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Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry
Author(s) -
Adamczyk Maceij,
Gebler John C.,
Wu Jiang
Publication year - 2001
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.394
Subject(s) - chemistry , biotinylation , phosphoserine , chromatography , mass spectrometry , threonine , trypsin , serine , tandem mass spectrometry , chemical modification , phosphorylation , phosphopeptide , biochemistry , affinity chromatography , protein mass spectrometry , peptide , enzyme
A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S ‐(2‐mercaptoethyl)cysteinyl or β‐methyl‐ S ‐(2‐mercaptoethyl)cysteinyl residues by β‐elimination/1,2‐ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity‐isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of α‐casein, β‐casein, and ovalbumin. The technique has potential for adaptations to proteome‐wide analysis of protein phosphorylation. Copyright © 2001 John Wiley & Sons, Ltd.