Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow

Rebekah L. Gundry, Melanie Y. White, Christopher I. Murray, Lesley A. Kane, Qin Fu, Brian A. Stanley, Jennifer E. Van Eyk

Research output: Contribution to journalArticle

116 Scopus citations

Abstract

This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be considered when analyzing the MS data to ensure stringent and robust protein identifications and characterization.

Original languageEnglish (US)
Pages (from-to)10.25.1-10.25.23
JournalCurrent Protocols in Molecular Biology
Issue numberSUPPL. 88
DOIs
StatePublished - 2009

Keywords

  • In-gel digestion
  • In-solution digestion
  • Mass spectrometry
  • Peptide desalting proteomics

ASJC Scopus subject areas

  • Molecular Biology

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