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The evolution of the SARS-CoV2 coronavirus spike S-protein is studied using a mass spectrometry based protein phylogenetic approach. A study of a large dataset comprising sets of peptide masses derived from over 3000 proteins of the SARS-CoV2 virus shows that the approach is capable of resolving and correctly displaying the evolution of the major variants of concern. Using these numerical datasets, through a pairwise comparison of sets of proteolytic peptide masses for each protein, the tree is built without the need for the sequence data itself or any sequence alignment. In the same analysis, single point mutations are calculated from peptide mass differences of different protein sets and these are displayed at the branch nodes on the tree. The tree topology is found to be consistent with that generated using conventional sequence-based phylogenetics by a manual visualisation and using a tree comparison algorithm. The mass tree resolves major variants of the virus and displays non-synonymous mutations, calculated based on the mass data alone, on the tree that enable protein evolution to be charted and tracked along interconnected branches. Tracking the evolution of the SARS-CoV2 coronavirus S-protein is of particular importance given its role in the attachment of the virus to host cells ahead of viral replication. [ABSTRACT FROM AUTHOR]

The accurate determination of disulfide bonds for protein identification is in high demand. In this study, a simple electrochemical-mass spectrometry (EC-MS) method that possesses advantages of real-time information, simultaneous disulfide bond electroreduction and tagging was developed. In this EC-MS, an ITO glass corner functions as a counter electrode and spray system, and allows the direct sampling of the droplet-scale reacting solution in real-time. The application of this method was successfully demonstrated by electrochemical reduction of oxidized glutathione (GSSG) with one disulfide bond as well as insulin with multiple disulfide bonds. The preferred electroreduction of intermolecular-bonded disulfides for insulin has been observed and the intramolecular bond was not favored. Moreover, simultaneously tagging the formed thiol residues from electroreduction of GSSG using electrogenerated intermediates such as dopamine orthoquinone (DQ) and benzoquinone (Q) was performed. A proof-of-concept was also demonstrated with a large molecule, β-lactoglobulin A. The relationship between signal strength and operating parameters was also studied. This method successfully detected the reduction reaction of the disulfide bond in the polypeptide and protein. The detection limit (S/N ≥ 3) is 0.398 μg mL−1. These results suggest that this EC-MS platform can count cysteine moieties in proteins using a single drop of sample and in real-time and is promising for protein identification experiments. [ABSTRACT FROM AUTHOR]

O-Linked N-acetylglucosamine (O-GlcNAc) is an emerging post-translational modification (PTM) of proteins. Analysis of O-GlcNAc modification using mass spectrometry (MS) is often problematic because of the low stoichiometry of the modification. In this study, we developed a new method for enriching O-GlcNAc-modified peptides using reversible hydrazide chemistry. O-GlcNAc-modified peptides were first labeled with N-azidoacetylgalactosamine (GalNAz) using gatactosyltransferase-T1 (Y289L) enzyme. The azide group on the GalNAz residue was then reacted with 3-ethynylbenzaldehyde via copper-catalyzed Huisgen 1,3-cycloaddition “click reaction” to form an aromatic aldehyde group of glycopeptides. Aromatic aldehyde-derivatized glycopeptides were enriched by reversible hydrazone formation with hydrazide resin. Reaction conditions for each step, especially for the click reaction, were optimized to achieve complete reaction without significant side reactions. This method was validated using a tryptic digest of bovine α-crystallin, which is an O-GlcNAc-modified glycoprotein. The developed method was also applied to structure-specific enrichment of N-linked glycopeptides having non-reducing terminal GlcNAc residues. All materials and chemicals required for this method are commercially available and there is no need to prepare any special reagents, facilitating the introduction of this method in any laboratory. [ABSTRACT FROM AUTHOR]