Tailor-Made Magnetic Fe3O4@mTiO2 Microspheres with a Tunable Mesoporous Anatase Shell for Highly Selective and Effective Enrichment of Phosphopeptides

Abstract

Selective enrichment of phosphoproteins or phosphopeptides from complex mixtures is essential for MS-based phosphoproteomics, but still remains a challenge. In this article, we described an unprecedented approach to synthesize magnetic mesoporous Fe₃O₄@mTiO₂ microspheres with a well-defined core/shell structure, a pure and highly crystalline TiO₂ layer, high specific surface area (167.1 m²/g), large pore volume (0.45 cm³/g), appropriate and tunable pore size (8.6–16.4 nm), and high magnetic susceptibility. We investigated the applicability of Fe₃O₄@mTiO₂ microspheres in a study of the selective enrichment of phosphopeptides. The experiment results demonstrated that the Fe₃O₄@mTiO₂ possessed remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), large enrichment capacity (as high as 225 mg/g, over 10 times as that of the Fe₃O₄@TiO₂ microspheres), extreme sensitivity (the detection limit was at the fmol level), excellent speed (the enrichment can be completed in less than 5 min), and high recovery of phosphopeptides (as high as 93%). In addition, the high magnetic susceptibility allowed convenient separation of the target peptides by magnetic separation. These outstanding features give the Fe₃O₄@mTiO₂ composite microspheres high benefit for mass spectrometric analysis of phosphopeptides.