β-Elimination coupled with strong cation-exchange chromatography for phosphopeptide analysis.

RATIONALE: Since the last decade, mass spectrometry (MS) has become an essential technique for phosphoprotein analysis. Formidable analytical challenges of MS for phosphoprotein study are both the low abundance of phosphopeptides and the lack of an unambiguous diagnostic fragment ion for identification of phospho residues. These challenges can be met by a charge-based isolation of β-elimination products after tryptic digestion using diagonal strong cation-exchange chromatography.

METHODS: β-Elimination combined with diagonal strong cation-exchange chromatography (BE/2SCX) was used for the enrichment of phosphorylated peptides prior to a mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (MS/MS). Bovine α-casein (≥70% purity) was used as a model protein.

RESULTS: Conditions for β-elimination were optimized to maximize the efficiency of the reaction. With a β-elimination, all four model phosphopeptides from enolase (yeast) were correctly identified. The application of the BE/2SCX enrichment strategy for the analysis of β-elimination products of α-casein (bovine) allowed the identification of 11 phosphorylated products.

CONCLUSIONS: The introduction of a BE/2SCX-based enrichment step prior to LC/MS/MS analysis of β-elimination products facilitates the identification of phosphopeptides. Copyright © 2016 John Wiley & Sons, Ltd.