ANALYZING PROTEOMICS

RESULTS

SEPARATING THE WHEAT

FROM THE CHAFF

Amit Kumar Yadav5th Dec 2012

Course B

INTERPRETING THE RESULTS

1. Significance assessment of

a Peptide-Spectrum

match (PSM)

2.Controlling error rates in a high through-

put experiment

3. Inferring the proteins

SIGNIFICANCE ASSESSMENT OF

PEPTIDE ASSIGNMENTS

I found some matches. Are they correct?

MS/MS DATABASE SEARCH

Understanding and Exploiting Peptide Fragment Ion Intensities Using Experimental and Informatic Approaches

Ashley C. Gucinski, Eric D. Dodds, Wenzhou Li, and Vicki H. Wysocki. Methods in Molecular Biology , Vol.604

WHAT’S THE PROBABILITY?

Computational Methods for Mass Spectrometry Proteomics I. Eidhammer, K. Flikka, L. Martens and S.-O. Mikalsen

Threshold score

FALSE DISCOVERY RATE

DECOY DATABASE

• e.g. ABCDEFGHIJ

Database Sequence (target)

• e.g. JIHGFEDCBA

Reversed Sequence (decoy)

• e.g. JDIBGEAFCH

Randomized Sequence (decoy)

DECOY DATABASE TYPES

Comparison of Novel Decoy Database Designs for Optimizing Protein Identification Searches Using ABRF sPRG2006 Standard MS/MS

Data Sets. Luca Bianco, Jennifer A. Mead, and Conrad Bessant, J. Proteome Res., 2009, 8 (4), 1782-1791

IS DECOY A GOOD NULL MODEL?50

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Nu

mb

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hit

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Score

Target

Decoyp- value?

e- value?

FALSE DISCOVERY RATE

Targ

et

Deco

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So

rted

Sco

res

Threshold score

FALSE DISCOVERY RATE

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Peptide mass (Mr)

1 % FDR

Target

Decoy

5 % FDR

q- value?

Threshold

score

FDR FORMULA

Concatenated target-decoy

search

• FDR= 2 x #decoy / # ( target + decoy )

Separate target and decoy search

• FDR= #decoy / #target

target

decoy

FLEXIFDR

Yadav AK, Kumar D, Dash D (2012) Learning from Decoys to Improve the Sensitivity and Specificity of Proteomics Database Search

Results. PLoS ONE 7(11): e50651

PROTEIN INFERENCE

…but I wanted to know the proteins in my sample?

…I have the peptides, but which proteins did

they come from?

Interpretation of Shotgun Proteomic Data:The Protein Inference Problem

Alexey I. Nesvizhskii and Ruedi Aebersold, MCP , July 11, 2005

PROTEIN INFERENCE PROBLEM

Interpretation of Shotgun Proteomic Data:The Protein Inference Problem

Alexey I. Nesvizhskii and Ruedi Aebersold, MCP , July 11, 2005

PROTEIN GROUPING

Interpretation of Shotgun

Proteomic Data:The Protein

Inference Problem

Alexey I. Nesvizhskii and

Ruedi Aebersold, MCP , July

11, 2005

QUANTITATION

How much of a protein is present?

QUANTITATION: SILAC

1. Achieving In-Depth Proteomics Profiling by Mass -ACS CHEMICAL BIOLOGY VOL.2 NO.1 • 39–52 • 2007

2. Proteomics: a pragmatic perspective, Nature biotechnology volume 28 number 7 july 2010

APPLICATIONS OF PROTEOMICS

Proteomics: a pragmatic perspective, Nature biotechnology volume 28 number 7 july 2010

Different -omics sciences describe many levels of biomolecular organization –but if used in isolation may give misleading inferences about the system !