Issue 5, 2010

An integrated global strategy for cell lysis, fractionation, enrichment and mass spectrometric analysis of phosphorylated peptides

Abstract

Recently, the field of phosphoproteomics has progressed to the point where thousands of protein phosphorylations can be analyzed simultaneously and used to address significant biological questions. However, several challenges still exist in current LC-MS/MS-based phosphoproteomics methods. Among these are the increased dynamic range of phosphoproteomics samples (due to low stoichiometry of most protein phosphorylations), insufficient inhibition of phosphatase activity, and neutral losses which occur during phosphopeptide fragmentation by MSn. Here we present an improved method, free of conventional phosphatase inhibitors, for sample treatment to minimize phosphatase activity and improve the efficiency of phosphopeptide enrichment. We also present a solution-based IEF method for phosphopeptide fractionation and explore the utility of various fragmentation methods for identifying phosphopeptides and localizing phosphorylation sites.

Graphical abstract: An integrated global strategy for cell lysis, fractionation, enrichment and mass spectrometric analysis of phosphorylated peptides

Supplementary files

Article information

Article type
Paper
Submitted
07 Aug 2009
Accepted
12 Jan 2010
First published
03 Feb 2010

Mol. BioSyst., 2010,6, 822-829

An integrated global strategy for cell lysis, fractionation, enrichment and mass spectrometric analysis of phosphorylated peptides

L. D. Rogers, Y. Fang and L. J. Foster, Mol. BioSyst., 2010, 6, 822 DOI: 10.1039/B915986J

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