Zirconium-Cation-Immobilized Core/Shell (Fe3O4@Polymer) Microspheres as an IMAC Material for the Selective Enrichment of Phosphopeptides

Immobilized metal affinity chromatography (IMAC) is a widely employed method for the enrichment of phosphopeptides from complex proteolytic digests prior to mass spectrometric analysis. In this work, a novel IMAC material, zirconium-phosphate (Zr4+-PO3)-modified magnetic Fe3O4/GMA-co-EDMA (core/shell) (Zr4+-Fe3O4@polymer) microspheres, has been synthesized in a facile manner and used for the selective capture of phosphopeptides from protein tryptic digests for mass spectrometry analysis. The enrichment conditions were optimized using a protein digest solution of beta-casein. By virtue of a thick and biocompatible poly(GMA-co-EDMA) shell, high tolerance of contaminants (salt, nonphosphopeptide) was demonstrated for the use of Zr4+-Fe3O4@polymer microspheres for the highly selective enrichment of phosphopeptides from a protein tryptic solution that contained a high concentration of NaCl (6.2 M) or urea (8,M) and a high ratio of nonphosphoprotein (BSA) to phosphoprotein (alpha-casein) (1:500). The performance of the Zr4+-Fe3O4@polymer microspheres was further successfully examined by the phosphoproteome analysis of mouse liver lysate.