Quantitative in vitro kinase reaction as a guide for phosphoprotein analysis by mass spectrometry

A simple calculation using the radioactive decay of P-32 incorporated into a protein during in vitro kinase reactions is described that allows the ove rall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated. Prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in a complex mixture followed by tandem mass spectrometry (MS/MS) to locate the phospho rylated residue within the phosphopeptide, such calculations are predictive of the chances for successful characterization by these methods. An exampl e of estimating the stoichiometry of peptide phosphorylation will be presen ted along with calculations that predict when adequate phosphopeptide is pr esent in any given spot on the thin-layer chromatography (TLC) plates used for two-dimensional phosphopeptide (2DPP) mapping to allow extraction and c omplete characterization by MS/MS. Copyright (C) 2000 John Wiley & Sons, Lt d.