Fe 3O 4@Al 2O 3 magnetic core–shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Abstract

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 Fe 3O 4@Al 2O 3 magnetic core–shell microspheres with phosphopeptides has been developed. With a well-defined core–shell structure, the Fe 3O 4@Al 2O 3 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 Fe 3O 4@Al 2O 3 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 Fe 3O 4@Al 2O 3 magnetic core–shell microspheres over Fe 3+-immobilized magnetic silica microspheres, commercial Fe 3+–IMAC (immobilized metal affinity chromatography) resin, and TiO 2 beads. Finally, the Al 2O 3 coated Fe 3O 4 microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe 3O 4@Al 2O 3 magnetic core–shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.