title: Characterization of multi-phosphopeptides by muHPLC-ESI-MS/MS with alkaline phosphatase treatment.
abstract: The detection of phosphopeptides, especially multi-phosphopeptides, by tandem electrospray ionization mass spectrometry (ESI-MS/MS) is a great challenge due to their low abundance and the poor ionization efficiency of samples. In our recent study, a strategy was proposed for the analysis of trace multi-phosphopeptides which combined selective enrichment of phosphorylated peptides by TiO2 and dephosphorylation by alkaline phosphatase (AP). After separation by muHPLC, the profiles of enriched peptides before and after AP treatment were compared, and the additional peaks appearing in the latter case hinted at the existence of multi-phosphopeptides. Subsequently, an incomplete dephosphorylation reaction was performed to partially remove the phosphate groups so that the phosphorylation sites of the multi-phosphopeptides might be estimated. Through analysis of the digests of beta-casein and extracted proteins of bovine milk, more information on the multi-phosphopeptides was obtained by muHPLC-ESI-MS/MS than that obtained without AP treatment, which demonstrated that such a strategy might supply some potential information about trace multi-phosphopeptides lost in shotgun analysis.
authors.affiliation: National Chromatographic Research & Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Jicheng
authors.initials: J
authors.lastname: Duan
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Weibing
authors.initials: W
authors.lastname: Zhang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D010748:Phosphopeptides; D000469:Alkaline Phosphatase