3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified Magnetic Graphene Oxide Nanocomposites for Human Plasma Glycoproteins Enrichment under Physiological Conditions.

Boronate affinity materials have been successfully used for the selective recognition of glycoproteins. However, by such materials, the large-scale glycoproteins enrichment from human plasma under physiological conditions is rarely reported. In this work, 3-carboxybenzoboroxole (CBX) functionalized polyethylenimine (PEI) modified magnetic graphene oxide nanocomposites were synthesized. Benefitting from the low pKa value of CBX (∼6.9) and PEI dendrimer-assisted multivalent binding, the Freundlich constant (KF) for the adsorption of horseradish peroxidase (HRP) was 3.0-7.3 times higher than that obtained by previous work, displaying the high enrichment capacity. Moreover, PEI could improve the hydrophilicity of nanocomposites and reduce nonglycoprotein adsorption. Therefore, such nanocomposites were successfully applied to the analysis of human plasma glycoproteome under physiological conditions, and the identified glycoproteins number and recognition selectivity was increased when compared to the results obtained by previous boronic acid-functionalized particles (Sil@Poly(APBA-co-MBAAm)) under common alkaline condition (137 vs 78 and 67.8% vs 57.8%, respectively). In addition, thrombin (F2), an important plasma glycoprotein, labile under alkaline conditions, was specifically identified by our method, demonstrating the great promise of such nanocomposites in the deep-coverage glycoproteome analysis.

Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling.

Exosomes are secreted nanovesicles shed by almost all kinds of cells. Recently, increased interest has been focused on these extracellular vesicles as natural carriers transporting biological contents for intercellular communication. However, current isolation techniques, such as ultracentrifugation, are not convenient and often require specialized equipment. Herein, we describe a polyethylene glycol (PEG)-based approach, which could permit facile, low-cost and effective isolation of exosomes from cell culture supernatant. High-resolution electron microscopes clearly visualized the size and morphology of isolated exosome aggregates, implying the mechanism of PEG-based precipitation. Combined with tandem mass spectrometry analysis, 6299 protein groups encoded by 5120 genes were successfully characterized from HeLa cell culture supernatant, including numerous exosome proteins which could overlap 97% of the Top 100 exosome marker proteins recorded in the ExoCarta database, as well as a series of low-abundance cytokines and biomarkers. Furthermore, we found a higher ratio of neo-cleavage sites in proteins identified from exosomes compared with cellular proteins, revealing the potential roles of exosomes in accumulation and transportation of protein degradation intermediates.