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One‐source peptide/phosphopeptide standards for accurate phosphorylation degree determination
Author(s) -
Hahn Bettina,
Böhm Martin,
Raia Valentina,
Zinn Nico,
Möller Peter,
Klingmüller Ursula,
Lehmann WolfDieter
Publication year - 2011
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000569
Subject(s) - phosphopeptide , peptide , phosphorylation , chemistry , chromatography , protein phosphorylation , analyte , combinatorial chemistry , biochemistry , protein kinase a
Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site‐specific protein phosphorylation degree determination by nanoLC‐ESI‐MS/MS. A stable isotope‐labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one‐source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one‐source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.