Abstract: A novel detection method for the analysis of multi-phosphopeptides using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry (μHPLC-ESI-MS/MS) was proposed by the dephosphorylation treatment of alkaline phosphatase (AP). After the selective enrichment by a microcolumn packed with TiO2, phosphopeptides from the tryptic digests of β-casein were dephosphorylated by AP. Through the removal of phosphate groups, the detection of multi-phosphopeptides according to the non-phosphorylated ones was achieved by ESI-MS/MS. By comparing the chromatograms before and after the AP treatment, mono-phosphopeptides were identified based on the relative molecular mass (Mr) difference of 80. Furthermore, since more peaks appeared after the treatment, the existence of multi-phosphopeptides was proven. By controlling the treatment procedure, the partial dephosphorylation of multi-phosphopeptides was performed, and the multi-phosphorylated sites of the digest of β-casein were found to be on serine residues at the possible sites of 17, 18 and 19.

Key words: alkaline phosphatase , electrospray ionization tandem mass spectrometry (ESI-MS/MS), multi-phosphopeptide, phosphorylation site, microcolumn high performance liquid chromatography (μHPLC)