Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry

Consecutive enzymatic: reactions of analytes which are affinity bound to im mobilized metal ion beads with subsequent direct analysis of the products b y matrix-assisted laser desorption/ ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta -casein and alpha-casein, as well as one phosphopeptide from a kinase react ion mixture. Agarose loaded with either Fe3+ or Ca3+ was used to isolate ph osphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+- loaded agarose was used rn isolate phosphopeptides. The selectivity for mon ophosphorylated peptides, however, was better with Fe3+ loaded agarose. Thi s technique is easy to use and has the ability to analyze extremely complic ated phosphopeptide mixtures. Moreover, it eliminates the need for prior hi gh-performance liquid chromatography separation or radiolabeling, thus grea tly simplifying the sample preparation. (J Am Soc Mass Spectrom 2000, 11, 2 73-282) (C) 2000 American Society for Mass Spectrometry.