WG2 - SRM Group11-12- 2012

1616 Genes on Chromosome 16

B-lymphocyte T-lymphocyte Leukocyte Epithelium Fibroblast0

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Protein coding genes (n=862)

Entr

ies

More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.

• Characterize the transcriptome to define the actual chr16’s gen set expressed and most importantly which genes are not expressed in the three selected cell types.

• Characterize the proteome in detail. • Expression, purification, and characterization of 22 unknown proteins available in NAPPA collection.• Development of SRM quantitative assays.

Proteins (862) will be distributed in known (562) and unknown groups (300) and the study will be performed along 3 years, starting in 2012 (214 proteins).

Protein packs will be assigned to each of the 8 MRM groups (24), including equal proportion of known (20, with different range of theoretical difficulty) and unknown proteins (4).

Unknown proteins will be first searched by targeted discovery (TD) and relevant information resulting from this studies will be used to design MRM assays. TD groups (9) will analyze 54 proteins the first year.

Each group will use the master sample (this might require the use of synthetic peptides during the refinement steps). TD will be done in all three selected cell lines. Regarding MRM, a pool of the three cell lines will be used for the 20 known proteins. Decision on using a pool or a particular cell line for the unknown proteins will be taken in light of the results from TD.

Once developed, the assays will be validated by 2 additional groups. Finally, the quantitative assay will be set up using heavy peptides.

• Definition of Bioinformatic standards and SOPs.• Definition of Biobanking strategy, standards... in collaboration with the Spanish Biobanking Platform.

1616 Workplan proposal

160 known Proteins, 8 groups → 20 proteins/groupGroup SRM Results

F. Vivanco-HNP x

M. Sánchez del Pino-UV

F.J. Blanco-INIBIC x

S. Barcelò-IACS x

F. Canals-VHIO x

F.J. Corrales-CIMA x

C. Gil-UCM-PCM x

J.P. Albar-CNB

Sample types : MCF7/ CCD18/ Ramos/ HUH7/ Plasma/ TC28

Instruments: 4000 QTRAP, 5500 QTRAPColumns: PepMap100, C18, 3um 100A, 75umx15cm (3)

PepMapRSLC, C18, 2um 100A, 75umx15cmEksigent Chrome XP C18 3 µm,, 0,075 x 150 mm Onyx Monolithic C18, 150x0.1mm

Gradients: 300 nL/min 35-85 min

PROTEINS ASSIGNED - 2012

• Results for 90 proteins

• 1775 assayed peptides

• 413 observed peptides

• 157 verified peptides

PROTEINS

565

160

90 86

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600

Chr16 Assigned SRM Evaluated SRM Results

# Pr

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Template results MRM_121127.xlsx

Assigned Laboratory

Protein Uniprot

CodeGENE NAME Protein Name

# Assayed Peptides

# Observed Peptides1

# Verified peptides 2

INIBIC Q14694 USP10 Ubiquitin carboxyl-terminal hydrolase 10 25 14 10INIBIC Q6P2E9 EDC4 Enhancer of mRNA-decapping protein 4 (Autoantigen Ge-1) (Autoantigen RCD-8) 25 13 8CIMA Q96Q15 SMG1 Serine/threonine-protein kinase SMG1 (SMG1) 83 12CIMA Q9UQ35 SRRM2 Serine/arginine repetitive matrix protein 2 (SRRM2) 71 12INIBIC Q86TD4 SRL Sarcalumenin 26 12 7INIBIC Q96BJ8 ELMO3 Engulfment and cell motility protein 3 19 11 3INIBIC Q7Z6E9 RBBP6 E3 ubiquitin-protein ligase RBBP6 28 11 8CIMA Q3L8U1 CHD9 Chromodomain-helicase-DNA-binding protein 9 (CHD9) 55 10HNP Q13509 TBB3 Tubulin beta-3 chain 21 10

INIBIC Q4G176 ACSF3 Acyl-CoA synthetase family member 3 24 10 9INIBIC O43809 NUDT21 Cleavage and polyadenylation specificity factor subunit 5 tor Im 25 kDa subunit) 13 10 10HNP P00738 HPT haptoglobin 22 9VHIO Q9NUU7 DDX19A DEAD (Asp-Glu-Ala-As) box polypeptide 19A 22 8 8INIBIC Q1ED39 C16orf88 Protein C16orf88 (Testis-specific gene 118 protein) 18 8 6INIBIC Q9NUI1 DECR2 Peroxisomal 2,4-dienoyl-CoA reductase 12 8 5INIBIC P31146 CORO1A Coronin-1A (Coronin-like protein A) 16 7 7INIBIC P49711 CTCF Transcriptional repressor CTCF 21 7 4INIBIC Q9BVA0 KATNB1 Katanin p80 WD40-containing subunit B1 25 7 3INIBIC Q12789 GTF3C1 General transcription factor 3C polypeptide 1 (TF3C-alpha) 23 7 5VHIO Q08AM6 VAC14 Vac14 homolog 32 6 6CIMA Q8IWR0 ZC3H7A Zinc finger CCCH domain-containing protein 7A (ZC3H7A) 20 6

PCM/UCM P49411 TUFM Tu translation elongation factor, mitochondrial [Source:HGNC Symbol;Acc:12420] 10 6 0INIBIC O75081 CBFA2T3 Protein CBFA2T3 (MTG8-related protein 2) 23 6 4VHIO Q86W42 THOC6 THO complex 6 homolog 15 5 5VHIO Q93009 USP7 ubiquitin specific peptidase 7 42 5 5CIMA P49588 AARS Alanine--tRNA ligase, cytoplasmic (AARS) 20 5CIMA P19784 CSNK2A2 Casein kinase II subunit alpha' (CSNK2A2) 9 5CIMA Q9Y613 FHOD1 FH1/FH2 domain-containing protein 1 (FHOD1) 30 5CIMA Q9Y4R8 TELO2 Telomere length regulation protein TEL2 homolog (TELO2) 26 5

PCM/UCM Q15155 NOMO1 NODAL modulator 1 [Source:HGNC Symbol;Acc:30060] 19 5 0PCM/UCM P15880 RPS2 ribosomal protein S2 [Source:HGNC Symbol;Acc:10404] 12 5 0PCM/UCM Q15393 SF3B3 splicing factor 3b, subunit 3, 130kDa [Source:HGNC Symbol;Acc:10770] 17 5 0

HNP Q12931 TRAP1 Heat shock protein 75 kDa, mitochondrial 36 5IACS P55291 Cadherin-15 (Cadherin-14) (Muscle cadherin) (M-cadherin) 27 5 0

Proteins

1) 2 or more coeluting transitions observed2) Identified by IDA (QTrap MSMS) or >3 Coeluting transitions matching observed MSMS spectrum (shotgun/targeted analysis)3) Peptides verified by 3 different laboratories

Protein Uniprot Code GENE NAME Peptide sequence

Proteotypic (Yes/No) 4 Isoform specific?

Observed MSMS shotgun

experiments (Yes/No)

# Transitions

Observed Retention

time

Relative Retention

time

Verified IDA

(Yes/No) 1

Verified MSMS

(Yes/No)S 2

Observed in Ramos

(Yes/No)

Observed in CCD18

(Yes/No)

Observed in MCF7

(Yes/No)

Observed in others

(comma separated) Instrument Column

Q1ED39 C16orf88 AADSLQQNLQR Yes 2 17.57 No Yes Yes No 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm P35637 FUS AAIDWFDGK No Yes 3 20.14 Yes Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm P07741 APT AAIGLLAR Yes Yes 4 8.6 No Yes No Yes 4000QTRAP Onyx Monolithic C18, 150x0.1mmP34059 GALNS AALLTGR no no No 3 17.9 yes no no no HUH7 1 QTRAP5500P31146 CORO1A AAPEASGTPSSDAVSR Yes 4 8.86 Yes Yes Yes No 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm P04075 ADDGRPFPQVIK Yes Yes Yes 3 44.7 Yes Yes No Yes Yes 4000QTRAPP61421 ATP6V0D1 ADDYEQVK Yes 5 17.68 Yes Yes TC28 1 QTRAP 5500 Chrom XP, 3C18-CL-120, 3 um, 120 A, 75umx15cm Q86TD4 SRL ADLIFVVFDPTK No 2 26.08 Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm P49411 TUFM AEAGDNLGALVR YES YES YES 5 NO NO YES 1 QTRAP 5500P00505 GOT2 AEAQIAAK YES YES NO 3 NO NO YES 1 QTRAP 5500Q96Q15 SMG1 AEYAVAK yes no No 4 17.0 yes no no no HUH7 1 QTRAP5500P61421 ATP6V0D1 AFIITINSFGTELSK Yes 2 11.83 No Yes TC28 1 QTRAP 5500 Chrom XP, 3C18-CL-120, 3 um, 120 A, 75umx15cm Q7Z6E9 RBBP6 AIDDSSASISLAQLTK Yes 3 9.2 No No Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm Q13509 TBB3 AILVDLEPGTMDSVR No No 7 20.6 Yes Yes Yes Yes 4000QTRAP Onyx Monolithic C18, 150x0.1mmQ86TD4 SRL AITQELPGLLGSLGLGK No 3 24.34 Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm P55291 AIVLAQDDASQPR Yes Yes No protein xls 2 64.3 No No Yes No No 4000QTRAPQ6P2E9 EDC4 ALAEGQQR No 3 15.68 Yes Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm Q4G176 ACSF3 ALAFGDR Yes 4 12.6 Yes Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm O43809 NUDT21 ALFAVPK Yes 2 14.27 No Yes Yes No 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm Q12788 ALLLAQWAWQEGSVTR Yes Yes Yes 2 85.2 No No Yes No Yes 4000QTRAPP80365 HSD11B2 ALLQLLRSDLR YES YES NO 4 NO NO YES NO NO 1 QTRAP 5500P80404 ALLTGLLDLQAR Yes Yes Yes 3 73.1 Yes Yes No Yes Yes 4000QTRAPP49588 AARS ALNEALK yes yes Yes 5 14.0 yes yes yes yes HUH7 1 QTRAP5500Q6P2E9 EDC4 ALQDVQIR Yes 3 11.01 Yes Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm Q1ED39 C16orf88 ALQEEIDR No 3 20.02 Yes Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm P20701 ALSC[CAM]NVSSPIFK Yes Yes No 2 65.0 No No No Yes Yes 4000QTRAPQ13509 TBB3 ALTVPELTQQMFDAK No No 5 26.3 No Yes Yes Yes 4000QTRAP Onyx Monolithic C18, 150x0.1mmO75081 CBFA2T3 ANLPLLQR No 2 21.43 Yes Yes Yes TC28 1 QTRAP 5500 Acclaim PepMap100, C18, 3um 100A, 75umx15cm Q13951 APMILNGVC[CAM]VIWK Yes Yes Yes 3 76.5 No Yes No Yes Yes 4000QTRAP

Peptides

Template results MRM_121127.xlsx

Protein Uniprot Code GENE NAME Peptide sequence Q1 m/z Q3 m/z

Parent Charge

Fragment Type

Fragment Charge CE Lab Instrument

Q1ED39 C16orf88 AADSLQQNLQR 622.32 786.42 2 y6 34 INIBIC QTRAP5500Q1ED39 C16orf88 AADSLQQNLQR 622.32 899.51 2 y7 34 INIBIC QTRAP5500P35637 FUS AAIDWFDGK 511.75 652.31 2 y5 34 INIBIC QTRAP5500P35637 FUS AAIDWFDGK 511.75 767.34 2 y6 34 INIBIC QTRAP5500P35637 FUS AAIDWFDGK 511.75 880.42 2 y7 34 INIBIC QTRAP5500P07741 APT AAIGLLAR 392.756 359.240 2 y3 1 22.00 HNP 4000QTRAPP07741 APT AAIGLLAR 392.756 472.320 2 y4 1 22.00 HNP 4000QTRAPP07741 APT AAIGLLAR 392.756 529.350 2 y5 1 22.00 HNP 4000QTRAPP07741 APT AAIGLLAR 392.756 642.430 2 y6 1 22.00 HNP 4000QTRAPP34059 GALNS AALLTGR 351.220 630.390 2 y6 1 20.00 CIMA QTRAP5500P34059 GALNS AALLTGR 351.220 527.320 2 b6 1 20.00 CIMA QTRAP5500P34059 GALNS AALLTGR 351.220 446.270 2 y4 1 20.00 CIMA QTRAP5500P31146 CORO1A AAPEASGTPSSDAVSR 751.86 818.4 2 y8 40 INIBIC QTRAP5500P31146 CORO1A AAPEASGTPSSDAVSR 751.86 1134.54 2 y12 40 INIBIC QTRAP5500P31146 CORO1A AAPEASGTPSSDAVSR 751.86 919.45 2 y9 40 INIBIC QTRAP5500P31146 CORO1A AAPEASGTPSSDAVSR 501.57 818.4 3 y8 30 INIBIC QTRAP5500P04075 ADDGRPFPQVIK 671.859 984.600 2 y8 1 39.00 IACS 4000QTRAPP04075 ADDGRPFPQVIK 671.859 828.500 2 y7 1 36.00 IACS 4000QTRAPP04075 ADDGRPFPQVIK 448.242 828.500 3 y7 1 22.00 IACS 4000QTRAPP61421 ATP6V0D1 ADDYEQVK 484.22 781.37 2 y6 28 INIBIC QTRAP5500P61421 ATP6V0D1 ADDYEQVK 484.22 666.35 2 y5 28 INIBIC QTRAP5500P61421 ATP6V0D1 ADDYEQVK 484.22 896.4 2 y7 28 INIBIC QTRAP5500P61421 ATP6V0D1 ADDYEQVK 484.22 821.33 2 b7 28 INIBIC QTRAP5500P61421 ATP6V0D1 ADDYEQVK 484.22 722.26 2 b6 28 INIBIC QTRAP5500Q86TD4 SRL ADLIFVVFDPTK 682.88 805.45 2 y7 37 INIBIC QTRAP5500Q86TD4 SRL ADLIFVVFDPTK 682.88 952.51 2 y8 37 INIBIC QTRAP5500P49411 TUFM AEAGDNLGALVR 593.315 515.330 2 y5 1 PCM/UCM QTRAP 5500P49411 TUFM AEAGDNLGALVR 593.315 985.543 2 y10 1 PCM/UCM QTRAP 5500P49411 TUFM AEAGDNLGALVR 593.315 914.505 2 y9 1 PCM/UCM QTRAP 5500P49411 TUFM AEAGDNLGALVR 593.315 628.414 2 y6 1 PCM/UCM QTRAP 5500P49411 TUFM AEAGDNLGALVR 593.315 742.457 2 y7 1 PCM/UCM QTRAP 5500

Transitions

Template results MRM_121127.xlsx

Proteotypic (%) Isoform specific (%) Observed MSMS shotgun experiments

N 258* 214* 413

Yes 221 (85.65%) 141 (65.88%) 165 (39.95%)

No 37 (14.35%) 73 (34.11%) 248 (60.05%)

From 413 observed peptides

*No Data for all the 413 observed peptides

Verified IDA (%)

Verified MSMS (%)

Total Verified

N 413 268* 413

Yes 213(51.57%) 128 (46.76%) 236 (57.14%)

No 200 (48.43%) 140 (52.23%) 177 (42.86%)

PEPTIDES – OBSERVED /VERIFIED

02468

101214161820

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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# Observed Peptides

86 Proteins

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# Verified Peptides

33 Proteins

PEPTIDES/PROTEIN

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16

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# Peptids

Ramos

CCD18

MCF7

81 prot evaluated66 in RAMOS48 in CCD1863 in MCF7

Peptides Observed in the cell Line

RAMOS CCD18 MCF7

Yes 192 107 183

No 151 99 71

Not evaluated 70 207 159

PEPTIDES/PROTEIN – Sample type

TC28 Plasma HUH7

#pep (prot) Evaluated 158 (20) 31 (5) 88 (18)

#pep (prot) Observed 134 (20) 9 (1) 56 (16)

Other Samples

1360 Transitions

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2 3 4 5 6 7 8 9 11

#Pep

tides

#Transitions

TRANSITIONS/ PEPTIDE

DISCUSSION POINTS

1. Peptide /Transitions verification criteria. IDA, MSMS # transitions2. Proteotypic peptides – Isoforms3. Scoring “goodness” for SRM4. Optimization of measurement conditions. Gradients – CE5. Use of Standards for relative Retention Time evaluation6. Use of (Interna) Standard for relative intensity measurement7. Selection of proteins for recombinant expression8. Selection of synthetic peptides for SRM assay development9. Selection of labeles synthetic peptides for quantitative assays10. Unknown- low abundant protein : detection limits? Fractionation? 11. Multi- laboratory validation12. Next Protein Assignment (when, number, unknown, samples…)

THANK YOU !!!