1. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 1 Proteored Multilab Experiment 5 QUANTITATIVE PROTEOMICS 4 proteins spiked in E. coli protein matrix Progenesis LC-MS assisted label-free analysis

2. Labs Reporting Data Universidad de Alicante (Qual-Quant) Hospital Universitario Instituto de Oncologia Vall d’Hebron (Qual-Quant) Centro de Biología Molecular “Severo Ochoa” (Qual: 72 Ecoli + 2 spiked) 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 2

3. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 3 APPROACH LAB1-LAB2 Sample processing and Data acquisition –Liquid digestion (iodoacetamide, trypsin) –nLC-nESI-IT –5 - 4 runs of sample A, 4 runs of sample B Label-free analysis with Progenesis LC-MS –LC-MS plot alignment –Whole-feature selection – feature abundance >1E+06 –Mascot search (2mclv, Ox(M), Deam(QN), 1.2Da – 1.4 Da parent, 0.6Da – 0.7 Da fragment) –Peptide match validation: score > 25, matching E. coli or spiked protein

4. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 4 Reference LC-MS run selection

5. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 5 LC-MS runs alignment

6. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 6 Feature filtering

7. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 7 Stats-based feature selection

8. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 8 Selected feature stats

9. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 9 Peptide search: mgf export, xml import

10. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 10 Peptide filtering

11. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 11 Protein view - conflict resolution

12. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 12 Report configuration

13. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 13 Web report

14. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 14 Average and %CV of A/B ratios of groups of E. coli proteins identified-quantified with only…

15. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 15 Deviation of average abundance LAB1 ALDOA MYG CYC 25fm 200fm 1000fm

16. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 16 Deviation of average abundance LAB2 ALDOA BSA/MYG CYC 25fm 1fm/200fm 1000fm

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18. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 18

19. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 19 Proteins stats LAB1 Number of E Coli proteins id. (total) 67 Number of E Coli single hit- proteins id14 Number of Spiked proteins id.3 ALDOA MYG CYC 25fm 200fm 1000fm Accession Peptide count Confidence score Anova (p)*A/B dtn - prep CYC_HORSE7287.880.00530640.6 – 0.67 MYG_EQUBU4236.170.000681575.5 – 2.6 ALDOA_RABIT3152.120.000280943.8 – 2.0 BSA_BOVIN K2C1_HUMAN -4-4 NOT 192.23 FOUND 0.269338461.4

20. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 20 Proteins stats LAB2 Number of E Coli proteins id. (total) 128 Number of E Coli single hit- proteins id74 Number of Spiked proteins id.4 Accession Peptide count Confidence score Anova (p)*A/B dtn - prep CYC_HORSE5285.880.00000670.57 – 0.67 MYG_EQUBU3244.410.000106172.18 – 2.6 ALDOA_RABIT134.810.90080340.99 – 2.0 BSA_BOVIN3174.110.01052591.22 – 0.2 ALDOA BSA/MYG CYC 25fm 1fm/200fm 1000fm

21. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 21

22. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 22 Accession concentration (fmol/microg) concentration /10 (pg/microg) Peptide count CYC_HORSE 100012007 MYG_EQUBU 2003404 ALDOA_RABIT 25983 BSA_BOVIN 16.62-3 LINEAR MODEL Suitable for interpolation

23. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 23 Accession concentration (fmol/microg) concentration /10 (pg/microg) Peptide count CYC_HORSE 100012007 MYG_EQUBU 2003404 ALDOA_RABIT 25983 BSA_BOVIN 16.60 LOGARITHMIC MODEL Suitable for extrapolation?

24. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 24 Accession concentration (fmol/microg) concentration /10 (pg/microg) Peptide count CYC_HORSE 100012007 MYG_EQUBU 2003404 ALDOA_RABIT 25983 BSA_BOVIN 16.61 EXPONENTIAL MODEL Suitable for inter- and extrapolation?

25. 16 Marzo 2010WG1&WG2 Meeting - CIC Salamanca 25 CONCLUSIONS LC-MS label free method was successfully used to quantitate relative abundance of spiked proteins across samples in a matrix of medium complexity Low resolution instruments (ESI-IT) produced reasonably accurate results Some spiked proteins can be in or below the LOD of the used instruments (inclusion lists) A thresold of 3 unique peptides seem to be needed for achieving an acceptable dispersion (p.ex. CV< 20%) of protein abundance data used for quantitation