One-source peptide/phosphopeptide standards for accurate phosphorylation degree determination.
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.
Molecular Structure Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Bettina
B
Hahn
Martin
M
Böhm
Valentina
V
Raia
Nico
N
Zinn
Peter
P
Möller
Ursula
U
Klingmüller
Wolf-Dieter
WD
Lehmann
D002853:Chromatography, Liquid; D006689:Hodgkin Disease / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D040901:Proteomics; D012015:Reference Standards; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D050800:STAT6 Transcription Factor / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D010748:Phosphopeptides; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D025521:Tumor Suppressor Proteins