P78-T Solid-Phase Strategy for Phosphorylation/ Glycosylation Site Mapping

Chemically induced β-elimination of phosphate from serine and threonine coupled with Michael addition has emerged as a chemical strategy to address both the ion suppression and the gas-phase lability of the phosphate group. In previous work, we have adopted the chemistry to solid-phase derivatization on C18 ZipTip pipette tips using barium hydroxide as elimination base and 2-amino-ethanethiol as nucleophile.1 The utility of the protocol for improved phosphopeptide detection by signal enhancement was demonstrated with low-level amounts of tryptic protein digests, and the resultant increased MS/MS spectral information content greatly facilitated mapping of the site of phosphorylation. In this report, we have focused on chemistry optimization of O-phospho and O-GlcNAc modified peptides reported as resistant to β-elimination, i.e., those containing the modified residues followed by proline. Conclusive mapping of these phosphorylation sites has become increasingly important in view of the fact that phosphorylated Ser/Thr-Pro motifs are substrates for prolyl cis/trans isomerase Pin1.2 Similarly, unambiguous site determination of O-GlcNAc modifications has more recently attracted considerable interest because of the global and often site-specific reciprocal relationship between O-GlcNAc and O-phosphate in many cellular responses.3 We have used a panel of model peptides to define the optimal reaction conditions for both concurrent and consecutive elimination/Michael addition reactions and employed carbon/C18 mixed-phase ZipTips to afford efficient binding of small hydrophilic peptides.