Identification of In Vivo Protein Phosphorylation Sites with Mass Spectrometry
Protein phosphorylation, principally on Ser, Thr, and Tyr residues, is one of the most common post-translational cellular regulatory mechanisms (1, 2, 3, 4). Phosphorylation can modulate enzyme activity, alter its affinity to other proteins, and transmit signals through kinase cascades that often are branched and interactive. To understand the molecular basis of these regulatory mechanisms and to dissect signal-transduction pathways, it is necessary to determine the sites that are phosphorylated in vivo. Mass spectrometry is in the process of becoming the method of choice for the identification of protein phosphorylation sites (5). This mass spectrometric approach offers at least four major advantages over the conventional biochemical and genetic approaches. First, it is accurate. There is no ambiguity once the phosphorylation site is identified through mass spectrometric sequencing of the phosphopeptide. Second, it is fast. The cycle of identifying phosphorylation sites is typically a few days. Third, it does not require prior knowledge of phosphorylation of the protein of interest. Phosphorylation sites can be identified even for proteins that the consensus phosphorylation sites of their up stream kinases are not known. Fourth, it does not require 32P labeling.
KeywordsBurner HPLC Acetonitrile Electrophoresis Bicarbonate
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