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β‐Elimination coupled with strong cation‐exchange chromatography for phosphopeptide analysis
Author(s) -
Buncherd Hansuk,
Roseboom Winfried,
Chokchaichamnankit Daranee,
Sawangareetrakul Phannee,
Phongdara Amornrat,
Srisomsap Chantragan,
Jong Luitzen,
Svasti Jisnuson
Publication year - 2016
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7606
Subject(s) - chemistry , phosphopeptide , chromatography , phosphoprotein , mass spectrometry , ion chromatography , phosphoproteomics , tandem mass spectrometry , peptide , phosphorylation , protein phosphorylation , biochemistry , protein kinase a
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.