Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a novel competitive substitution strategy
Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ions coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells.
Sample Processing Protocol
The Ti4+-IMAC adsorbents were home-made according to the work of Houjiang Zhou. 1 mg Ti4+-IMAC adsorbents were firstly incubated with 200 μl 10 mM phosphate salt solution (orthophosphate, β-glycerophosphate or pyrophosphate) for 30 min and stepwise washed with 200 mM NaCl and Milli-Q water. The multi-phosphopeptides enrichment procedures were similar to the traditional strategies. Briefly, the peptides mixture was incubated with phosphate ions pre-treated affinity adsorbents with a ratio of 1:10 (w/w) for 30 min in the sample loading buffer (40% ACN and 3% TFA). Then the non-specific adsorbed peptides on the adsorbents surfaces were washed away by washing buffer 1 (50% ACN 6% TFA and 200 mM NaCl) and washing buffer 2 (30% ACN and 0.1% TFA) in sequential. Finally, the multi-phosphopeptides were eluted from the affinity adsorbents by 10% NH3•H2O and lyophilized.
Data Processing Protocol
All the data of the LC-MS/MS were searched by Maxquant (version 126.96.36.199) against a human protein database obtained from Uniprot (201312). Trypsin was selected as the specific enzyme with up to 3 miss cleavages. The phosphorylation of serine (S), threonine (T) and tyrosine (Y) and the oxidation of methionine (M) were set as variable modifications. Meanwhile, the carbamidomethylation of cysteine (C) was set as fixed modification. Phosphopeptides with the false discovery rate <0.01 were accepted for confident identifications with a minimum score of 20.
Liu Z, Wang F, Chen J, Zhou Y, Zou H. Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a competitive substitution strategy. J Chromatogr A. 2016 Jul 20. pii: S0021-9673(16)30964-5 PubMed: 27470094