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

5. Unphosphorylated Form (inactive) Di-phosphorylated Form (active) Structures of MAP kinase B. J. Canagarajah et al., 1997, Cell 90:859

7. y1 – y5b12 – b16

8. ?X

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

10. 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

11. SIQDLTVTGTEPGQVSSR + p vs

12. SpIQDLTVTGTEPGQVSSR SIQDLTpVTGTEPGQVSSR SIQDLTVTpGTEPGQVSSR SIQDLTVTGTpEPGQVSSR SIQDLTVTGTEPGQVSpSR SIQDLTVTGTEPGQVSSpR

13. Ascore Spectrum Model Only looks for B/Y ions Either it’s there or it’s not

14. 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

15. Spectrum Peak Depth

17. 1 peak per 100 m/z 2 peaks per 100 m/z 3 peaks per 100 m/z 4 peaks per 100 m/z 5 peaks per 100 m/z 6 peaks per 100 m/z Spectrum Peak Depth

18. 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

19. 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)

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

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

22. 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

23. SpIQDLTVTGTEPGQVSSR SIQDLTpVTGTEPGQVSSR

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

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

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

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

28. FELNDDYPSLPSMGWASp FELNDDYPSLPSpMGWAS

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

30. 32.025.60.37.2 Δ18.4

31. 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