Qualitative and quantitative analysis of phosphopeptides with immobilized metal ion affinity chromatography enrichment, stable isotope labeling by amino acids in cell culture and nano-liquid chromatography-tandem mass spectrometry

Abstract

The progression of the cell cycle in eukaryote cells is tightly regulated by protein phosphorylation in order to ensure genetic integrity. Quantitative evaluation of phosphorylation changes during the cell cycle is obtained with titanium dioxide (TiO₂) enrichment coupled with stable isotope labeling and mass spectrometry (MS). However, information about multiply phosphorylated peptides is insufficient because of the low recovery of multiply phosphorylated peptides enriched and quantified with this strategy. In this study, simultaneous identification and quantitation of phosphopeptides, especially multiply phosphorylated peptides, were performed based on immobilized metal ion affinity chromatography (IMAC), stable isotope labeling by amino acids in cell culture (SILAC) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). Direct nano-ESI-MS/MS and nano-LC-MS/MS demonstrated complementary properties for phosphopeptide detection. A total of 355 phosphorylation sites from 268 phosphopeptides were identified and quantified. More than 30% of phosphopeptides were multiply phosphorylated ones. Up to 40 phosphopeptides were up-regulated and 24 phosphopeptides were down-regulated during the cell cycle. The method demonstrated itself as a feasible tool for quantitative analysis of phosphopeptides, especially multiply phosphorylated peptides from complex samples.