Highly selective enrichment of multi-phosphopeptides by polyurea-modified macroporous adsorption resin via hydrogen-bond-mediated interaction.

The selective enrichment of phosphopeptides plays a crucial role in enhancing food nutritional value and developing functional foods. However, the identification of low-abundance multi-phosphopeptides is a formidable challenge as the extreme hydrophilicity and low ionization efficiency of the multi-phosphopeptides restrict in-depth research of multisite phosphorylation processes. To address this issue, a novel hydrogen-bond-mediated functional material (MAR@PUP) was facilely designed and fabricated , with epoxy group-containing macroporous adsorption resin (MAR) as the matrix, via a "one-pot" reaction of p-phenylene diisocyanate (PDI) and tris(2-aminoethyl)amine (TAEA). The resulting MAR@PUP leverages the polyurea-based groups on its surface to achieve selective enrichment of mono-/multi-/total phosphopeptides by modulating the strength of multiple hydrogen bond interactions under varying conditions, with particularly high efficiency in multi-phosphopeptides enrichment. Furthermore, nine multi-phosphopeptides were successfully identified from 5 µg of actual nonfat milk samples, which verified the feasibility of MAR@PUP material for practical sample analysis and its promising application potential. This research provides a novel and practical strategy for the efficient enrichment and accurate determination of multi-phosphopeptides in food chemistry.