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.

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification.

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the online integration with nano-high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoHPLC-ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.

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.

Hydrogen-bond interaction assisted branched copolymer HILIC material for separation and N-glycopeptides enrichment.

Hydrophilic interaction chromatography (HILIC) has attracted increasing attention in recent years due to its efficient application in the separation of polar compounds and the enrichment of glycopeptides. However, HILIC materials are still of weak hydrophilicity and thereby present weak retention and selectivity. In this work, branched copolymer modified hydrophilic material Sil@Poly(THMA-co-MBAAm), with high hydrophilicity and unique "claw-like" polyhydric groups, were prepared by "grafting from" thiol-ene click reaction. Due to the abundant functional groups provided by branched copolymer, the material showed excellent retention for nucleosides, necleobases, acidic compounds, sugars and peptides. Furthermore, Sil@Poly(THMA-co-MBAAm) was also applied for the N-glycosylation sites profiling towards the digests of the mouse brain, and 1997N-glycosylated peptides were identified, corresponding to 686 glycoprotein groups. Due to the assisted hydrogen-bond interaction, the selectivity for glycopeptide enrichment in the real sample reached 94.6%, which was the highest as far as we know. All these results indicated that such hydrogen-bond interaction assisted branched copolymer HILIC material possessed great potential for the separation and large scale glycoproteomics analysis.

Polyethyleneimine-modified graphene oxide nanocomposites for effective protein functionalization.

A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g(-1)), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions.