Fast and efficient phosphopeptide enrichment using TiO_(2) coated magnetic beads and its application to Drosophila Kc167 phosphoproteomic analysis

摘要

We have demonstrated a fast and selective phophopeptide enrichment method using TiO_(2) magnetic beads (Merck KGaA) from tests with various standard mixtures and Drosophila Kc167 cell lysate. The addition of DHB (Glycolic acid) in loading condition is not needed to obtain a high phophopeptide specificity. With this work flow, hundreds of phosphorylation sites can be identified from a single enrichment and single LC-MS run using low amount of Kc167 cell lysate (around 50 ug without any pre-fractionation procedure). A specificity of more than 90percent can be achieved. This protocol can easily be automated for large scale phosphorylation analysis. Even though this TiO_(2) magnetic beads method is proved to be highly compatible with complex real sample, for a complete phosphoproteomic analysis, we still need better sample preparation, further optimization and combination of different methods.