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 (Zr 4+ -PO 3 )-modified magnetic Fe 3 O 4 /GMA- co -EDMA (core/shell) (Zr 4+ -Fe 3 O 4 @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 β-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 Zr 4+ -Fe 3 O 4 @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 (α-casein) (1:500). The performance of the Zr 4+ -Fe 3 O 4 @polymer microspheres was further successfully examined by the phosphoproteome analysis of mouse liver lysate.