Highly selective and rapid enrichment of phosphorylated peptides using gallium oxide-coated magnetic microspheres for MALDI-TOF-MS and nano-LC-ESI-MS/MS/MS analysis

In this work, we present, to our knowledge, the first demonstration of the utility of iron oxide magnetic microspheres coated with gallium oxide for the highly selective enrichment of phosphopeptide prior to mass spectrometric analysis. These microspheres that we prepared not only have a shell of gallium oxide, giving them a high-trapping capacity for the phosphopeptides, but also their magnetic property enables easy isolation by positioning an external magnetic field. Tryptic digest products of phosphoproteins including beta-casein, ovalbumin, casein, as well as five protein mixtures were used as the samples to exemplify the feasibility of this approach. In very short time (only 0.5 min), phosphopeptides sufficient for characterization by MALDI-TOF-MS were selectively enriched by the Ga2O3-coated Fe3O4 microspheres. The performance of the Ga2O3-coated Fe3O4 microspheres were further compared with Fe3+-immobilized magnetic silica microspheres, commercial Fe3+-IMAC resin, and TiO2 beads for enrichment of peptides originating from tryptic digestion of beta-casein and BSA with a molar ratio of 1:50, and the results proved a stronger selective ability of Ga2O3-coated Fe3O4 microspheres over the other materials. Finally, the Ga2O3-coated Fe3O4 microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. All results show that Ga2O3-coated Fe3O4 microsphere is an effective material for selective isolation and concentration of phosphopeptides.