Nanopores are single-molecule sensors used in nucleic acid analysis, whereas their applicability towards full protein identification has yet to be demonstrated. Here, we show that an engineered Fragaceatoxin C nanopore is capable of identifying individual proteins by measuring peptide spectra that are produced from hydrolyzed proteins. Using model proteins, we show that the spectra resulting from nanopore experiments and mass spectrometry share similar profiles, hence allowing protein fingerprinting. The intensity of individual peaks provides information on the concentration of individual peptides, indicating that this approach is quantitative. Our work shows the potential of a low-cost, portable nanopore-based analyzer for protein identification.
Extracellular polysaccharides and glycoproteins of pathogenic bacteria assist in adherence, autoaggregation, biofilm formation, and host immune system evasion. As a result, considerable research in the field of glycobiology is dedicated to study the composition and function of glycans associated with virulence, as well as the enzymes involved in their biosynthesis with the aim to identify novel antibiotic targets. Especially, insights into the enzyme mechanism, substrate binding, and transition-state structures are valuable as a starting point for rational inhibitor design. An intriguing aspect of enzymes that generate or process polysaccharides and glycoproteins is the level of processivity. The existence of enzymatic processivity reflects the need for regulation of the final glycan/glycoprotein length and structure, depending on the role they perform. In this Review, we describe the currently reported examples of various processive enzymes involved in polymerization and transfer of sugar moieties, predominantly in bacterial pathogens, with a focus on the biochemical methods, to showcase the importance of studying processivity for understanding the mechanism.
Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis.
Glycosylation is a ubiquitous process that is universally conserved in nature. The various products of glycosylation, such as polysaccharides, glycoproteins, and glycolipids, perform a myriad of intra- and extracellular functions. The multitude of roles performed by these molecules is reflected in the significant diversity of glycan structures and linkages found in eukaryotes and prokaryotes. Importantly, glycosylation is highly relevant for the virulence of many bacterial pathogens. Various surface-associated glycoconjugates have been identified in bacteria that promote infectious behavior and survival in the host through motility, adhesion, molecular mimicry, and immune system manipulation. Interestingly, bacterial glycosylation systems that produce these virulence factors frequently feature rare monosaccharides and unusual glycosylation mechanisms. Owing to their marked difference from human glycosylation, bacterial glycosylation systems constitute promising antibacterial targets. With the rise of antibiotic resistance and depletion of the antibiotic pipeline, novel drug targets are urgently needed. Bacteria-specific glycosylation systems are especially promising for antivirulence therapies that do not eliminate a bacterial population, but rather alleviate its pathogenesis. In this review, we describe a selection of unique glycosylation systems in bacterial pathogens and their role in bacterial homeostasis and infection, with a focus on virulence factors. In addition, recent advances to inhibit the enzymes involved in these glycosylation systems and target the bacterial glycan structures directly will be highlighted. Together, this review provides an overview of the current status and promise for the future of using bacterial glycosylation to develop novel antibacterial strategies.
Selective modification of carbohydrate residues in glycopeptides is highly relevant as a tool in glycobiology. In particular, oxidation allows for subsequent ligation with a label or handle and can be effectuated enzymatically or chemically. Chemical oxidation of carbohydrate residues in glycopeptides is nearly invariably done using periodate cleavage, leading to aldehydes. In this work, we applied palladium-catalyzed oxidation for the same purpose. The catalyst, [(neocuproine)PdOAc] 2 OTf 2 , developed for the site-selective oxidation of mono-and oligosacchar-ides on preparative scale, proved suitable for the oxidation of glucose residues in a series of glucopeptides. Careful optimization of the reaction conditions is necessary to get acceptable conversions without excessive over-oxidation of amino acid side-chains, in particular threonine. The resulting carbonyl function can be used for an oxime-ligation to biotin. A protocol for the analysis of the products using mass spectrometry is also reported.
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