Fast fabrication and modification of polyoctahedral silsesquioxane-containing monolithic columns via two-step photo-initiated reactions and their application in proteome analysis of tryptic digests

A fast and robust approach was developed to fabricate and modify hybrid monolithic columns via two-step photo-initiated reactions. At first, acrylopropyl polyoctahedral silsesquioxane (acryl-POSS) and 3-(triallyl silyl) propyl acrylate (TAPA) were chosen as precursors to synthesize poly (POSS-co-TAPA) monolithic column (monolith I) via photo-initiated free-radical polymerization within 10 min, which left lots of allyl groups on the surface of monolith. Secondly, two thiol-containing compounds, penicillamine and 1-octadecanethiol (ODT), were introduced to modify the prepared poly (POSS-co-TAPA) column via photo-initiated thiol-ene click reaction within 20 min. Finally, three resulting monolithic columns were applied to separate phenolic, anilines and antibiotics mixtures. These mixtures were baseline-separated on the monolith modified with penicillamine (monolith II), exhibiting better selectivity than both pristine monolith I and that modified with ODT (monolith HI). Additionally, these columns were further used for separation of tryptic digest of HeLa cells by cLC-MS/MS. The 5071 unique peptides mapped to 2442 proteins were identified from HeLa cells digest on monolith H, which were superior over those on monolith III, but slightly lower than those on monolith I. These results demonstrated that these POSS-containing columns exhibited great separation ability for complex samples.