Mapping Sites of Protein Phosphorylation by Mass Spectrometry Utilizing a Chemical-Enzymatic Approach:  Characterization of Products from α-S1Casein Phosphopeptides

A novel chemical-enzymatic approach was developed to facilitate identification of phosphorylation sites in isolated phosphoproteins. ESI−TOF mass spectrometry was used to characterize products from the chemical-enzymatic cleavage of specific phosphorylation sites in bovine α-S1 casein and synthetic phosphopeptides containing substitutions at a single phosphorylation site. Further refinements to this approach for identification of protein phosphorylation sites and its utility for the quantification of phosphopeptides by isotope-dilution mass spectrometry are presented. Keywords: chemical-enzymatic • phosphorylation • 2-aminoethanethiol • isotope dilution mass spectrometry • phosphoproteins • phosphopeptides • quantification