In this study, we developed a centrifugation assisted solid phase extraction (SPE) method for the selective enrichment of phosphopeptides using a new Ti4+-IMAC material synthesized in-house. This new material has the feature of big size and large specific surface area which makes it more suitable to enrich phosphopeptides in a SPE way. The spin tips loaded with the Ti4+-IMAC material were applied to enrich phosphopeptides from the complex protein digests. It was found that phosphopeptides can be specifically enriched from tryptic digest of bovine serum albumin and β-casein at a molar ratio up to 1000:1. And about 4700 unique phosphorylated peptides can be identified with the specificity as high as 99% from the tryptic digest of HeLa cell proteins. This tip was demonstrated to have good column-to-column reproducibility. Furthermore, it is fitted to analyze minute amount of sample. Compared with the conventional solution method, the SPE method facilitated the rapid and complete separation of the material with solution, which making it a time-saving and convenient method for phosphopeptide enrichment. Compared with the commercial TiO2 material, this new materials yielded much more phosphopeptide identifications and much higher enrichment specificity.
Keywords: Immobilized titanium (IV) ion affinity chromatography; Liquid chromatography–tandem mass spectrometry; Phosphopeptide enrichment; Protein phosphorylation; Solid phase extraction.
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