Fe3O4@Al2O3 magnetic core–shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe3O4@Al2O3 magnetic core–shell microspheres with phosphopeptides has been developed. With a well-defined core–shell structure, the Fe3O4@Al2O3 magnetic core–shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe3O4@Al2O3 magnetic core–shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins β-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of β-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe3O4@Al2O3 magnetic core–shell microspheres over Fe3+-immobilized magnetic silica microspheres, commercial Fe3+–IMAC (immobilized metal affinity chromatography) resin, and TiO2 beads. Finally, the Al2O3 coated Fe3O4 microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe3O4@Al2O3 magnetic core–shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.