Boronate Avidity Assisted by Dendrimer-like Polyhedral Oligomeric Silsesquioxanes for a Microfluidic Platform for Selective Enrichment of Ubiquitination and Glycosylation

A new strategy of improving boronate avidity with good accessibility of sites was suggested by utilizing a dendrimer-like structure of boron materials based on octavinyl-polyhedral oligomeric silsesquioxanes (Ov-POSS). 3-(Acrylamido)phenylboronic acid (AAPBA) was used as a functional monomer and ethylene glycol dimethacrylate (EDMA) and Ov-POSS as cross-linkers. The resulting Ov-POSS cross-linked boron monolith exhibited 27 times stronger affinity for glycoproteins than the Ov-POSS-free monolith. Importantly, the bonding strength of the poly(AAPBA-co-Ov-POSS-co-EDMA) monolith to the glycoproteins with multiple sugars, horseradish peroxidase (HRP) was 4 orders of magnitude higher than that of the single cis-diol-containing compound. The resulting monolith was used as a part of a microfluidic platform for online processing of the protein extracts from mouse liver, which integrated five functions, including protein grading, denaturation, enzymatic hydrolysis, and enrichment of glycopeptides and ubiquitin-modified peptides. The sample processing time can be reduced by nearly half compared to the offline method. Moreover, 86.7% of glycopeptides and 75% of glycoproteins were newly identified after treatment. All of the results indicated that the synergistic strategy of Ov-POSS cross-linking can significantly improve trace glycosylation's binding capacity and enrichment performance. The microfluidic platform developed may provide a promising technical tool for automated, high-efficiency, high-throughput analysis for post-translational modification proteomics.