Ascore Explained
description
Transcript of Ascore Explained
Ascore ExplainedBrian C. Searle
Proteome Software Inc. Portland, Oregon USA
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
Search Engines
Search Engines
Unphosphorylated Form(inactive)
Di-phosphorylated Form(active)
Structures of MAP kinase
B. J. Canagarajah et al., 1997, Cell 90:859
y1 – y5 b12 – b16
? X
Ascore is interested in telling you just how LITTLE you know
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
SIQDLTVTGTEPGQVSSR + pvs
SpIQDLTVTGTEPGQVSSR
SIQDLTpVTGTEPGQVSSR
SIQDLTVTpGTEPGQVSSR
SIQDLTVTGTpEPGQVSSR
SIQDLTVTGTEPGQVSpSR
SIQDLTVTGTEPGQVSSpR
Ascore Spectrum Model
•Only looks for B/Y ions•Either it’s there or it’s not
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
Spectrum Peak Depth
Spectrum Peak Depth
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
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
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)
1 2 3 4 5 6 7 8 9 100
20
40
60
80
100
120
140
160
S1T6T8T10S16S17
Number of Peaks per 100 m/z
Pepti
de S
core
Spectrum Peak Depth
1 2 3 4 5 6 7 8 9 100
20
40
60
80
100
120
140
160
S1T6T8T10S16S17
Number of Peaks per 100 m/z
Pepti
de S
core Δ54.2
Spectrum Peak Depth
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
SpIQDLTVTGTEPGQVSSR
SIQDLTpVTGTEPGQVSSR
SpIQDLTVTGTEPGQVSSR
SIQDLTpVTGTEPGQVSSR
Pep Score= 43.0
Pep Score= 4.7
Final Ascore = Δ38.3
Ascore Precision
Beausoleil SA, et al Nat Biotechnol. 2006 Oct;24(10):1285-92.
Ascore Precision
Peptide Score Difference = +20
P-value Difference of 10-2
Best configuration is two orders of magnitude more likely
Ascore Precision
Beausoleil SA, et al Nat Biotechnol. 2006 Oct;24(10):1285-92.
FELNDDYPSLPSMGWASp
FELNDDYPSLPSpMGWAS
FELNDDYPSLPSMGWASp
FELNDDYPSLPSpMGWAS
Pep Score= 1.8
Pep Score= 0.4
Final Ascore = Δ1.4
32.0 25.6 0.37.2
Δ18.4
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