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ProteoRed WG1-WG2 Meeting Salamanca, March,16th 2010

PME5: Quantitative LC-MS differential analysis F. Canals. ProteoRed WG1-WG2 Meeting Salamanca, March,16th 2010. THANK YOU!. Joan Josep Bech Núria Colomé Marta Monge. Salvador Martínez de Bartolomé Alex Campos … All participants in the study. LC-MS . => Isotopic labeling, Label free.

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ProteoRed WG1-WG2 Meeting Salamanca, March,16th 2010

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  1. PME5: Quantitative LC-MS differential analysis F. Canals ProteoRed WG1-WG2 Meeting Salamanca, March,16th 2010

  2. THANK YOU! Joan Josep Bech Núria Colomé Marta Monge Salvador Martínez de Bartolomé Alex Campos … All participants in the study

  3. LC-MS => Isotopic labeling, Label free 2008 MULTICENTRIC STUDY MDA-MB-468 Br.Cancer Cells Treated Cells (+ EGF 24 h) Untreated Cells Sample 2 Sample 1 2DE-DIGE 4 + 4 technical replicates Protein Identification: 30 top differential protein spots > 1.5 fold p < 0.05 Image analysis: - NLD - LUDESI - GE

  4. PROTEORED ASSAY 2008 Gel-free quantitative approaches

  5. PROTEORED ASSAY 2008

  6. 1 4 2 1 1 1 1 1 2 2 4 laboratories 3 laboratories 2 laboratories Proteins reported as differential

  7. UPF UPV BPG UCM PCBM PCBQ Proteins Identified by LC/MS/MS

  8. “Centralized” analysis of data • “Collective” analysis of “pooled” data • Next Multicentric Experiment on (Quantitative) LC-MS experiment

  9. PME09 • 1- Run LC-MS/MS of a sample of “medium” complexity in triplicate • -> evaluate reproducibility -> Different protocols – SOP ? 2- 2a) Two standard mixtures A, B: Protein 1 50 fmol A1-B1 -> 1 : 5 Protein 2 500 fmol A2-B2 -> 1 : 2 Protein 3 5 pmol A3-B3 -> 1.5:1 2b) A, B + Matrix (medium complexity) -> Run triplicates 2a, 2b ? Matrix -> Bacterial lysate?, Fraction cell lysate (supplied?) SOP-> Include Fractionation ? Labeling – Label Free -> assign # labs? LC gradient Search parameters

  10. Matrix: Bacteriophage T4 capside proteins: ~35 proteins • > adequate complexity for single LC-MS runs - > replicas • Samples: A , B • > 4 level spiked proteins – 3 orders of magnitude • > relative abundance 0.38 - 5 Per 1 mg T4 proteins

  11. PME5 • Distribute ~100 mg each sample A,B • Isotope label (i-TRAQ, ICPL, O18…) or label-free relative quantitation A/B • -> minimum of 4 replicas: evaluate variability, quantitation accuracy… • 2DE-DIGE? • Provide unified database (E.Coli + spiked proteins). Inter-Lab comparisons. • Report using MIAPE document generator

  12. E. Coli sample preparation Cytoplasmatic fraction SCX A9 A10 A11 A13 A14 B4 B5 B7 B9 B11 100-300 proteins Id 10/40 fractions Off-gel fractionation: 902 prots

  13. MSMS Efficiency IT Q-TOF TOF/TOF OT

  14. Protein Identification IT Q-TOF TOF/TOF OT

  15. Protein Id Peptide Id IT Q-TOF TOF/TOF OT

  16. Quantification

  17. Ratio variability ICPL ITRAQ TMT LF DIGE False positives ?

  18. Quantitation Results 1000/1500 fmol / mg

  19. Quantitation Results 520/200 fmol / mg

  20. Quantitation Results 50/25 fmol / mg

  21. Quantitation Results 5/1 fmol / mg

  22. MRM

  23. Conclusions • Sample issues: Complexity of matrix, stability, solubility… • Consistent quantitafication down to 25 fmol/ug. 1fmol level? • Accuracy of “theoretical values”?  SRM measurements • CVs 10-20%. False differential protein Id.? • Reporting data!

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