Phosphopeptide Enrichment by Covalent Chromatography After Solid Phase Derivatization of Protein Digests on Reversed...

The isolation of the phosphopeptide constituents from phosphoprotein digests is prerequisite to facilitate the mass spectrometric characterization of phosphorylation events. Here, we describe a chemical proteomics approach which combines solid phase derivatization of phosphoprotein digests with phosphopeptide enrichment by covalent chromatography. The use of the solid phase support for derivatization ensures for speed and completeness of reactions. The isolates proved highly suitable for mapping of the sites of phosphorylation by collisionally induced dissociation (CID). The method combines robustness with simplicity of operation using equipment available in biological laboratories, and may be readily extended to map the sites of O-glycosylation.