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

• DOI: 10.1073/pnas.232735599
• Publication Date: 2003-02-04T17:24:41Z
• Authors: Débora Bonenfant; Tobias Schmelzle; Estela Jacinto; José L. Crespo; Thierry Mini; Michael N. Hall; Paul Jenoe
• Keywords: Phosphopeptide; Dephosphorylation; Phosphoproteomics; Protein phosphatase 1

Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes the extent of is lacking. Here, we present quantitate occurring response stimulus. The consists three steps: ( i ) enzymatic digestion H 2 16 O or isotopically enriched 18 label individual pools differentially phosphorylated proteins; ii affinity selection phosphopeptides from combined digests by immobilized metal-affinity chromatography; iii dephosphorylation with alkaline phosphatase allow for quantitation matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied this strategy analysis yeast nitrogen permease reactivator kinase involved target rapamycin signaling pathway. Alteration at Ser-353 Ser-357 could be easily assessed quantitated both wild-type cells treated lacking SIT4 responsible dephosphorylating protein. described here allows relative level proteins, thus yielding information critical understanding regulation complex cascades.