1. Laboratori de Proteòmica Vall 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

2. 1D- SDS-PAGE 12% - 100  g each sample Trypsin digestion  In solution – 100  g Ramos TCA/Acetone 5.93 mg/mL MCF7 4.92 mg/mL CCD18 7.30 mg/mL SAMPLE PREPARATION  In gel – 15 bands : ~ 6.7  g / band A: RamosMWB: MCF7C: CCD18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 75 100 50 37

3. 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

4. Proteins to be assayed by SRM in our lab 20 Known Proteins 6 Unknown Proteins

5. Phase 1: Selection of 5 known + 1 unknown proteins All observed in MCF7 in MaxQuant database (11 cell lines, M.Mann) In Silico MRM Pilot Software Repositories data SRM Atlas (Peptide Atlas) MaxQuant database Shotgun data 1D Gel In solution digestion Previous Experimental data + SRM Method Generation

6. 6 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:

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

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

9.  In worksheet “Filtered_List”: selected transitions according to specific criteria from TSV table in the right format for Analyst software

10. SRM Methods Exp. data derived MethodIn Silico derived Methods MRM Pilot generated methods for each protein 4 transitions / peptide 20-30 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  m column; 300 nl/min; 90 min gradient

11. Ramos MCF7 CCD18 Solution Digests SRM - ANALYSIS Exp. data derived Method 6 x In Silico derived Methods Exp. data derived Method CREBBP 265 kDa Band 1 VAC14 87 kDa Band 3 LMF1 65 kDa Band 4 In Silico derived Methods Bands 1-7

12. SRM - RESULTS “KNOWN” PROTEINS

13. SRM - RESULTS “KNOWN” PROTEINS

14. SRM - RESULTS “KNOWN” PROTEINS “UNKNOWN” PROTEIN

15. 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 ?

16. Laboratori de Proteòmica Vall dHebron Institut d’Oncologia Núria Colomé Joan Josep Bech Luna Martín Adelina Acosta ACKNOWLEDGMENTS UCTS Fátima Nuñez Ricardo Gonzalo M. Ángeles Artaza Proteomics Laboratory

17. Thank You ! Laboratori de Proteòmica Vall dHebron Institut d’Oncologia