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Ascore Explained

Ascore Explained. Brian C. Searle Proteome Software Inc. Portland, Oregon USA Brian.Searle@ProteomeSoftware.com A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Beausoleil SA, Villén J, Gerber SA, Rush J, Gygi SP.

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Ascore Explained

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  1. Ascore Explained Brian C. Searle Proteome Software Inc. Portland, Oregon USA Brian.Searle@ProteomeSoftware.com A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Beausoleil SA, Villén J, Gerber SA, Rush J, Gygi SP. Nat Biotechnol. 2006 Oct;24(10):1285-92. Creative Commons Attribution

  2. Search Engines

  3. Search Engines

  4. Structures of MAP kinase Unphosphorylated Form (inactive) Di-phosphorylated Form (active) B. J. Canagarajah et al., 1997, Cell90:859

  5. y1 – y5 b12 – b16

  6. ? X

  7. Ascore is interested in telling you just how LITTLE you know

  8. Ascore Outline • Build fragmentation model for each peptide • Normalize spectrum at 10 different peak depths • Determine the best peak depth • Calculate Ascore at that peak depth on only relevant peaks

  9. SIQDLTVTGTEPGQVSSR + p vs

  10. SpIQDLTVTGTEPGQVSSRSIQDLTpVTGTEPGQVSSRSIQDLTVTpGTEPGQVSSRSIQDLTVTGTpEPGQVSSRSIQDLTVTGTEPGQVSpSRSIQDLTVTGTEPGQVSSpRSpIQDLTVTGTEPGQVSSRSIQDLTpVTGTEPGQVSSRSIQDLTVTpGTEPGQVSSRSIQDLTVTGTpEPGQVSSRSIQDLTVTGTEPGQVSpSRSIQDLTVTGTEPGQVSSpR

  11. AscoreSpectrum Model • Only looks for B/Y ions • Either it’s there or it’s not

  12. Ascore Outline • Build fragmentation model for each peptide • Normalize spectrum at 10 different peak depths • Determine the best peak depth • Calculate Ascore at that peak depth on only relevant peaks

  13. Spectrum Peak Depth

  14. Spectrum Peak Depth

  15. Spectrum Peak Depth 1 peak per 100 m/z 4 peaks per 100 m/z 2 peaks per 100 m/z 5 peaks per 100 m/z 3 peaks per 100 m/z 6 peaks per 100 m/z

  16. Ascore Outline • Build fragmentation model for each peptide • Normalize spectrum at 10 different peak depths • Determine the best peak depth • Calculate Ascore at that peak depth on only relevant peaks

  17. Binomial Distribution Scorer • N = # of B/Y peaks in fragmentation model • n = # of matched peaks to actual spectrum • p = prior (e.g. 6 peaks per 100 m/z = 0.06) • Peptide Score = -10*log(P)

  18. Spectrum Peak Depth Peptide Score Number of Peaks per 100 m/z

  19. Spectrum Peak Depth Δ54.2 Peptide Score Number of Peaks per 100 m/z

  20. Ascore Outline • Build fragmentation model for each peptide • Normalize spectrum at 10 different peak depths • Determine the best peak depth • Calculate Ascore at that peak depth on only relevant peaks

  21. SpIQDLTVTGTEPGQVSSRSIQDLTpVTGTEPGQVSSR

  22. SpIQDLTVTGTEPGQVSSR Pep Score= 43.0 SIQDLTpVTGTEPGQVSSR Pep Score= 4.7 Final Ascore = Δ38.3

  23. Ascore Precision Beausoleil SA, et al Nat Biotechnol. 2006 Oct;24(10):1285-92.

  24. Ascore Precision Peptide Score Difference = +20 P-value Difference of 10-2 Best configuration is two orders of magnitude more likely

  25. Ascore Precision Beausoleil SA, et al Nat Biotechnol. 2006 Oct;24(10):1285-92.

  26. FELNDDYPSLPSMGWASpFELNDDYPSLPSpMGWAS

  27. FELNDDYPSLPSMGWASp Pep Score= 1.8 • FELNDDYPSLPSpMGWAS Pep Score= 0.4 Final Ascore = Δ1.4

  28. 32.0 25.6 7.2 0.3 Δ18.4

  29. Conclusions • Software like Ascore is critical for scientifically useful publication • MCP Philadelphia guidelines (Paris 2) require acknowledgement of ambiguity • It’s just as important to know what you don’t know

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