Fabrication of hydrophilic titanium (IV)-immobilized polydispersed microspheres via inverse suspension polymerization for enrichment of phosphopeptides in milk.

It is prerequisite to efficient extraction of phosphopeptides in bottom-up strategy for protein phosphorylation research. A kind of Ti-immobilized polydispersed phosphate-rich microsphere was fabricated via inverse suspension polymerization by employing vinylphosphonic acid (VPA) and N,N-methylenebisacrylamide (MBA) as functional monomer and crosslinker, respectively. The resulting microsphere demonstrated excellent sensitivity (as low as 10 fmol), selectivity (the mass ratio of β-casein to BSA digests is 1/500), and adsorption capacity (up to 200 mg g). What's more, 113 unique phosphopeptides assigned to 25 unique phosphoproteins were indiscriminately identified from 5 μL of pasteurized milk digest, exhibiting great performance in capturing phosphopeptides. In this approach, only two steps were required to synthesize Ti-IMAC (Ti-Immobilized metal affinity chromatography). Compared with other methods required multistep modifying process, this strategy is simple and time-saving, offering a prospect of pilot production and commercialization. It is expected that the application of IMAC in milk and other food samples will still make it possible to unravel the huge complexity of the Foodome in the near future.