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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

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Search Engines

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

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y1 – y5b12 – b16

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?X

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Ascore is interested in telling you just how LITTLE you know

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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

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SIQDLTVTGTEPGQVSSR + p vs

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SpIQDLTVTGTEPGQVSSR SIQDLTpVTGTEPGQVSSR SIQDLTVTpGTEPGQVSSR SIQDLTVTGTpEPGQVSSR SIQDLTVTGTEPGQVSpSR SIQDLTVTGTEPGQVSSpR

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

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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

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Spectrum Peak Depth

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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

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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

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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)

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

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

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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

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SpIQDLTVTGTEPGQVSSR SIQDLTpVTGTEPGQVSSR

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

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

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

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

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FELNDDYPSLPSMGWASp FELNDDYPSLPSpMGWAS

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

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32.025.60.37.2 Δ18.4

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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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