Quantitation of changes in protein phosphorylation: a simple method based on stable isotope labeling and mass spectrometry.

ABSTRACT: Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied

SUBMITTER: Bonenfant D 

PROVIDER: S-EPMC298695 | biostudies-literature | 2003 Feb

REPOSITORIES: biostudies-literature