A protocol combining immobilized metal ion affinitychromatography and
-elimination with concurrent Michaeladdition has been developed for enhanced analysis ofprotein phosphorylation. Immobilized metal ion affinitychromatography was initially used to enrich for phosphorylated peptides.
-Elimination, with or without concurrentMichael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound tothe chromatography resin. Derivatization of the phosphatefacilitated the precise determination of phosphorylationsites by MALDI-PSD/LIFT tandem mass spectrometry,avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ionaffinity chromatography and
-elimination with concurrentMichael addition in this manner circumvented severalinherent disadvantages of the individual methods. Inparticular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to
-elimination/Michael addition and (ii) the elution of peptides fromthe chromatography resin as derivatized phosphopeptidesdistinguished them from unphosphorylated species thatwere also retained. The chemical derivatization of phosphopeptides greatly increased the information obtainedduring peptide sequencing by mass spectrometry. Thecombined protocol enabled the detection and sequencingof phosphopeptides from protein digests at low femtomoleconcentrations of initial sample and was employed toidentify novel phosphorylation sites on the cell adhesionprotein p120 catenin and the glycoprotein fetuin.