Characterization of multi-phosphopeptides by μHPLC–ESI-MS/MS with alkaline phosphatase treatment

✍ Scribed by Hui Wang; Jicheng Duan; Lihua Zhang; Zhen Liang; Weibing Zhang; Yukui Zhang

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: English
• Weight: 783 KB
• Volume: 31
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200700445

✦ Synopsis

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 TiO~2~ and dephosphorylation by alkaline phosphatase (AP). After separation by μHPLC, 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 β‐casein and extracted proteins of bovine milk, more information on the multi‐phosphopeptides was obtained by μHPLC–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.