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

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...

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Bibliographic Details
Published inCurrent protocols in molecular biology (Print) Vol. Chapter 10; p. Unit10.25
Main Authors Gundry, Rebekah L, White, Melanie Y, Murray, Christopher I, Kane, Lesley A, Fu, Qin, Stanley, Brian A, Van Eyk, Jennifer E
Format Journal Article
LanguageEnglish
Published United States 01.10.2009
Subjects
Mass Spectrometry - methods
Peptides - analysis
Peptides - isolation & purification
Proteins - analysis
Proteins - isolation & purification
Proteins - metabolism
Proteomics - methods
Specimen Handling - methods
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Summary: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.
ISSN:1934-3647
DOI:10.1002/0471142727.mb1025s88