Phosphoproteomic analysis with a solid-phase capture-release-tag approach

A comprehensive study of global phosphorylation events in biological systems is critical. We report a chemistry-based capture-release-tag method for isolation of complex phospho-Ser/Thr-containing peptides by liquid beta-elimination combined with solid-phase Michael addition. The free thiol groups of 6-(mercapto-acetylamino)-hexanoic acid functionalized resin are used as immobilized Michael donors to capture dehydro-serine/threonine peptides. After an acid-mediated release step, phospho-peptides are labeled with a 6-(2-mercapto-acetylamine)-hexanoic amide tag at phosphorylated sites. We applied this method to analyze the phosphorylation status of microtubule-associated proteins. We find that a CDK5 substrate microtubule-associated protein 2 (MAP2) is phosphorylated on residues that are within a homologous region of Tau. The chemical method corroborates previous results and suggests that Tau and MAP2 may contain a CDK5 phosphorylation motif.