Design and fabrication of reusable core-shell composite microspheres based on nanodiamond for selective enrichment of phosphopeptides.

A kind of core-shell composite microsphere (CM) with nano-on-micro structure was synthesized via grafting amine-modified nanodiamonds onto the surface of monodisperse nonporous polymeric microsphere. In this way, the agglomeration of nanodiamond particles in the solution was avoided. After modification with pyrogallol groups, CM could chelate titanium ions (Ti 4+ ) and thus be utilized as immobilized metal affinity chromatography (IMAC) sorbent to enrich phosphopeptides from biological samples. The resulting Ti 4+ -CM exhibited high enrichment efficiency and specificity to phosphopeptides. A total of 106 of unique phosphopeptides mapped to 29 phosphoproteins were clearly identified from 5 µL of a milk digest after enrichment. Owing to the strong chelation between Ti 4+ and pyrogallol ligands, the Ti 4+  is not released from the sorbent after completion of the enrichment process. As a result, the Ti 4+ -CM sorbent could be reused, and no significant loss of enrichment efficiency occurred even on the fourth run employing a β-casein digest as the sample. The strategy adopted presents a new way to prepare a high-performance reusable IMAC sorbent.