2_HUMAN P01344 85 33 3 3 1 3 2 4 2 3 2 3 9 2 1 2 1 1 2 3 1 1 3 4 3 1 4 2 1 7 2 1 2 3 3EGF_HUMAN P01133 97 30 6 2 8 6 1 3 9 1 1 1 14 1 1 1 2 1 1 1 1 2 1 1 1 2 22 2 1 1 1 2CYC_HUMAN P99999 79 26 4 2 6 3 5 3 1 5 5 1 4 2 2 1 4 1 5 3 5 1 1 2 4 1 2 6FABPH_HUMAN P05413 107 24 6 5 11 4 2 2 8 3 6 2 2 1 1 6 7 4 1 1 4 7 5 4 6 9LALBA_HUMAN P00709 40 19 6 2 5 2 2 2 5 1 2 1 1 1 1 1 2 1 2 1 2TNFA_HUMAN P01375 33 15 1 1 2 6 1 3 3 2 2 4 2 1 1 2 2UB2E1_HUMAN P51965 40 14 3 2 6 1 3 2 2 1 3 4 3 2 4 4

RG ObseA1AG1_HUMAN P02763 92 31 6 6 8 8 4 1 3 4 6 3 2 1 5 1 1 3 1 2 6 1 1 2 1 1 1 1 1 5 5 1 1RG ObseTETN_HUMAN P05452 84 29 3 2 12 2 2 5 4 6 3 1 2 2 1 4 1 5 2 1 1 1 2 6 2 2 1 3 1 6 1RG ObseSBP1_HUMAN Q13228 97 20 6 14 2 2 11 3 4 2 5 3 8 1 6 1 5 5 3 1 2 13RG ObseHBD_HUMAN P02042 62 15 10 8 7 3 1 1 1 11 3 1 5 6 2 1 2Epith K2C1_HUMAN P04264 149 11 8 10 2 22 17 11 10 47 12 3 7

IGF1B_HUMAN P05019 39 10 7 5 4 8 1 3 4 5 1 1Epith K1C10_HUMAN P13645 87 9 6 7 8 13 10 27 2 7 7Epith K1C9_HUMAN P35527 86 8 8 3 14 6 14 29 1 11RG ObseGST26_SCHJA P08515 73 7 9 14 6 7 16 11 10Epith DESP_HUMAN P15924 54 7 15 13 5 14 4 2 1RG ObseKAIN_HUMAN P29622 24 6 11 3 2 4 2 2

CO8G_HUMAN P07360 16 6 4 3 3 1 2 3GSTA2_HUMAN P09210 15 6 5 2 1 4 2 1

RG ObseK22E_HUMAN P35908 33 5 4 2 13 2 12RG ObseMUC_HUMAN P01871 20 5 9 2 1 6 2Ig IGHA1_HUMAN P01876 14 5 6 1 2 4 1

RASN_HUMAN P01111 11 4 2 2 6 1RG ObseCAP7_HUMAN P20160 4 4 1 1 1 1Epith HORN_HUMAN Q86YZ3 16 3 6 6 4

PRDX2_HUMAN P32119 12 3 2 2 8RASK_HUMAN P01116 11 3 6 2 3

RG ObseKAC_HUMAN P01834 9 3 4 3 2SYHH_HUMAN P49590 8 3 2 1 5

Epith DCD_HUMAN P81605 7 3 3 3 1VPS11_HUMAN Q9H270 6 3 1 3 2HSPB1_HUMAN P04792 6 3 2 1 3Q92468_HUMAN Q92468 4 3 2 1 1S100P_HUMAN P25815 3 3 1 1 1

Epith Q8N175_HUMANQ8N175 41 2 22 19FETA_HUMAN P02771 26 2 20 6CO3_HUMAN P01024 24 2 6 18TYRO3_HUMAN Q06418 21 2 2 19RASH_MSVNS P23175 16 2 8 8

Epith PLAK_HUMAN P14923 15 2 11 4Epith K2C6A_HUMAN P02538 12 2 7 5

UBE2I_MESAU O09181 10 2 7 3Q566M9_HUMANQ566M9 9 2 8 1S10A9_HUMAN P06702 8 2 5 3APOH_HUMAN P02749 7 2 6 1SPTN5_HUMAN Q9NRC6 6 2 3 3Q567Q0_HUMANQ567Q0 6 2 5 1

RG ObseBLVRB_HUMAN P30043 6 2 4 2THIO_PONPY Q5R9M3 5 2 3 2PHF7_HUMAN Q9BWX1 5 2 1 4GSTA3_HUMAN Q16772 4 2 3 1Q14015_HUMAN Q14015 4 2 3 1LEG8_HUMAN O00214 3 2 1 2GSTA5_HUMAN Q7RTV2 3 2 2 1CALL5_HUMAN Q9NZT1 3 2 1 2APOA1_HUMAN P02647 3 2 2 1A1AG2_HUMAN P19652 3 2 2 1PADI1_HUMAN Q9ULC6 2 2 1 1NGF_HUMAN P01138 2 2 1 1KPYM_HUMAN P14618 2 2 1 1KCRB_HUMAN P12277 2 2 1 1

Ig HS90A_HUMAN P07900 2 2 1 1CO4_HUMAN P01028 2 2 1 1MADI_HUMAN O95405 29 1 29

Epith K2M2_SHEEP P15241 26 1 26Q7Z6L1_HUMAN Q7Z6L1 25 1 25ZO2_HUMAN Q9UDY2 24 1 24POL_HV2KR Q74120 24 1 24NALP1_HUMAN Q9C000 23 1 23IRF5_HUMAN Q13568 22 1 22Y143_HUMAN Q14156 21 1 21

Epith Q4VAQ2_HUMANQ4VAQ2 21 1 21KCRM_PIG Q45EW9 21 1 21AACT_DICDI P05095 21 1 21ZN519_HUMAN Q8TB69 20 1 20UBR1_HUMAN Q8IWV7 20 1 20UVRA_BORBU O51777 19 1 19SPOT_MYCPN P75386 19 1 19PMPC_CHLTR O84419 19 1 19NFRA_ECOLI P31600 18 1 18STX3_HUMAN Q13277 17 1 17RRPO_ROTPC P26190 17 1 17Q9BVL8_HUMANQ9BVL8 17 1 17Q9NZJ2_HUMANQ9NZJ2 16 1 16Q6TN97_HUMANQ6TN97 16 1 16Q53HR9_HUMANQ53HR9 16 1 16ACCN1_HUMAN Q16515 16 1 16SIAT6_HUMAN Q11203 15 1 15CETP_HUMAN P11597 15 1 15UBIQ_CHICK P62973 13 1 13SGK2_HUMAN Q9HBY8 13 1 13EMIL1_HUMAN Q9Y6C2 13 1 13Q8N872_HUMANQ8N872 12 1 12Q8IUN5_HUMAN Q8IUN5 12 1 12Q24575_DROME Q24575 12 1 12PHYIP_HUMAN Q92561 12 1 12PDIA1_HUMAN P07237 12 1 12DYN1_HUMAN Q05193 12 1 12APOE_HUMAN P02649 12 1 12TTHY_HUMAN P02766 11 1 11S3TC1_HUMAN Q8TE82 11 1 11

Ig Q6PIL0_HUMAN Q6PIL0 11 1 11Q5IST6_MACFA Q5IST6 11 1 11PPIA_PAPAN P62941 11 1 11PE2R3_HUMAN P43115 11 1 11RS27A_HUMAN P14978 10 1 10Q5JR20_HUMAN Q5JR20 10 1 10NT5_HUMAN P34130 10 1 10MYLK2_RABIT P07313 10 1 10

Epith K2C5_HUMAN P13647 10 1 10Epith K1C18_HUMAN P05783 10 1 10

A1AT_HUMAN P01009 10 1 10(Sarcoma Oncoge(na) 10 1 10Q96MT1_HUMANQ96MT1 9 1 9Q96M97_HUMANQ96M97 9 1 9Q7Z505_HUMAN Q7Z505 9 1 9Q7L8K4_HUMAN Q7L8K4 9 1 9Q6ZW98_HUMANQ6ZW98 9 1 9Q6UIR8_MACMUQ6UIR8 9 1 9Q4KLL6_RAT Q4KLL6 9 1 9M10L1_HUMAN Q9BXT6 9 1 9EFNA4_HUMAN P52798 9 1 9ZBT12_HUMAN Q9Y330 8 1 8STK3_HUMAN Q13188 8 1 8SPTN4_HUMAN Q9H254 8 1 8Q9BZ64_HUMANQ9BZ64 8 1 8PTGDS_HUMAN P41222 8 1 8PPIA_CRIGR P14851 8 1 8OTC_HUMAN P00480 8 1 8CLUS_HUMAN P10909 8 1 8CAN2_HUMAN P17655 8 1 8ATP7A_HUMAN Q04656 8 1 8UBIQ_DROME P68198 7 1 7SYNE2_HUMAN Q8WXH0 7 1 7RNF17_HUMAN Q9BXT8 7 1 7RASH_MOUSE Q61411 7 1 7

Ig Q9UL77_HUMANQ9UL77 7 1 7Q9BZW7_HUMANQ9BZW7 7 1 7Q9BZ55_HUMANQ9BZ55 7 1 7Q96AA2_HUMANQ96AA2 7 1 7Q6ZNM7_HUMANQ6ZNM7 7 1 7

Ig Q6GMW7_HUMAQ6GMW7 7 1 7Epith KRHB6_HUMAN O43790 7 1 7

KAD5_HUMAN Q9Y6K8 7 1 7COX2_HUMAN P00403 7 1 7CAS1_BOVIN P02662 7 1 7BPAEA_HUMAN Q94833 7 1 7TAGL2_HUMAN P37802 6 1 6SRPX_HUMAN P78539 6 1 6Q9BR65_HUMANQ9BR65 6 1 6Q7JPZ2_DROMEQ7JPZ2 6 1 6

Epith Q15151_HUMAN Q15151 6 1 6MAP2_HUMAN P11137 6 1 6CSAD_HUMAN Q9Y600 6 1 6CAB39_HUMAN Q9Y376 6 1 6UB2E2_HUMAN Q96LR5 5 1 5TAF5_HUMAN Q15542 5 1 5Q9NS87_HUMANQ9NS87 5 1 5Q8WZ42_HUMANQ8WZ42 5 1 5Q3B792_HUMAN Q3B792 5 1 5PRDX4_HUMAN Q13162 5 1 5PPB1_HUMAN P05187 5 1 5PPAC_HUMAN P24666 5 1 5NEDD8_MOUSE P29595 5 1 5MKL1_HUMAN Q969V6 5 1 5LIFR_HUMAN P42702 5 1 5LAM1_HUMAN P20700 5 1 5

Epith K1C14_HUMAN P02533 5 1 5Ig IGHG1_HUMAN P01857 5 1 5

HBB2_PANLE P18988 5 1 5E41L2_HUMAN O43491 5 1 5

Epith DSG1_HUMAN Q02413 5 1 5CSF1_HUMAN P09603 5 1 5C560_HUMAN Q99643 5 1 5ANF_HUMAN P01160 5 1 5TAGL3_HUMAN Q9UI15 4 1 4SCCA1_HUMAN P29508 4 1 4Q8NF33_HUMANQ8NF33 4 1 4

Epith Q6EIY9_CANFA Q6EIY9 4 1 4Q5T937_HUMAN Q5T937 4 1 4Q5T794_HUMAN Q5T794 4 1 4Q5JUW8_HUMANQ5JUW8 4 1 4Q5D862_HUMANQ5D862 4 1 4Q4R4P0_MACFA Q4R4P0 4 1 4Q2YDB4_HUMANQ2YDB4 4 1 4Q14215_HUMAN Q14215 4 1 4O60744_HUMAN O60744 4 1 4NALP7_HUMAN Q8WX94 4 1 4MYOZ3_HUMAN Q8TDC0 4 1 4

Epith KRA3A_SHEEP P02443 4 1 4Epith K2C6B_HUMAN P04259 4 1 4

HTF4_HUMAN Q99081 4 1 4HBA_BOVIN P01966 4 1 4G6PI_HUMAN P06744 4 1 4G3P2_HUMAN P04406 4 1 4F19A3_HUMAN Q7Z5A8 4 1 4DDG_ECOLI P76522 4 1 4CYC_EQUBU P68096 4 1 4CDC2_HUMAN P06493 4 1 4ACTB_HUMAN P60709 4 1 4A1BG_HUMAN P04217 4 1 41433G_HUMAN P61981 4 1 4ZN189_HUMAN O85720 3 1 3VILI_HUMAN P09327 3 1 3TOP1_HUMAN P11387 3 1 3SPTA2_HUMAN Q13813 3 1 3S106_HUMAN P06703 3 1 3Q8WYN9_HUMA Q8WYN9 3 1 3Q862G1_BOVIN Q862G1 3 1 3Q5T0X9_HUMANQ5T0X9 3 1 3

Ig Q5EFE6_HUMANQ5EFE6 3 1 3Ig Q567P1_HUMAN Q567P1 3 1 3

Q17001_ARTSF Q17001 3 1 3PCD15_HUMAN Q96QU1 3 1 3PARG_HUMAN Q86W56 3 1 3O76045_HUMAN O76045 3 1 3HNRPQ_HUMAN O60506 3 1 3HBG2_HUMAN P69892 3 1 3HBE_HUMAN P02100 3 1 3H13_HUMAN P16402 3 1 3GPIX_HUMAN P14770 3 1 3CYTC_HUMAN P01034 3 1 3CE005_HUMAN Q9NYF5 3 1 3BICD1_HUMAN Q96G01 3 1 3ACTA_HUMAN P62736 3 1 3Y0182_HUMAN Q14687 2 1 2TTC3_HUMAN P53804 2 1 2TNFA_CAPHI P13296 2 1 2SODC_PONPY Q8HXQ4 2 1 2SMD_HUMAN P62318 2 1 2S10A8_HUMAN P05109 2 1 2RETBP_CHICK P41263 2 1 2

Ig Q96BB9_HUMANQ96BB9 2 1 2Q8WYN8_HUMA Q8WYN8 2 1 2Q8NA76_HUMANQ8NA76 2 1 2Q6JPJ6_9BACT Q6JPJ6 2 1 2

Ig Q6GMY2_HUMANQ6GMY2 2 1 2Ig Q6GMW4_HUMAQ6GMW4 2 1 2

Q569L2_HUMAN Q569L2 2 1 2Q53SJ7_HUMAN Q53SJ7 2 1 2Q53GA7_HUMANQ53GA7 2 1 2Q4ZG21_HUMANQ4ZG21 2 1 2Q4ZG02_HUMANQ4ZG02 2 1 2Q15670_HUMAN Q15670 2 1 2Q01720_HUMAN Q01720 2 1 2PLMN_HUMAN P00747 2 1 2PKN3_HUMAN Q6P5Z2 2 1 2OXRP_HUMAN Q9Y4L1 2 1 2OTOF_HUMAN Q9HC10 2 1 2NMDE1_HUMAN Q12879 2 1 2MYH10_HUMAN P35580 2 1 2MBP_HUMAN P02686 2 1 2MACF1_HUMAN Q9UPN3 2 1 2LEG7_HUMAN P47929 2 1 2

Epith KAP61_SHEEP Q02958 2 1 2HYEP_HUMAN P07099 2 1 2CO1A1_CANFA Q9XSJ7 2 1 2CDKL5_HUMAN O76039 2 1 2CDC5L_HUMAN Q99459 2 1 2BIG2_HUMAN Q9Y6D5 2 1 2ANK3_HUMAN Q12955 2 1 2XYLT1_HUMAN Q86Y38 1 1 1VMAT2_HUMAN Q05940 1 1 1UTRO_HUMAN P46939 1 1 1

sPRG2006Protein

Identification Legend

1 - 23 - 5> 5

Labs

sPRG2006(49)419

Proteins identifiedby only 1 lab(362) (86%)

Proteins identifiedby 2 or more labs

(58) (14%)

www.abrf.org/sprg

ABRF


Full sPRG2006 HeatMap available at www.abrf.org/sprg

Source Entry Accession Total Unique

# ofLabs

7770

8v29

504v

7207

965

215

9816

658

941

2911

527

960v

1701

742

457

1287

446

013

0715

2608

192

616

2245

521

079

1008

537

266

9733

0v53

908

7752

674

643

6256

227

406

0014

422

069

9850

640

003

2551

906

511

1499

710

6200

011

641

5195

854

601

9174

172

791

9675

105

013

6908

926

402

1898

400

700

8165

8834

823

258

4788

620

4763

1v53

017v

7078

829

105

2101

310

266v

7148

914

474

3850

158

696

F475

501

903

2862

910

812

3196

053

178

2134

7943

691

7418

720

014

1389

532

344

3956

339

440

1508

6691

919

3218

1R

0001

RR

0002

RR

0003

RR

0004

RR

0005

R

sPRG2006 Proteins: 48 48 47 46 46 46 46 45 45 45 45 44 44 44 44 43 43 43 43 42 42 41 41 40 40 40 38 37 37 37 37 36 36 36 36 36 35 35 34 34 33 32 32 32 31 30 30 30 30 29 29 27 27 26 26 25 24 24 24 24 23 21 19 19 18 17 16 15 9 6 5 4 1 0 49 47 49 48 48Unique Peptides: 808 497 883 646 576 568 562 712 702 547 442 520 477 349 419 631 347 222 306 261 340 185 381 462 219 570 195 160 118 177 338 190 36 221 198 290 328 410 224 235 109 80 253 100 337 156 158 122 371 111 213 222 383 128 95 283 317 191 70 162 311 77 137 179 18 156 193 117 101 13 5 23 1 0 381 408 317 390 645

Median Unique: 12 6 13 9 9 10 9 11 12 10 7 9 8 6 6 10 5 4 5 5 6 3 6 8 5 8 4 3 2 4 7 4 1 4 4 5 8 8 5 5 2 1 7 3 6 4 4 4 7 3 6 7 8 4 2 8 5 6 2 4 12 3 6 9 1 9 11 7 12 2 1 6 1 0 6 6 4 6 8Mean Unique 17 10 19 14 13 12 12 16 16 12 10 12 11 8 10 15 8 5 7 6 8 5 9 12 5 14 5 4 3 5 9 5 1 6 6 8 9 12 7 7 3 3 8 3 11 5 5 4 12 4 7 8 14 5 4 11 13 8 3 7 14 4 7 9 1 9 12 8 11 2 1 6 1 0 8 9 6 8 13

Incorrect (Only lab): 0 0 5 0 0 0 8 0 0 0 0 0 0 1 6 1 2 2 3 2 4 0 2 5 6 12 1 0 3 7 8 0 0 1 2 17 0 17 0 6 3 0 2 4 3 0 3 6 9 0 4 0 8 4 22 0 2 7 11 25 1 3 0 1 20 7 1 3 4 1 4 17 11 32 0 0 0 0 0

sPRG50 ALBU_HUMAN P02768 2102 76 55 53 70 55 50 49 46 67 60 42 48 39 38 36 42 69 51 26 20 26 31 28 39 36 14 40 17 20 10 14 41 15 1 14 15 13 20 39 24 36 12 7 21 8 46 8 25 8 48 11 24 10 63 8 12 13 34 25 7 20 24 12 11 19 1 8 10 12 12 6 8 26 28 27 26 53CATA_HUMAN P04040 1153 74 41 16 41 26 28 27 26 38 25 30 23 26 22 16 23 26 15 12 21 12 15 10 20 23 3 25 10 9 6 12 21 12 1 18 8 23 14 21 19 11 8 5 16 4 21 7 3 7 14 5 9 10 6 5 7 15 28 14 7 7 17 3 11 7 1 10 10 11 1 13 21 11 16 48ANT3_HUMAN P01008 1100 74 32 21 39 27 22 12 25 31 35 29 21 19 22 14 17 23 11 12 21 9 12 5 21 23 12 27 10 10 5 4 17 13 1 16 9 20 18 26 10 10 6 4 10 4 20 10 5 5 19 6 11 13 16 8 6 19 20 15 3 6 18 10 18 1 13 13 8 15 1 12 15 12 15 32GELS_HUMAN P06396 1190 73 42 24 40 36 25 34 39 34 31 28 26 26 28 19 18 42 10 10 17 13 14 7 17 28 8 23 6 5 6 3 14 12 1 14 15 20 25 22 7 1 5 3 14 6 8 10 7 5 18 3 9 11 22 9 5 23 22 9 8 20 11 11 15 12 11 8 13 1 14 21 15 15 36TRFE_HUMAN P02787 1898 71 64 46 75 56 45 46 45 51 37 47 35 56 43 25 41 50 25 18 22 23 33 20 30 38 46 16 8 6 12 30 13 1 9 27 26 27 35 24 19 4 11 28 8 34 15 32 11 4 19 26 23 20 4 39 48 26 4 15 27 8 11 1 10 26 7 20 27 26 25 29 40SYH_HUMAN P12081 1066 70 44 22 54 22 33 13 31 30 24 33 21 25 17 18 21 31 18 11 16 13 19 3 12 26 9 27 9 5 8 7 18 5 1 9 6 14 7 27 7 2 7 1 12 1 17 6 3 4 6 4 12 18 5 5 21 36 13 2 3 14 2 14 1 16 18 29 16 7 19 36TAU_HUMAN P10636 1059 70 28 22 29 29 28 38 27 24 35 18 18 28 17 16 19 36 15 11 14 11 9 7 22 20 5 22 6 6 4 6 15 12 1 12 11 16 18 18 10 13 4 2 7 5 34 4 5 6 35 4 12 11 32 7 4 35 13 7 11 15 3 11 1 11 4 8 13 21 14 24KCRM_HUMAN P06732 1224 68 35 22 40 25 33 25 30 49 33 23 17 32 24 15 18 33 25 11 13 13 21 8 16 24 18 23 13 11 8 6 12 1 11 15 19 22 20 14 17 8 6 16 8 17 11 11 19 8 16 24 21 10 22 24 19 21 11 16 1 12 24 7 17 14 19 9 14 24GSTP1_HUMAN P09211 426 68 14 6 16 9 7 9 8 24 13 10 12 8 9 6 9 12 5 2 9 3 6 4 6 10 3 10 3 1 2 3 6 4 1 6 2 5 10 6 5 6 1 2 7 2 12 3 2 4 4 2 1 7 4 6 7 2 1 11 2 6 6 9 1 6 9 6 6 7CYB5_HUMAN P00167 322 68 8 6 13 6 6 4 7 11 10 6 6 5 5 5 6 5 4 5 4 5 6 4 6 8 2 4 4 4 2 4 4 4 1 3 5 4 5 5 7 2 1 1 5 3 2 4 3 2 6 7 6 3 2 5 1 4 5 7 4 1 5 7 5 3 4 4 3 8TRFL_HUMAN P02788 1252 67 46 51 45 45 23 37 27 28 41 15 23 18 15 15 22 38 22 11 13 13 23 12 21 29 8 30 9 9 4 10 14 11 1 10 1 2 9 27 11 27 5 5 16 6 24 8 10 48 7 14 2 40 5 15 14 2 4 8 14 7 6 6 26 35 27 31 51PRDX1_HUMAN Q06830 549 67 21 11 23 13 16 13 13 14 18 16 9 11 12 7 7 14 6 4 4 7 11 5 9 13 9 14 4 3 4 6 3 1 5 3 13 11 10 6 4 3 1 10 4 4 6 2 8 4 6 5 10 6 6 3 5 2 12 15 1 12 5 9 8 7 5 7 10LEP_HUMAN P41159 353 66 8 4 13 7 8 8 8 7 16 7 7 8 7 8 4 15 6 4 5 5 4 3 7 3 5 5 3 2 3 4 6 6 1 4 6 5 7 5 4 8 3 1 1 14 4 4 5 3 3 3 4 2 2 4 2 1 5 4 4 5 7 7 5 4 3 7MYG_HUMAN P02144 439 65 17 7 22 12 19 14 10 14 8 6 11 9 6 5 8 9 4 8 4 6 3 10 9 3 7 2 3 4 4 8 3 1 4 4 5 8 5 2 4 3 10 3 1 2 10 2 3 14 9 2 1 10 6 2 9 3 5 1 14 7 7 6 3 6 12CAH2_HUMAN P00918 712 64 22 20 23 19 16 12 16 35 32 14 14 17 15 9 16 24 11 6 12 8 10 9 19 10 4 10 7 5 5 8 6 1 13 3 9 12 10 10 8 5 6 5 9 5 13 5 11 3 24 4 7 5 1 9 6 8 9 6 2 11 15 14 14 15CAH1_HUMAN P00915 554 64 14 12 18 13 11 12 13 23 24 15 15 13 9 9 12 14 12 5 5 8 8 6 12 10 4 12 6 5 3 10 4 1 7 3 5 8 8 10 9 2 1 10 5 2 4 14 4 6 7 13 4 7 10 1 9 1 6 7 5 7 20 4 4 13UBE2C_HUMAN O00762 428 64 17 3 17 9 11 14 13 12 11 12 9 6 12 9 8 4 6 7 6 8 8 3 11 8 6 7 6 2 4 9 3 1 8 8 5 10 3 3 5 1 4 12 2 5 2 5 3 2 5 4 8 1 1 3 9 10 1 8 1 9 5 5 6 12HBB_HUMAN P68871 425 64 14 6 13 8 12 10 11 16 15 10 7 10 11 10 12 6 3 8 6 6 4 9 12 4 4 2 8 7 4 1 6 5 8 8 4 7 8 1 4 7 3 1 2 2 4 8 4 3 7 2 1 8 5 3 7 4 4 8 1 8 8 7 7 11UBIQ_HUMAN P62988 493 63 10 5 10 10 3 6 7 19 24 10 5 9 4 12 1 4 3 4 4 3 6 90 4 3 1 2 5 1 3 6 4 6 7 3 12 3 3 7 2 3 4 2 5 6 17 1 2 1 5 47 8 5 5 5 16 3 2 5 5 7 4 7 7BID_HUMAN P55957 383 63 11 3 13 8 7 13 6 12 14 7 12 6 9 8 17 10 6 6 6 5 5 7 4 5 7 3 5 4 6 11 8 1 8 3 5 10 3 4 6 2 1 1 10 4 4 4 3 3 6 1 1 6 4 3 4 1 7 5 6 8 6 3 6HBA_HUMAN P69905 288 63 11 7 9 6 7 6 10 6 14 9 7 4 5 4 5 7 5 3 7 1 4 2 10 5 5 6 3 2 1 4 8 3 1 4 4 2 4 4 3 1 2 2 3 1 6 2 2 3 5 1 4 2 1 2 6 9 4 4 6 3 1 5 5CATD_HUMAN P07339 521 62 16 8 23 14 13 13 11 21 12 13 9 12 12 14 9 10 7 3 7 7 10 1 8 10 9 13 6 4 2 3 12 3 1 2 4 11 12 10 5 2 2 8 5 1 2 4 4 5 7 7 13 3 6 12 11 1 7 14 12 12 11 12CATG_HUMAN P08311 452 62 15 8 14 14 15 11 13 15 14 10 9 15 15 6 4 8 7 6 2 5 9 2 5 9 3 7 5 2 4 5 8 3 1 4 2 4 12 5 2 2 7 3 10 7 3 4 2 6 11 2 6 2 1 11 1 7 11 7 13 10 12 16PPIA_HUMAN P62937 378 61 13 9 12 14 14 11 9 17 11 11 9 11 7 7 10 15 6 2 6 4 5 2 5 5 12 7 2 2 3 10 1 6 7 5 7 10 2 2 4 1 9 1 2 2 3 6 2 2 6 3 1 6 1 4 4 1 6 5 4 7 7RASH_HUMAN P01112 422 60 16 10 19 14 12 12 11 11 12 11 11 11 13 7 9 12 10 4 8 6 6 4 9 8 4 12 5 1 4 4 4 1 2 2 6 7 12 4 5 2 2 6 3 5 4 3 3 3 1 8 9 4 1 9 1 11 6 4 8 10LYSC_HUMAN P61626 255 58 11 8 10 10 10 7 6 14 5 7 4 9 7 2 5 5 2 4 3 3 2 2 7 5 4 2 2 1 5 1 1 1 4 4 4 2 2 1 1 4 3 1 2 11 1 1 5 2 9 2 1 3 1 5 2 4 11 4GSTA1_HUMAN P08263 368 57 18 4 28 12 16 13 12 9 5 12 2 13 15 3 6 4 1 2 2 5 4 1 13 9 7 5 2 3 7 4 1 4 6 8 1 3 4 4 4 3 2 5 10 4 2 11 7 2 1 10 9 1 3 8 3 8 7SODC_HUMAN P00441 247 57 12 6 8 9 6 10 8 5 4 7 7 4 4 4 12 6 1 1 5 2 1 5 7 5 2 16 5 1 1 1 3 3 4 6 4 1 1 1 3 2 6 1 2 2 1 8 1 2 5 1 4 4 5 1 3 2 6NEDD8_HUMAN Q15843 181 57 6 6 6 5 2 6 6 4 2 5 2 2 5 3 3 8 3 3 3 2 3 1 3 5 3 1 2 1 2 6 1 1 2 4 3 3 2 1 2 3 3 1 6 1 2 7 2 1 2 3 6 1 2 2 4 3 4ANXA5_HUMAN P08758 438 55 21 5 26 7 12 5 9 11 10 20 6 9 9 3 17 3 5 1 3 2 4 2 4 9 7 16 3 6 2 1 2 4 7 8 11 1 6 5 1 7 5 12 2 10 13 2 19 1 10 12 11 12 7 11 21CO5_HUMAN P01031 159 52 10 4 4 8 3 4 5 6 5 4 3 3 2 11 4 1 1 3 2 2 2 4 2 3 2 1 1 3 1 1 1 2 1 11 1 1 1 2 1 5 2 1 7 1 1 1 4 1 2 1 4 3PDGFB_HUMAN P01127 259 50 12 4 11 7 9 10 2 6 13 9 5 6 6 5 4 8 5 4 3 9 5 5 4 6 2 3 1 2 8 2 5 3 3 2 1 6 1 4 1 1 13 8 1 4 3 4 3 6 8 6UBE2I_HUMAN P63279 241 49 11 4 12 10 9 2 8 6 11 10 5 8 7 5 5 3 3 5 7 1 3 3 6 4 8 4 2 2 3 4 2 3 7 4 1 1 2 5 4 4 7 2 2 3 3 5 4 5 6THIO_HUMAN P10599 169 49 6 2 7 8 6 8 4 4 7 6 5 4 4 4 2 3 2 1 5 2 2 4 4 1 1 6 1 1 2 1 3 6 1 1 3 1 1 2 1 2 1 5 1 4 5 3 5 3 8NQO2_HUMAN P16083 208 47 11 8 14 9 2 9 5 5 13 5 5 1 4 5 1 9 3 4 3 2 1 7 7 3 4 1 2 2 4 1 1 2 3 2 1 1 1 3 5 5 2 2 6 6 4 4 10CRP_HUMAN P02741 147 42 7 3 7 5 4 2 3 4 6 8 3 7 3 3 2 2 1 3 3 4 1 3 5 3 5 3 1 1 4 1 3 5 2 1 3 2 1 5 3 3 4 8SUMO1_HUMAN P63165 133 42 6 3 4 6 6 4 3 4 3 3 3 2 5 2 2 3 1 1 2 4 1 6 4 3 3 1 1 2 1 1 2 2 3 7 1 2 5 2 4 3 5 7NQO1_HUMAN P15559 184 39 10 8 15 7 5 6 9 9 1 4 1 4 4 4 1 1 3 2 2 4 8 4 7 4 3 1 3 1 5 4 3 4 1 1 6 8 2 4 15B2MG_HUMAN P61769 113 39 6 4 1 8 2 5 4 3 4 4 3 3 2 2 2 2 4 3 1 4 4 2 1 3 1 2 1 1 2 3 1 4 11 1 1 1 2 2 3IGF1A_HUMAN P01343 106 35 7 2 4 5 5 9 5 5 2 4 1 4 1 1 1 4 3 3 4 3 1 1 4 1 3 1 1 6 1 1 4 3 2 2 2RETBP_HUMAN P02753 122 34 9 4 6 9 5 4 5 5 2 5 5 6 2 3 2 2 2 2 3 1 2 2 1 2 2 2 3 4 1 1 7 3 4 6IL8_HUMAN P10145 85 34 3 4 4 6 3 4 3 3 6 3 1 2 1 2 1 1 1 3 2 1 4 1 1 1 2 3 1 2 6 1 2 1 4 2IGF2_HUMAN P01344 85 33 3 3 1 3 2 4 2 3 2 3 9 2 1 2 1 1 2 3 1 1 3 4 3 1 4 2 1 7 2 1 2 3 3EGF_HUMAN P01133 97 30 6 2 8 6 1 3 9 1 1 1 14 1 1 1 2 1 1 1 1 2 1 1 1 2 22 2 1 1 1 2CYC_HUMAN P99999 79 26 4 2 6 3 5 3 1 5 5 1 4 2 2 1 4 1 5 3 5 1 1 2 4 1 2 6FABPH_HUMAN P05413 107 24 6 5 11 4 2 2 8 3 6 2 2 1 1 6 7 4 1 1 4 7 5 4 6 9LALBA_HUMAN P00709 40 19 6 2 5 2 2 2 5 1 2 1 1 1 1 1 2 1 2 1 2TNFA_HUMAN P01375 33 15 1 1 2 6 1 3 3 2 2 4 2 1 1 2 2UB2E1_HUMAN P51965 40 14 3 2 6 1 3 2 2 1 3 4 3 2 4 4

sPRG2006 Protein IdentificationLegend: 1 - 2 3 - 5 > 5

ABRF


Proteins Identified by 2 or More Labs

Full sPRG2006 HeatMap available at www.abrf.org/sprg

Legend: 1 - 2 3 - 5 > 5

RG ObseA1AG1_HUMAN P02763 92 31 6 6 8 8 4 1 3 4 6 3 2 1 5 1 1 3 1 2 6 1 1 2 1 1 1 1 1 5 5 1 1RG ObseTETN_HUMAN P05452 84 29 3 2 12 2 2 5 4 6 3 1 2 2 1 4 1 5 2 1 1 1 2 6 2 2 1 3 1 6 1RG ObseSBP1_HUMAN Q13228 97 20 6 14 2 2 11 3 4 2 5 3 8 1 6 1 5 5 3 1 2 13RG ObseHBD_HUMAN P02042 62 15 10 8 7 3 1 1 1 11 3 1 5 6 2 1 2Epith K2C1_HUMAN P04264 149 11 8 10 2 22 17 11 10 47 12 3 7

IGF1B_HUMAN P05019 39 10 7 5 4 8 1 3 4 5 1 1Epith K1C10_HUMAN P13645 87 9 6 7 8 13 10 27 2 7 7Epith K1C9_HUMAN P35527 86 8 8 3 14 6 14 29 1 11RG ObseGST26_SCHJA P08515 73 7 9 14 6 7 16 11 10Epith DESP_HUMAN P15924 54 7 15 13 5 14 4 2 1RG ObseKAIN_HUMAN P29622 24 6 11 3 2 4 2 2

CO8G_HUMAN P07360 16 6 4 3 3 1 2 3GSTA2_HUMAN P09210 15 6 5 2 1 4 2 1

RG ObseK22E_HUMAN P35908 33 5 4 2 13 2 12RG ObseMUC_HUMAN P01871 20 5 9 2 1 6 2Ig IGHA1_HUMAN P01876 14 5 6 1 2 4 1

RASN_HUMAN P01111 11 4 2 2 6 1RG ObseCAP7_HUMAN P20160 4 4 1 1 1 1Epith HORN_HUMAN Q86YZ3 16 3 6 6 4

PRDX2_HUMAN P32119 12 3 2 2 8RASK_HUMAN P01116 11 3 6 2 3

RG ObseKAC_HUMAN P01834 9 3 4 3 2SYHH_HUMAN P49590 8 3 2 1 5

Epith DCD_HUMAN P81605 7 3 3 3 1VPS11_HUMAN Q9H270 6 3 1 3 2HSPB1_HUMAN P04792 6 3 2 1 3Q92468_HUMAN Q92468 4 3 2 1 1S100P_HUMAN P25815 3 3 1 1 1

Epith Q8N175_HUMANQ8N175 41 2 22 19FETA_HUMAN P02771 26 2 20 6CO3_HUMAN P01024 24 2 6 18TYRO3_HUMAN Q06418 21 2 2 19RASH_MSVNS P23175 16 2 8 8

Epith PLAK_HUMAN P14923 15 2 11 4Epith K2C6A_HUMAN P02538 12 2 7 5

UBE2I_MESAU O09181 10 2 7 3Q566M9_HUMANQ566M9 9 2 8 1S10A9_HUMAN P06702 8 2 5 3APOH_HUMAN P02749 7 2 6 1SPTN5_HUMAN Q9NRC6 6 2 3 3Q567Q0_HUMANQ567Q0 6 2 5 1

RG ObseBLVRB_HUMAN P30043 6 2 4 2THIO_PONPY Q5R9M3 5 2 3 2PHF7_HUMAN Q9BWX1 5 2 1 4GSTA3_HUMAN Q16772 4 2 3 1Q14015_HUMAN Q14015 4 2 3 1LEG8_HUMAN O00214 3 2 1 2GSTA5_HUMAN Q7RTV2 3 2 2 1CALL5_HUMAN Q9NZT1 3 2 1 2APOA1_HUMAN P02647 3 2 2 1A1AG2_HUMAN P19652 3 2 2 1PADI1_HUMAN Q9ULC6 2 2 1 1NGF_HUMAN P01138 2 2 1 1KPYM_HUMAN P14618 2 2 1 1KCRB_HUMAN P12277 2 2 1 1

Ig HS90A_HUMAN P07900 2 2 1 1CO4_HUMAN P01028 2 2 1 1

IGF1A_HUMAN

MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM

IGF1B_HUMAN

MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGWPKTHPGGEQKEGTEASLQIRGKKKEQRREIGSRNAECRGKKGK

IGF1A_HUMAN

GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

IGF1B_HUMAN

GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

ABRF


Isoform Issue

Revised Guideline 7.

• Identical peptide sequences can occur as elements of multiple unique protein sequences due to biological variation.

• Shotgun proteomics (proteolytic digestion of protein mixtures) introduces the complication of loss of connectivity between peptides and their protein precursors.

• Assignment of peptide sequences results in two outcomes; distinct peptides that map to only one protein sequence or shared peptides that map to more than one protein sequence.

• Detection of shared peptides introduces an uncertainty bioinformatics redundancy versus physical redundancy.

• The apparent ambiguity in peptide assignment requires reporting of a protein group.

Molecular and Cellular Proteomics WorkshopParis - May 2005

ABRF


Sean Seymour - de novo sequencing/DB searching tutorial session - Tuesday 11 am

INCORRECT PROTEINS (Non-Epithelial, Non-IgG, Proteins detected by only one lab)

MADI_HUMAN O95405 29 1 29Q7Z6L1_HUMAN Q7Z6L1 25 1 25ZO2_HUMAN Q9UDY2 24 1 24POL_HV2KR Q74120 24 1 24NALP1_HUMAN Q9C000 23 1 23IRF5_HUMAN Q13568 22 1 22Y143_HUMAN Q14156 21 1 21KCRM_PIG Q45EW9 21 1 21AACT_DICDI P05095 21 1 21ZN519_HUMAN Q8TB69 20 1 20UBR1_HUMAN Q8IWV7 20 1 20UVRA_BORBU O51777 19 1 19SPOT_MYCPN P75386 19 1 19PMPC_CHLTR O84419 19 1 19NFRA_ECOLI P31600 18 1 18STX3_HUMAN Q13277 17 1 17RRPO_ROTPC P26190 17 1 17Q9BVL8_HUMAN Q9BVL8 17 1 17Q9NZJ2_HUMAN Q9NZJ2 16 1 16Q6TN97_HUMAN Q6TN97 16 1 16Q53HR9_HUMAN Q53HR9 16 1 16ACCN1_HUMAN Q16515 16 1 16SIAT6_HUMAN Q11203 15 1 15CETP_HUMAN P11597 15 1 15UBIQ_CHICK P62973 13 1 13SGK2_HUMAN Q9HBY8 13 1 13EMIL1_HUMAN Q9Y6C2 13 1 13Q8N872_HUMAN Q8N872 12 1 12Q8IUN5_HUMAN Q8IUN5 12 1 12Q24575_DROME Q24575 12 1 12PHYIP_HUMAN Q92561 12 1 12PDIA1_HUMAN P07237 12 1 12DYN1_HUMAN Q05193 12 1 12APOE_HUMAN P02649 12 1 12TTHY_HUMAN P02766 11 1 11S3TC1_HUMAN Q8TE82 11 1 11Q5IST6_MACFA Q5IST6 11 1 11PPIA_PAPAN P62941 11 1 11PE2R3_HUMAN P43115 11 1 11RS27A_HUMAN P14978 10 1 10Q5JR20_HUMAN Q5JR20 10 1 10NT5_HUMAN P34130 10 1 10MYLK2_RABIT P07313 10 1 10A1AT_HUMAN P01009 10 1 10(Sarcoma Oncogen (na) 10 1 10Q96MT1_HUMAN Q96MT1 9 1 9Q96M97_HUMAN Q96M97 9 1 9Q7Z505_HUMAN Q7Z505 9 1 9Q7L8K4_HUMAN Q7L8K4 9 1 9Q6ZW98_HUMAN Q6ZW98 9 1 9Q6UIR8_MACMU Q6UIR8 9 1 9Q4KLL6_RAT Q4KLL6 9 1 9M10L1_HUMAN Q9BXT6 9 1 9EFNA4_HUMAN P52798 9 1 9ZBT12_HUMAN Q9Y330 8 1 8STK3_HUMAN Q13188 8 1 8SPTN4_HUMAN Q9H254 8 1 8Q9BZ64_HUMAN Q9BZ64 8 1 8PTGDS_HUMAN P41222 8 1 8PPIA_CRIGR P14851 8 1 8OTC_HUMAN P00480 8 1 8CLUS_HUMAN P10909 8 1 8CAN2_HUMAN P17655 8 1 8ATP7A_HUMAN Q04656 8 1 8UBIQ_DROME P68198 7 1 7SYNE2_HUMAN Q8WXH0 7 1 7RNF17_HUMAN Q9BXT8 7 1 7RASH_MOUSE Q61411 7 1 7Q9BZW7_HUMAN Q9BZW7 7 1 7Q9BZ55_HUMAN Q9BZ55 7 1 7Q96AA2_HUMAN Q96AA2 7 1 7Q6ZNM7_HUMAN Q6ZNM7 7 1 7KAD5_HUMAN Q9Y6K8 7 1 7COX2_HUMAN P00403 7 1 7CAS1_BOVIN P02662 7 1 7BPAEA_HUMAN Q94833 7 1 7TAGL2_HUMAN P37802 6 1 6SRPX_HUMAN P78539 6 1 6Q9BR65_HUMAN Q9BR65 6 1 6Q7JPZ2_DROME Q7JPZ2 6 1 6MAP2_HUMAN P11137 6 1 6CSAD_HUMAN Q9Y600 6 1 6CAB39_HUMAN Q9Y376 6 1 6

ABRF


Incorrect IDs: High Peptide CountLegend: 1 - 2 3 - 5 > 5

sPRGR0001R 49 381 6 8R0002R 47 408 6 9R0003R 49 317 4 6R0004R 48 390 6 8R0005R 48 645 8 13

Lab Proteins % Incorrect Accuracy Overall Peptides Median Mean77708 48 98% 0 100% 98% 808 11.5 16.829504 48 98% 0 100% 98% 497 6.0 10.472079 47 96% 5 90% 87% 883 13.0 18.865215 46 94% 0 100% 94% 646 9.0 14.098166 46 94% 0 100% 94% 576 9.0 12.558941 46 94% 0 100% 94% 568 9.5 12.329115 46 94% 8 85% 80% 562 8.5 12.227960 45 92% 0 100% 92% 712 11.0 15.817017 45 92% 0 100% 92% 702 12.0 15.642457 45 92% 0 100% 92% 547 10.0 12.212874 45 92% 0 100% 92% 442 7.0 9.846013 44 90% 0 100% 90% 520 8.5 11.80715 44 90% 0 100% 90% 477 8.0 10.826081 44 90% 1 98% 88% 349 5.5 7.992616 44 90% 6 88% 79% 419 5.5 9.522455 43 88% 1 98% 86% 631 10.0 14.721079 43 88% 2 96% 84% 347 5.0 8.110085 43 88% 2 96% 84% 222 4.0 5.237266 43 88% 3 93% 82% 306 5.0 7.197330 42 86% 2 95% 82% 36 5.0 6.253908 42 86% 4 91% 78% 340 5.5 8.177526 41 84% 0 100% 84% 185 3.0 4.574643 41 84% 2 95% 80% 381 6.0 9.362562 40 82% 5 89% 73% 462 8.0 11.627406 40 82% 6 87% 71% 219 4.5 5.500144 40 82% 12 77% 63% 570 7.5 14.222069 38 78% 1 97% 76% 195 4.0 5.198506 37 76% 0 100% 76% 160 3.0 4.340003 37 76% 3 93% 70% 118 2.0 3.225519 37 76% 7 84% 63% 177 4.0 4.8

Unique PeptidessPRG50 Identified

Total Correct / (Correct + Incorrect)

% Identified xAccuracy

ABRF


06511 37 76% 8 82% 62% 338 7.0 9.114997 36 73% 0 100% 73% 190 4.0 5.31062000 36 73% 0 100% 73% 36 1.0 1.011641 36 73% 1 97% 71% 221 4.0 6.151958 36 73% 2 95% 70% 198 4.0 5.554601 36 73% 17 68% 50% 290 5.0 8.191741 35 71% 0 100% 71% 328 8.0 9.472791 35 71% 17 67% 48% 410 8.0 11.796751 34 69% 0 100% 69% 224 4.5 6.605013 34 69% 6 85% 59% 235 4.5 6.969089 33 67% 3 92% 62% 109 2.0 3.326402 32 65% 0 100% 65% 80 1.0 2.518984 32 65% 2 94% 61% 253 7.0 7.900700 32 65% 4 89% 58% 100 2.5 3.18165883487 31 63% 3 91% 58% 337 6.0 10.923258 30 61% 0 100% 61% 156 4.0 5.24788620 30 61% 3 91% 56% 158 3.5 5.347631 30 61% 6 83% 51% 122 3.5 4.153017 30 61% 9 77% 47% 371 7.0 12.470788 29 59% 0 100% 59% 111 3.0 3.829105 29 59% 4 88% 52% 213 6.0 7.321013 27 55% 0 100% 55% 222 7.0 8.210266 27 55% 8 77% 43% 383 8.0 14.271489 26 53% 4 87% 46% 128 4.0 4.914474 26 53% 22 54% 29% 95 2.0 3.738501 25 51% 0 100% 51% 283 8.0 11.358696 24 49% 2 92% 45% 317 5.0 13.2F4755 24 49% 7 77% 38% 191 6.0 8.001903 24 49% 11 69% 34% 70 2.0 2.928629 24 49% 25 49% 24% 162 3.5 6.710812 23 47% 1 96% 45% 311 12.0 13.531960 21 43% 3 88% 38% 77 3.0 3.753178 19 39% 0 100% 39% 137 6.0 7.2213479 19 39% 1 95% 37% 179 9.0 9.443691 18 37% 20 47% 17% 18 1.0 1.074187 17 35% 7 71% 25% 156 9.0 9.220014 16 33% 1 94% 31% 193 11.0 12.113895 15 31% 3 83% 26% 117 7.0 7.832344 9 18% 4 69% 13% 101 12.0 11.239563 6 12% 1 86% 10% 13 1.5 2.239440 5 10% 4 56% 6% 5 1.0 1.0150866 4 8% 17 19% 1.6% 23 5.5 5.891919 1 2% 11 8% 0.2% 1 1.0 1.032181 0 0% 32 0% 0% 0 0.0 0.0

Protein Identification Accuracy

Protein Identification Performance for 74 Labs

The performance of each lab is represented by a point defined by the fraction of all known proteins identified and the fraction of all reported identifications that were correct. Those labs with no false identifications fall along the upper axis.

0.0 0.2 0.4 0.6 0.8 1.00.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of ID

s C

orre

ct

Fraction of Proteins Identified (49 total)

ABRF


Number of Proteins Identified by Respondents

0-910-19

20-29

30-39

> 40

ABRF


35%

31%

31%

8%

8%

Labs detecting few er correct proteins tended to identify more incorrect proteins

0.02.04.06.08.0

10.012.014.0

>=40 30-39 20-29 10-19 0-9

Number of sPRG50 Proteins Identif ied

Aver

age

Inco

rrec

t Pr

otei

ns Id

entif

ied

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0

5

10

15

20

25

30

35

40

45

50

Num

ber o

f Lab

s

On-line 1D LC-MS/MS

On-line 2D LC-MS/MS

PMF TOF Off-line capillaryRP-HPLC +

MALDITOF/TOF

PMF TOF/TOF Off-line capillaryHPLC + static

nanospray

No Protein Separation1D PAGE2D PAGE

ABRF


Primary Method of Analysis

0

5

10

15

20

25

Num

ber o

f Lab

s Em

ploy

ing

3D ion trapFTICR

linear ion trap-FTICR

hybrid quadupole-linear ion trap

linear ion traplinear ion trap-orbitrap

ESI-quadrupole-TOF

MALDI-linear ion trap

MALDI-quadrupole-TOF

reflectron-equipped TOF

tandem TOF

Electrospray MALDI

Mass Spectrometers UsedABRF


Identification Accuracy Using Different Experimental ApproachesEach data point represents the instrument(s) used and the accuracy achieved, where

Accuracy = total correct identifications / (correct identifications + incorrect identifications)Brackets enclose range of scores for each method; slash indicates multiple instruments

1 20 40 60 80 100Separation performed at the protein level1D PAGE/LC-ESI-MS/MS [g e d a dd d g a g e]1D PAGE/MALDI-MS [i h h h]1D PAGE/ESI and MALDI [(a/h) x (a/i)] 2D PAGE/MALDI [(i/c) i]1D Protein HPLC/ESI-MS/MS [d e]

“Shotgun protomic” approachesLC-ESI-MS/MS [dg c d df gcg g dgdgc e gga]LC-MALDI-TOF/TOF [i i i i]LC-MALDI and ESI [(i/a) (i/g)]2D LC-ESI-MS/MS [a g d a g b]

Split sample and did both protein separation and “shotgun” techniquesShotgun + 1D PAGE [g g g a x i]Combination w/ 2D PAGE [(g/i) g] Truly miscellaneous [(g/h) a x a]

a=3D ion trap, b=FTICR, c=hybrid quadrupole-linear ion trap, d=linear ion trape=linear ion trap-FTICR, f=linear ion trap-orbitrap, g=quadrupole-TOFh=reflectron-equipped TOF, i=tandem TOF, x=other combination

0

10

20

30

40

50

60

MascotSequest

X!Tandem

ProteinLynx Global Server

Spectrum Mill

PEAKS

Search Engines Used for Protein Identification from Tandem Mass Spectra

Num

ber

of L

abor

ator

ies E

mpl

oyin

g

ABRF


Number of Proteins Correctly Identified Using Each Search Engine/Combination of Engines

Each data point represents the results from an individual lab

1 10 20 30 40 50Mascot x xx x xx xxx x xx x xxxx xxxxxxxSequest x x x xxx x xProteinLynx xxSpectrum Mill x xPMF/Sequest xPMF/X!Tandem xMascot/ProteinLynx x xMascot/Sequest x xMascot/ProteinLynx/X!Tandem xMascot/Protein Prospector x Mascot/X!Tandem/PEAKS x Mascot/Sequest/PEAKS xMascot/Pro ID xMascot/X!Tandem x xSequest/X!Tandem xSequest/Other xMascot/Spectrom Mill xMascot/Sequest/X!Tandem x

ABRF


Comparison of Observed vs Expected Unique Tryptic Peptides

10 10010

100

1000

NQO1

CYCFABPH

IGF1A

TRFEALBU

UBIQ

IGF2

LALBATNFA

UB2E1

CO5

Tota

l Uni

que

Pept

ides

Number of Tryptic Peptides (>5 AAs)

EGF

For each protein in the test mixture, the total number of reported unique peptides is compared to the maximum expected number of different simple tryptic peptides. Since an effort was made to prepare the proteins in eqimolar amounts (5 pmol), if all peptides were formed and detected with equal efficiency, a straight line would result. The origin of large dispersion is not clear, but outliers at the lower right are glycoproteins (CO5_HUMAN and EFG_HUMAN) while the highest yields are from secreted proteins (ALBU_HUMAN and TRFE_HUMAN). Other protein at the extremities are also labeled.

ABRF


0

20

40

60

80

100

120

140

160

180

0 20000 40000 60000 80000 100000 120000 140000

Molecular Weight

# of

tryp

tic p

eptid

esABRF


Theoretical number of tryptic peptides vs molecular weight

10

100

1000

10000

0 10 20 30 40 50 60 70 80

Number of Labs Identifying Protein

Num

ber o

f Uni

que

Pept

ides

Iden

tifed

Unique Peptides vs Successful IDsABRF


0.00

0.10

0.20

0.30

0.40

0.50

0.60

IGF2

UN

IQ IL8

THIO

B2M

G

LALB

A

FAB

PH

CY

B5

HB

B

MY

G

UB

E2I

RE

TBP

UB

2E1

PR

DX

1

GS

TP1

TNFA

CA

TG

CA

H2

AN

XA

5

KC

RM

SY

H

ALB

U

TRFE

TAU

EG

F

Protein (> increasing molecular weight)

# of

tryp

tic p

eptid

es/#

of I

Ds

ABRF


Protein Sequence Coverage:Molecular Weight

0.00

0.10

0.20

0.30

0.40

0.50

0.60

LALB

ACYB5

CO5SUMO1

BIDGSTP1

CATDSODC

SYHNQO2B2M

GFABPH

IGF2

CAH1KCRMUBE2C

CATAIG

F1ANEDD8

TRFLUB2E

1NQO1

IL8PDGFB

CATG

Protein (> isoelectric point)

# of

tryp

tic p

eptid

es/#

of I

Ds

ABRF


Protein Sequence Coverage:Isoelectric Point

0

2

4

6

8

10

12

14

16

18

less than1 hour

1 - 4 hours 4 - 8 hours 1 day 2 days 3 days 4 days 5days 6days 1 week more than1 week

I tried but Igave up

Time

Num

ber o

f Rep

sond

ents

ABRF


Time to Prepare and Analyze

0

5

10

15

20

25

30

less than 1 hour 1 hour 2 hours 3 hours 4 hours Other (please specify)

Time

Num

ber o

f Res

pond

ents

ABRF


Time to Complete Survey

0

2

4

6

8

10

12

14

16

18

20

less than1 hour

1 - 4 hours 4 - 8 hours 1 day 2 days 3 days 4 days 5days 6days 1 week more than1 week

I tried but Igave up

Time

Num

ber o

f Res

pond

ents

ABRF


Time for Data Analysis

69

2 2 1

50

17

6

10

10

20

30

40

50

60

70

80

Yes No Not Sure NR Yes No Not Sure NR

Num

ber o

f Res

pond

ents

ABRF


Utility

0

3

3234

20

0

5

10

15

20

25

30

35

40

Mindlessly simple Easy Just Right Challenging Too Difficult Impossible

Num

ber o

f Res

pond

ents

ABRF


Level of Difficulty

Conclusions

• A standard protein mixture has been developed that has broad usefulness for a variety of proteomics strategies.

• The fact that no single approach performed better than others may suggest that success is not so much method-dependent as possibly experience-, or technique-dependent (technique of the individual performing the analysis).

• Good results are achievable by a lab which does not necessarily have the latest instruments but optimizes variables within its control.

A BR F


• Many labs can reliably identify a large fraction of proteins at these concentrations with few false positive results.

• The nature of variability expected in a semi-complex protein mixture analysis will have to be dealt with in future standards design and analysis.

• We demonstrated in a tangible way, problems resulting from the myriad representations of proteins and lack of data representation standards.

• The study has led to a publicly available set of raw data files for analysis of a known protein mixture by a number of independent labs.

Conclusions (cont.)A BR F


Lilley LabJulie HowardRenata Feret

Markey LabJim MakuskyEwa Jankowska-

StevensWenyao Shi

Martin Lab

Stein LabLisa Kilpatrick Jeri Roth Sara Yang

Andrews LabAngela Walker John Strahler Jayson Falkner

Arnott LabAlexander James Arnott

Gawinowicz LabSusan Pollak

Lane LabJohn M. NeveuRenee RobinsonMarcelo Cantu

AcknowledgementsABRF


Sigma-AldrichGraham ScottGeorge LipscombJennifer WilliamsMichael Hadjisavas

Judy BolandJohn DapronMalaika DurhamJeffrey Turner

Steven CockrillJustin WildsmithBeth GeoppoKevin RayMark Tizzard

Phil Andrews - University of Michigan

David Arnott - Genentech, Inc.

Mary Ann Gawinowicz - Columbia University

Jeffrey A. Kowalak - National Institutes of Health (Chair)

William S. Lane - Harvard University (EB Liaison)

Kathryn S. Lilley - University of Cambridge

Larry Martin - East West University

Stephen E. Stein - NIST

Proteomics Standards ResearchGroup (sPRG) Members

ABRF


THANK YOUFor participating in the ABRF 2006

Proteomics Standards Research Group Study

ABRF


proteomics standards research group sprg2006: a proteomics ... · proteomics standards research...

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