Development of erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns for selective enrichment of phosphopeptides

In this study, a novel method for the highly selective enrichment of phosphopeptides using erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns is presented. Erbium phosphate was synthesized by precipitation from boiling phosphoric acid and incubated overnight in erbium chloride solutions. The resulting powder was embedded in a monolithic poly(glycidyl methacrylate/ethylene dimethacrylate) polymer. The monolith was synthesized in a spin column by radical polymerization. Erbium phosphate demonstrated a high affinity and selectivity for phosphopeptides due to the strong interaction of trivalent erbium ions with the phosphate groups of phosphopeptides. The high selectivity and performance of the designed spin columns were demonstrated by successfully enriching phosphopeptides from tryptically digested protein mixtures containing the model phosphoproteins α- and β-casein, bovine milk, and human saliva. By the implementation of several washing steps, unspecific components were removed and the enriched phosphopeptides were effectively eluted from the spin columns under alkaline conditions. The selective performance of the presented method was further demonstrated by the enrichment of two synthetic phosphopeptides, which were spiked in tryptically digested and dephosphorylated HeLa cell lysates at low ratios. Finally, the presented approach was compared to conventional phosphopeptide enrichment by titanium oxide and revealed higher recoveries for the erbium phosphate doped monoliths.