Laboratori de ProteòmicaVall d’Hebron Institut d’Oncologia

Francesc Canals

Madrid 28/08/2012

Human Proteome Project – CHR16

SRM Analysis of selected proteins (ABSciex 4000 QTRAP)

Vall d’Hebron Institute of Oncology

Vall d’Hebron University Hospital, Barcelona

1D- SDS-PAGE 12% - 100 mg each sample

Trypsin digestion

In solution – 100 mg

Ramos TCA/Acetone 5.93 mg/mLMCF7 4.92 mg/mLCCD18 7.30 mg/mL

SAMPLE PREPARATION

In gel – 15 bands :

~ 6.7 mg / band

A: RamosMW B: MCF7 C: CCD18

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SHOTGUN LC-MSMS Analysis MCF7 sample

Esquire HCT Ion trap

• 15 bands *2uL (~400ng)

1093 proteins identified

• 200ng In solution digestion

152 proteins identified

4 of “our” 20 known proteins to analyze observed in Gel-LCMS of MCF7

29 of 160 total known proteins to analyze observed in Gel-LCMS of MCF7

Protein Name/ Accession ensembl gene id

external gene id description

P69905 ENSG00000188536 HBA2 hemoglobin, alpha 2 [Source:HGNC Symbol;Acc:4824]P07911 ENSG00000169344 UMOD uromodulin [Source:HGNC Symbol;Acc:12559]P23141 ENSG00000198848 CES1 carboxylesterase 1 [Source:HGNC Symbol;Acc:1863]O43303 ENSG00000103540 CCP110 centriolar coiled coil protein 110kDa [Source:HGNC Symbol;Acc:24342]P35858 ENSG00000099769 IGFALS insulin-like growth factor binding protein, acid labile subunit [Source:HGNC Symbol;Acc:5468]Q92793 ENSG00000005339 CREBBP CREB binding protein [Source:HGNC Symbol;Acc:2348]P15559 ENSG00000181019 NQO1 NAD(P)H dehydrogenase, quinone 1 [Source:HGNC Symbol;Acc:2874]P69849 ENSG00000103226 NOMO3 NODAL modulator 3 [Source:HGNC Symbol;Acc:25242]O43294 ENSG00000140682 TGFB1I1 transforming growth factor beta 1 induced transcript 1 [Source:HGNC Symbol;Acc:11767]Q6P996 ENSG00000179889 PDXDC1 pyridoxal-dependent decarboxylase domain containing 1 [Source:HGNC Symbol;Acc:28995]Q93009 ENSG00000187555 USP7 ubiquitin specific peptidase 7 (herpes virus-associated) [Source:HGNC Symbol;Acc:12630]P15170 ENSG00000103342 GSPT1 G1 to S phase transition 1 [Source:HGNC Symbol;Acc:4621]Q9NPB6 ENSG00000102981 PARD6A par-6 partitioning defective 6 homolog alpha (C. elegans) [Source:HGNC Symbol;Acc:15943]Q15911 ENSG00000140836 ZFHX3 zinc finger homeobox 3 [Source:HGNC Symbol;Acc:777]Q15554 ENSG00000132604 TERF2 telomeric repeat binding factor 2 [Source:HGNC Symbol;Acc:11729]P63279 ENSG00000103275 UBE2I ubiquitin-conjugating enzyme E2I [Source:HGNC Symbol;Acc:12485]Q9NUU7 ENSG00000168872 DDX19A DEAD (Asp-Glu-Ala-As) box polypeptide 19A [Source:HGNC Symbol;Acc:25628]Q16775 ENSG00000063854 HAGH hydroxyacylglutathione hydrolase [Source:HGNC Symbol;Acc:4805]Q08AM6 ENSG00000103043 VAC14 Vac14 homolog (S. cerevisiae) [Source:HGNC Symbol;Acc:25507]Q86W42 ENSG00000131652 THOC6 THO complex 6 homolog (Drosophila) [Source:HGNC Symbol;Acc:28369]

Protein Name/Accessionensembl_gene_id external_gene_iddescriptionP51170 ENSG00000166828 SCNN1G sodium channel, nonvoltage-gated 1, gamma [Source:HGNC Symbol;Acc:10602]Q7L3S4 ENSG00000179965 ZNF771 zinc finger protein 771 [Source:HGNC Symbol;Acc:29653]Q96S06 ENSG00000103227 LMF1 lipase maturation factor 1 [Source:HGNC Symbol;Acc:14154]O00219 ENSG00000103044 HAS3 hyaluronan synthase 3 [Source:HGNC Symbol;Acc:4820]Q2QL34 ENSG00000156968 MPV17L MPV17 mitochondrial membrane protein-like [Source:HGNC Symbol;Acc:26827]Q9NVC3 ENSG00000103042 SLC38A7 solute carrier family 38, member 7 [Source:HGNC Symbol;Acc:25582]

Proteins to be assayed by SRM in our lab

20 Known Proteins

6 Unknown Proteins

Phase 1: Selection of 5 known + 1 unknown proteins

Q92793 ENSG00000005339 CREBBP CREB binding protein [Source:HGNC Symbol;Acc:2348]Q93009 ENSG00000187555 USP7 ubiquitin specific peptidase 7 (herpes virus-associated) [Source:HGNC Symbol;Acc:12630]Q08AM6 ENSG00000103043 VAC14 Vac14 homolog (S. cerevisiae) [Source:HGNC Symbol;Acc:25507]Q86W42 ENSG00000131652 THOC6 THO complex 6 homolog (Drosophila) [Source:HGNC Symbol;Acc:28369]Q9NUU7 ENSG00000168872 DDX19A DEAD (Asp-Glu-Ala-As) box polypeptide 19A [Source:HGNC Symbol;Acc:25628]Q96S06 ENSG00000103227 LMF1 lipase maturation factor 1 [Source:HGNC Symbol;Acc:14154]

All observed in MCF7 in MaxQuant database (11 cell lines, M.Mann)

In Silico

MRM Pilot SoftwareRepositories dataSRM Atlas (Peptide Atlas)MaxQuant database

Shotgun data1D GelIn solution digestion

Previous Experimental data

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SRM Method Generation

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Excel Macro_MRM_GenerateMethodList © Adelina Acosta

Macro that generates a transition table with the specific format of Analyst software (ABSciex 4000 QTRAP).

From SRMAtlas Data:

1. Copy the TSV table in Worksheet “TSV” 2. Run Macro

In worksheet “MRM_List”: all transitions from TSV table in the right format for Analyst software

In worksheet “Filtered_List”: selected transitions according to specific criteria

from TSV table in the right format for Analyst software

SRM Methods

Exp. data derived Method In Silico derived Methods

MRM Pilot generated methods for each protein

4 transitions / peptide20-30 peptides/ protein

SRM Atlas Data for all 6 proteins

Filtered using Excel Macro

+ Observed MSMS data : shotgun + MaxQuant

5 transitions / peptide 5 peptides/ protein

LC-MS conditions: 15 cm x 75 mm column; 300 nl/min; 90 min gradient

A: RamosMW B: MCF7 C: CCD18

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2

3

4

5

6

7

8

9

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A: RamosMW B: MCF7 C: CCD18

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RamosMCF7CCD18

Solution Digests

SRM - ANALYSIS

Exp. data derived Method

6 x In Silico derived Methods

Exp. data derived Method

CREBBP 265 kDa Band 1VAC14 87 kDa Band 3LMF1 65 kDa Band 4

In Silico derived Methods

Bands 1-7

Q92793 CREBBP SRM Atlas In Silico CCD1

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MCF

7

Ram

os

SRM Atlas In SilicoN Dif.

TransitionsDAFLTLAR 3 3QAASTSGPTPAASQALNPQAQK 4 x x x 4

In Solution Digestion Ramos Band A1

SRM - RESULTS

“KNOWN” PROTEINS

Ramos Band A2-3

Q93009 USP7 SRM Atlas In Silico CCD1

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MCF

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Ram

os

SRM AtlasN Dif.

Transitions

FDDDVVSR 5 x x x 5IIGVHQEDELLEC[160]LSPATSR 4 4IQDYDVSLDK 4 x 4LLEIVSYK 5 x x x 5LSEVLQAVTDHDIPQQLVER 4 4

In Solution Digestion

Q08AM6 VAC14 SRM Atlas In Silico CCD1

8

MCF

7

Ram

os

SRM Atlas In SilicoN Dif.

TransitionsDSGLYLK 3 3FDLVSFIPLLR 4 x x 4GPFIIR 3 3LAADPDPNVK 3 x 3 3QAEPTPDDALPK 4 4VAALEIEK 3 3

In Solution Digestion Ramos Band A3

Ramos Band A7

Q86W42 THOC6 SRM Atlas In Silico CCD1

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MCF

7

Ram

os

SRM AtlasN Dif.

Transitions

AFSLSF 1 x 1AVPLAVPLGQTEVFQALQR 4 x 4EVQTIEVYK 4 x 4HLLSAGDGEVK 5 5SPEVLSGGEDGAVR 5 9 x x x 5 9SSTPTTIFPIR 6 x x 6

In Solution Digestion

SRM - RESULTS

“KNOWN” PROTEINS

Ramos Band A1/2/4

Q9NUU7 DDX19A SRM Atlas In Silico CCD1

8

MCF

7

Ram

os

SRM AtlasN Dif.

Transitions

DGNPDNETYLHR 5 x 5DPNSPLYSVK 5 7 x x 5 7EEETLDTIK 4 x 4FYPELK 4 x 4LDTDDLDEIEK 2 5 x 5 5SNLVDNTNQVEVLQR 5 7 x x 5 8VLVTTNVC[CAM]AR 4 x 4 5YPQC[CAM]LC[CAM]LSPTYELALQTGK 3 x x x 3

In Solution Digestion

Q96S06 LMF1 SRM Atlas In Silico CCD1

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MCF

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Ram

osSRM Atlas In Silico

N Dif. Transitions

LLASDAEALSLLAHNPFAGRPPPR 2 x 2

In Solution Digestion Ramos Band A4

SRM - RESULTS

“KNOWN” PROTEINS

“UNKNOWN” PROTEIN

SRM -CONCLUSIONS

• Better results by In Silico prediction than SRM Atlas

• Limited shotgun data utility

• Confidence by: MSMS spectra, different cell lines

• Known Proteins: Enough peptides /protein

• 3-5 transitions/peptide => optimize transitions

• Unknown Proteins => fractionate ?

Laboratori de ProteòmicaVall dHebron Institut d’Oncologia

Núria ColoméJoan Josep BechLuna MartínAdelina Acosta

ACKNOWLEDGMENTS

UCTS

Fátima Nuñez

Ricardo GonzaloM. Ángeles Artaza

Proteomics Laboratory

Thank You !

Laboratori de ProteòmicaVall dHebron Institut d’Oncologia