Highly Specific Capture and Direct MALDI MS Analysis of Phosphopeptides by Zirconium Phosphonate on Self-Assembled Monolayers

The dynamic range and low stoichiometry of protein phosphorylation frequently demands the enrichment of phosphorylated peptides from protein digests prior to mass spectrometry. Several techniques have been reported in literature for phosphopeptide enrichment, including metal oxides such as TiO2 and ion metal affinity chromatography (IMAC). While the metal oxides have been used with reasonable success, IMAC has suffered from reduced selectivity and poor reproducibility. In this report, we present the first demonstration of the use of immobilized zirconium on a phosphonate-terminated self-assembled monolayer (SAM) for specific phosphopeptide capture and direct analysis by MALDI MS. By using the herein described functionalized-surface-based technology, efficient enrichment of phosphopeptides in different standard test systems such as α- or β-casein digests or synthetic phosphopeptides spiked in nonphosphorylated protein digest has been demonstrated. The limit of detection for a β-casein phosphopeptide was as...