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

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

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

5. PROTEORED ASSAY 2008 Gel-free quantitative approaches

6. PROTEORED ASSAY 2008

7. 1 1 1 1 2 4 1 2 2 1 4 laboratories 3 laboratories 2 laboratories Proteins reported as differential

8. Labsproteins% 123123 715 229 79 64.5 20.7 7.1 4444.0 5191.7 6222.0 UPFUPVBPGUCMPCBMPCBQ Proteins Identified by LC/MS/MS

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

10. 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 150 fmolA1-B1 -> 1 : 5 Protein 2500 fmolA2-B2 -> 1 : 2 Protein 35 pmolA3-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 PME09

11. - 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  g T4 proteins

12. Distribute ~100  g 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 PME5

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

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

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

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

18. Quantification

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

20. 1000/1500 fmol /  g Quantitation Results

21. 520/200 fmol /  g Quantitation Results

22. 50/25 fmol /  g Quantitation Results

23. 5/1 fmol /  g Quantitation Results

24. MRM

25. 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!