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Human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for cellular entry of SARS-CoV-2 into human host cells. hACE2 is heavily glycosylated and glycans on the receptor may play a role in viral binding. Thus, comprehensive characterization of hACE2 glycosylation could aid our understanding of interactions between the receptor and SARS-CoV-2 spike (S) protein, as well as provide a basis for the development of therapeutic drugs targeting this crucial interaction. Herein, 138 N-glycan compositions were identified, most of which are complex-type N-glycans, from seven N-glycosites of hACE2. Among them, 67% contain at least one sialic acid residue. At the level of glycopeptides, the overall quantification of sialylated glycan isomers observed on the sites N322 and N546 have a higher degree of NeuAc (α2–3)Gal (over 80.3%) than that of other N-glycosites (35.6–71.0%). In terms of O-glycans, 69 glycan compositions from 12 O-glycosites were identified, and especially, the C-terminus of hACE2 is heavily O-glycosylated. The terminal sialic acid linkage type of H1N1S1 and H1N1S2 are covered highly with α2,3-sialic acid. These findings could aid the investigation of the interaction between SARS-CoV-2 and human host cells. [ABSTRACT FROM AUTHOR]

Novel magnetic silica nanoparticles functionalized with layer-by-layer detonation nanodiamonds (dNDs) were prepared by coating single submicron-size magnetite particles with silica and subsequently modified with dNDs. The resulting layer-by-layer dND functionalized magnetic silica microspheres (Fe3O4@SiO2@[dND]n) exhibit a well-defined magnetite–core–silica–shell structure and possess a high content of magnetite, which endow them with high dispersibility and excellent magnetic responsibility. Meanwhile, dNDs are known for their high affinity and biocompatibility towards peptides or proteins. Thus, a novel convenient, fast and efficient pretreatment approach of low-abundance peptides or proteins was successfully established with Fe3O4@SiO2@[dND]n microspheres. The signal intensity of low-abundance peptides was improved by at least two to three orders of magnitude in mass spectrometry analysis. The novel microsphere also showed good tolerance to salt. Even with a high concentration of salt, peptides or proteins could be isolated effectively from samples. Therefore, the convenient and efficient enrichment process of this novel layer-by-layer dND-functionalized microsphere makes it a promising candidate for isolation of protein in a large volume of culture supernatant for secretome analysis. In the application of Fe3O4@SiO2@[dND]n in the secretome of hepatoma cells, 1473 proteins were identified and covered a broad range of pI and molecular weight, including 377 low molecular weight proteins. [ABSTRACT FROM AUTHOR]

Here we present the novel functionalization of detonation nanodiamond (dND) with amino-phenyl boronic acid (APBA) assisted by poly-l-lysine (PL) and PEG-diglycolic acid (PEG) (dND-p-APBA). With the assistance of biocompatible poly-l-lysine and PEG, dND-p-APBA particles show good dispersibility in aqueous solution and plentiful boronic acid functional groups on the surface, which make it a novel material promising for glycoproteome. The dND-p-APBA particles demonstrate highly specific and efficient capture of glycopeptides from complex samples. In comparison with direct (or traditional) analysis, the novel method shows better specificity for glycopeptides even when non-glycopeptides are 40 times more than glycopeptides. Besides that, the detection sensitivity of glycopeptides could be increased 50 times by the novel strategy. Additionally, the recovery of glycopeptides is up to 72.2% and the glycopeptides even can be sensitively specifically enriched and detected in the presence of 1 M sodium chloride or 0.1% sodium dodecyl sulfate. Furthermore, in combination with liquid chromatography electronspray ionization mass spectrometry (LC-ESI-MS), the novel approach was successfully applied to characterize one fraction of mouse liver, in which 40 different N-glycosylation peptides within 34 unique glycoproteins have been identified. Accordingly, the usefulness of the functionalized dND might be a promising efficient and specific analytical platform in glycoproteome analysis. [ABSTRACT FROM AUTHOR]

With an ultra-high surface area and abundant functional groups, graphene oxide (GO) provides an ideal substrate for the immobilization of trypsin. We demonstrated that trypsin could be immobilized on GO sheets assisted by polymers as molecular spacers to maintain the activity of the enzyme. And with the trypsin-linked GO as the enzyme immobilization probe, a novel microwave-assisted on-plate digestion method has been developed with subsequent analysis by MALDI-MS. The feasibility and performance of the digestion approach were demonstrated by the proteolysis of standard proteins. The results show that this novel approach substantially accelerated proteolysis and reduced the time required for traditional procedures involving on-plate enzymatic digestion and sample preparation prior to MALDI-MS analysis. The novel digestion approach is simple and efficient, offering great promise for high throughput protein identification.