Multisite phosphorylation of proteins plays an important role in signal transduction. However, it is difficult to describe precisely the mechanism of these phosphorylation cascades. In order to study the function of phosphorylated proteins in a deep-going way, enrichment protocol with high selectivity and coverage is urgent needed. In the present study Click TE-GSH, a novel HILIC material synthetized in our group, was used to enrich and sequentially elute phosphorylated peptides in different fractions. As a mixed-mode chromatographic material, Click TE-GSH exhibits both hydrophilic interaction and cation-exchange characteristics. To understand the retention mechanism, we firstly carried out investigation to study the influence of acetonitrile (ACN) concentration, solution pH value and salt concentration on the retention of phosphorylated peptides respectively. The results showed that phosphorylated peptides were eluted with low concentration of ACN and non-phosphorylated peptides were eluted with high concentration of ACN, which was in accordance with the hydrophilic retention characteristics of Click TE-GSH material. Meanwhile, pH value and salt concentration affected the retention of phosphorylated peptides, owing to the change of surface charge on the stationary phase. Under the optimized condition, mono-phosphorylated peptide, di-phosphorylated peptides and multiply phosphorylated peptides were enriched selectively. 6 mono-phosphorylated peptides, 2 di-phosphorylated peptides and 15 multiply phosphorylated peptides were effectively enriched and detected. By contrast, immobilized metal ion affinity chromatography (IMAC) was utilized to enrich phosphorylated peptides. 2 mono-phosphorylated peptides and 6 multiply phosphorylated peptides were identified. It is obvious the enrichment efficiency of Click TE-GSH was much higher than that of IMAC. The established method was validated with relatively complex sample, including peptide mixture of α-casein and bovine serum albumin (BSA) at the molar ratio of 1:1 and 1:10. In addition, Click TE-GSH was applied to enrich phosphorylated peptides in milk. 11 multiply phosphorylated peptides were successfully identified. The result proved the excellent selectivity of the method. Therefore, this optimized protocol has great potential for the enrichment of multiply phosphorylated peptides.

Key words: multiply phosphorylated peptides, Click TE-GSH, highly selective enrichment, mass spectrometry