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Proteored Multilab Experiment 5 QUANTITATIVE PROTEOMICS

Proteored Multilab Experiment 5 QUANTITATIVE PROTEOMICS. 4 proteins spiked in E. coli protein matrix Progenesis LC-MS assisted label-free analysis. Labs Reporting Data. Universidad de Alicante (Qual-Quant) Hospital Universitario Instituto de Oncologia Vall d’Hebron (Qual-Quant)

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Proteored Multilab Experiment 5 QUANTITATIVE PROTEOMICS

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

  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) WG1&WG2 Meeting - CIC Salamanca

  3. APPROACHLAB1-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 WG1&WG2 Meeting - CIC Salamanca

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

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

  6. Feature filtering WG1&WG2 Meeting - CIC Salamanca

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

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

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

  10. Peptide filtering WG1&WG2 Meeting - CIC Salamanca

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

  12. Report configuration WG1&WG2 Meeting - CIC Salamanca

  13. Web report WG1&WG2 Meeting - CIC Salamanca

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

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

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

  17. WG1&WG2 Meeting - CIC Salamanca

  18. WG1&WG2 Meeting - CIC Salamanca

  19. ALDOA MYG CYC 25fm 200fm 1000fm Proteins stats LAB1 WG1&WG2 Meeting - CIC Salamanca

  20. ALDOA BSA/MYG CYC 25fm 1fm/200fm 1000fm Proteins stats LAB2 WG1&WG2 Meeting - CIC Salamanca

  21. WG1&WG2 Meeting - CIC Salamanca

  22. LINEAR MODEL Suitable for interpolation WG1&WG2 Meeting - CIC Salamanca

  23. LOGARITHMIC MODEL Suitable for extrapolation? WG1&WG2 Meeting - CIC Salamanca

  24. EXPONENTIAL MODEL Suitable for inter- and extrapolation? WG1&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 WG1&WG2 Meeting - CIC Salamanca

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