ascore explained brian c. searle proteome software inc. portland, oregon usa...

Ascore ExplainedBrian 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

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