[Detection of multi-phosphopeptide sites using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry].
A novel detection method for the analysis of multi-phosphopeptides using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry (microHPLC-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 beta-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 stees of the digest of beta-casein were found to be on serine residues at the possible sites of 17, 18 and 19.
Wang
Hui
H
National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, the Chinese Academy of Sciences, Dalian 116023, China. huiwang@dicp.ac.cn
Duan
Jicheng
J
Yuang
Huiming
H
Zhang
Lihua
L
Zhang
Weibing
W
Zhang
Yukui
Y
D000469:Alkaline Phosphatase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010748:Phosphopeptides; D000469:Alkaline Phosphatase