Label-free protein quantitation using weighted spectral counting

Christine Vogel, Edward M. Marcotte

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Mass spectrometry (MS)-based shotgun proteomics allows protein identifications even in complex biological samples. Protein abundances can then be estimated from the counts of MS/MS spectra attributable to each protein, provided that one corrects for differential MS-detectability of the contributing peptides. We describe the use of a method, APEX, which calculates Absolute Protein EXpression levels based on learned correction factors, MS/MS spectral counts, and each protein's probability of correct identification. The APEX-based calculations consist of three parts: (1) Using training data, peptide sequences and their sequence properties, a model is built that can be used to estimate MS-detectability (O i) for any given protein. (2) Absolute abundances of proteins measured in an MS/MS experiment are calculated with information from spectral counts, identification probabilities and the learned O i-values. (3) Simple statistics allow for significance analysis of differential expression in two distinct biological samples, i.e., measuring relative protein abundances. APEX-based protein abundances span more than four orders of magnitude and are applicable to mixtures of hundreds to thousands of proteins from any type of organism.

Original languageEnglish (US)
Title of host publicationQuantitative Methods in Proteomics
EditorsMarcus Katrin
Pages321-341
Number of pages21
DOIs
StatePublished - 2012

Publication series

NameMethods in Molecular Biology
Volume893
ISSN (Print)1064-3745

Keywords

  • Label-free mass spectrometry
  • Protein expression
  • Quantitative proteomics
  • Spectral counting

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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