Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells

• TiO2/MHMSS and ZrAs–Fe3O4@SiO2 were used for sequential phosphopeptides enrichment.
• A novel strategy for the identification of phosphosites was established.
• We were able to identify 11,579 unique phosphosites using the method.

As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs–Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11,579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs–Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively.