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Snake venoms are comprised of bioactive proteins and peptides that facilitate severe snakebite envenomation symptoms. A comprehensive understanding of venom compositions and the subtle heterogeneity therein is important. While bottom-up proteomics has been the well-established approach to catalogue venom compositions, top-down proteomics has emerged as a complementary strategy to characterize venom heterogeneity at the intact protein level. However, top-down proteomics has not been as widely implemented in the snake venom field as bottom-up proteomics, with various emerging top-down methods yet to be developed for venom systems. Here, we have explored three main top-down mass spectrometry methodologies in a proof-of-concept study to characterize selected three-finger toxin and phospholipase A 2 proteoforms from the forest cobra ( Naja melanoleuca ) venom. We demonstrated the utility of a data-independent acquisition mode "MS E " for untargeted fragmentation on a chromatographic time scale and its improvement in protein sequence coverage compared to conventional targeted tandem mass spectrometry analysis. We also showed that protein identification can be further improved using a hybrid fragmentation approach, combining electron-capture dissociation and collision-induced dissociation. Lastly, we reported the promising application of multifunctional cyclic ion mobility separation and post-ion mobility fragmentation on snake venom proteins for the first time.

Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors including the length and composition of bases in the sequence. Here, different DNA base modifications have been explored, primarily using native mass spectrometry, in efforts to enable stronger binding between the triplex forming oligonucleotide (TFO) and duplex target sites. These modifications can also be used to overcome pyrimidine interruptions in the duplex sequence in promoter regions of genomes, to expand triplex target sequences for antigene therapies. Using model sequences with a single pyrimidine interruption, triplex forming oligonucleotides containing locked nucleic acid base modifications were shown to have a higher triplex binding propensity than DNA-only and dSpacer-containing TFOs. However, the triplex forming ability of these systems was limited by the competitive formation of multiple higher order assemblies. Triplex forming sequences that correspond to specific gene targets from the Pseudomonas aeruginosa genome were also investigated, with LNA-containing TFOs the only variant able to form triplex using these sequences. This work indicates the advantages of utilizing synthetically modified TFOs to form triplex assemblies in vivo for potential therapeutic applications and highlights the advantages of native mass spectrometry for the study of their formation.

Snakebite envenomation has been a long-standing global issue that is difficult to treat, largely owing to the flawed nature of current immunoglobulin-based antivenom therapy and the complexity of snake venoms as sophisticated mixtures of bioactive proteins and peptides. Comprehensive characterisation of venom compositions is essential to better understanding snake venom toxicity and inform effective and rationally designed antivenoms. Additionally, a greater understanding of snake venom composition will likely unearth novel biologically active proteins and peptides that have promising therapeutic or biotechnological applications. While a bottom-up proteomic workflow has been the main approach for cataloguing snake venom compositions at the toxin family level, it is unable to capture snake venom heterogeneity in the form of protein isoforms and higher-order protein interactions that are important in driving venom toxicity but remain underexplored. This review aims to highlight the importance of understanding snake venom heterogeneity beyond the primary sequence, in the form of post-translational modifications that give rise to different proteoforms and the myriad of higher-order protein complexes in snake venoms. We focus on current top-down proteomic workflows to identify snake venom proteoforms and further discuss alternative or novel separation, instrumentation, and data processing strategies that may improve proteoform identification. The current higher-order structural characterisation techniques implemented for snake venom proteins are also discussed; we emphasise the need for complementary and higher resolution structural bioanalytical techniques such as mass spectrometry-based approaches, X-ray crystallography and cryogenic electron microscopy, to elucidate poorly characterised tertiary and quaternary protein structures. We envisage that the expansion of the snake venom characterisation "toolbox" with top-down proteomics and high-resolution protein structure determination techniques will be pivotal in advancing structural understanding of snake venoms towards the development of improved therapeutic and biotechnology applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The use of direct injection ion mobility mass spectrometry (DI-IM-MS) to detect and identify betacyanin pigments in A. hortensis 'rubra' extracts was explored for the first time, with results compared to conventional LC-MS/MS analysis. The anti-inflammatory activities of leaf and seed extracts, alongside purified amaranthin and celosianin pigments, were investigated using a model of lipopolysaccharide (LPS)-activated murine macrophages. Extracts and purified pigments significantly inhibited the production of prostaglandin E 2 and NO by up to 90% and 70%, respectively, and reduced the expression of Il6, Il1b, Nos2, and Cox2. Leaf and seed extracts also decreased secretion of Il6 and Il1b cytokines and reduced protein levels of Nos2 and Cox2. Furthermore, extracts and purified pigments demonstrated potent dose-dependent radical scavenging activity in a cellular antioxidant activity assay (CAA) without any cytotoxic effects. Our research highlights the promising biological potential of edible, climate-resilient A. hortensis 'rubra' as a valuable source of bioactive compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Red-fleshed apple cultivars with an enhanced content of polyphenolic compounds have attracted increasing interest due to their promising health benefits. Here, we have analysed the polyphenolic content of young, red-fleshed apples (RFA) and optimised extraction conditions of phenolics by utilising natural deep eutectic solvents (NDES). We also compare the antioxidant, neuroprotective and antimicrobial activities of NDES- and methanol-extracted phenolics from young RFA. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was used for phenolics identification and quantification. Besides young RFA, ripe red-fleshed, young and ripe white-fleshed apples were analysed, revealing that young RFA possess the highest phenolic content (2078.4 ± 4.0 mg gallic acid equivalent/100 g), and that ripe white-fleshed apples contain the least amount of phenolics (545.0 ± 32.0 mg gallic acid equivalent/100 g). The NDES choline chloride-glycerol containing 40 % w/w H 2 O gave similar yields at 40 °C as methanol. In addition, the polyphenolics profile, and bioactivities of the NDES extract from young RFA were comparable that of methanol extracts. Altogether, our data show that NDES extracts of young RFA are a promising source of bioactive polyphenolics with potential applications in diverse sectors, e.g., for functional food production, smart material engineering and natural therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Australian elapid snake radiation (Hydrophiinae) has evolved in the absence of competition from other advanced snakes. This has resulted in ecological specialisation in Australian elapids and the potential for venom proteomes divergent to other elapids. We characterised the venom of the Australian elapid Vermicella annulata (eastern bandy bandy). The venom was analysed using a two-dimensional fractionation process consisting of reverse-phase high-performance liquid chromatography then sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by bottom-up proteomics. Resulting peptides were matched to a species-specific transcriptome and 87% of the venom was characterised. We identified 11 toxins in the venom from six families: snake venom metalloproteinases (SVMP; 24.2%; two toxins) that are class P-III SVMPs containing a disintegrin-like domain, three-finger toxins (3FTx; 21.6%; five toxins), kunitz peptides (KUN; 19.5%; one toxin), cysteine-rich secretory proteins (CRiSP; 18%; one toxin), and phospholipase A2 (PLA2; 4%; two toxins). The venom had low toxin diversity with five protein families having one or two toxins, except for 3FTx with five different toxins. V. annulata expresses an unusual venom proteome, with high abundances of CRiSP, KUN and SVMP, which are not normally highly expressed in elapid venoms. This unusual venom composition could be an adaptation to its specialised diet. BIOLOGICAL SIGNIFICANCE: Although the Australian elapid radiation represents the most extensive speciation event of elapids on any continent, with 100 terrestrial species, the venom composition of these snakes has rarely been investigated, with only five species currently characterised. Here we provide the venom proteome of a sixth species, Vermicella annulata. The venom of this species could be particularly informative from an evolutionary perspective, as it is an extreme dietary specialist, only preying on blind snakes (Typhlopidae). We show that V. annulata expresses a highly unusual venom for an elapid, due to the high abundance of the protein families SVMP, CRiSP, and KUN, which together make up 61% of the venom. When averaged across all species, a typical elapid venom is 82% PLA 2 and 3FTx. This is the second recorded instance of an Australian elapid having evolved highly divergent venom expression., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Deoxyribonucleic acid is a genetic biomacromolecule that contains the inherited information required to build and maintain a living organism. While the canonical duplex DNA structure is rigorously characterized, the structure and function of higher order DNA structures such as DNA triplexes are comparatively poorly understood. Previous literature has shown that these triplexes can form under physiological conditions, and native mass spectrometry offers a useful platform to study their formation and stability. However, experimental conditions including buffer salt concentration, pH, and instrumentation parameters such as ion mode can confound analysis by impacting the amount of triplex observed by mass spectrometry. Using model 30mer Y-type triplex sequences, we demonstrate the influence a range of experimental variables have on the detection of DNA triplex structures, informing suitable conditions for the study. When carefully considered conditions are used, mass spectrometry offers a powerful complementary tool for the analysis of higher order DNA assemblies.

Snake venoms consist of highly biologically active proteins and peptides that are responsible for the lethal physiological effects of snakebite envenomation. In order to guide the development of targeted antivenom strategies, comprehensive understanding of venom compositions and in-depth characterisation of various proteoforms, often not captured by traditional bottom-up proteomic workflows, is necessary. Here, we employ an integrated 'omics' and intact mass spectrometry (MS)-based approach to profile the heterogeneity within the venom of the forest cobra (Naja melanoleuca), adopting different analytical strategies to accommodate for the dynamic molecular mass range of venom proteins present. The venom proteome of N. melanoleuca was catalogued using a venom gland transcriptome-guided bottom-up proteomics approach, revealing a venom consisting of six toxin superfamilies. The subtle diversity present in the venom components was further explored using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to intact MS. This approach showed a significant increase in the number of venom proteoforms within various toxin families that were not captured in previous studies. Furthermore, we probed at the higher-order structures of the larger venom proteins using a combination of native MS and mass photometry and revealed significant structural heterogeneity along with extensive post-translational modifications in the form of glycosylation in these larger toxins. Here, we show the diverse structural heterogeneity of snake venom proteins in the venom of N. melanoleuca using an integrated workflow that incorporates analytical strategies that profile snake venom at the proteoform level, complementing traditional venom characterisation approaches., Competing Interests: Declaration of Competing Interest Chia-De Ruth Wang reports financial support was provided by Australian Government. Alix Harlington reports financial support was provided by Australian Government., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Naja nivea ( N. nivea ) is classed as a category one snake by the World Health Organization since its envenomation causes high levels of mortality and disability annually. Despite this, there has been little research into the venom composition of N. nivea , with only one full venom proteome published to date. Our current study separated N. nivea venom using size exclusion chromatography before utilizing a traditional bottom-up proteomics approach to unravel the composition of the venom proteome. As expected by its clinical presentation, N. nivea venom was found to consist mainly of neurotoxins, with three-finger toxins (3FTx), making up 76.01% of the total venom proteome. Additionally, cysteine-rich secretory proteins (CRISPs), vespryns (VESPs), cobra venom factors (CVFs), 5'-nucleotidases (5'NUCs), nerve growth factors (NGFs), phospholipase A2s (PLA 2 ), acetylcholinesterases (AChEs), Kunitz-type serine protease inhibitor (KUN), phosphodiesterases (PDEs), L-amino acid oxidases (LAAOs), hydrolases (HYDs), snake venom metalloproteinases (SVMPs), and snake venom serine protease (SVSP) toxins were also identified in decreasing order of abundance. Interestingly, contrary to previous reports, we find PLA 2 toxins in N. nivea venom. This highlights the importance of repeatedly profiling the venom of the same species to account for intra-species variation. Additionally, we report the first evidence of covalent protein complexes in N. nivea venom, which likely contribute to the potency of this venom.

A highly sought after reaction in chemical synthesis is the activation of unactivated carbon-hydrogen bonds. We demonstrate the hydroxylation of fatty acids using an engineered thermostable archaeal cytochrome P450 enzyme. By replacing a seven amino acid section of the I-helix, the nicotinamide cofactor-dependent monooxygenase was converted into a hydrogen peroxide using peroxygenase, enabling the efficient biocatalytic oxidation of C-H bonds at room temperature to 90 °C.

Ortho-phthalaldehyde (OPA) is used as a high-level disinfectant for reusable medical devices in healthcare settings. The ACGIH recently adopted a Threshold Limit Value-Surface Limit (TLV-SL; 25 µg/100 cm2) for OPA surface contamination to prevent induction of dermal and respiratory sensitization following dermal exposure. However, there is no current validated method to measure OPA surface contamination. This study aimed to develop a standardized approach for sample collection and quantitative determination of OPA from work surfaces for use in risk assessment practices. The reported method utilises readily available commercial wipes to collect surface samples coupled with direct detection of OPA via liquid chromatography time of flight mass spectrometry (LC-ToF-MS). This approach avoided complex derivatization steps commonly required for the analysis of aldehydes. Method evaluation was conducted in accordance with the Occupational Safety and Health Administration (OSHA) surface sampling guidelines. Overall recoveries of 25 µg/100 cm2 of OPA from stainless steel and glass surfaces were 70% and 72%, respectively. The reported LOD for this method was 1.1 µg/sample and the LOQ was 3.7 µg/sample. OPA remained stable on the sampling medium for up to 10 days, when stored at 4 °C. The method was demonstrated in a workplace surface assessment at a local hospital sterilising unit, successfully detecting OPA on work surfaces. This method is intended to supplement airborne exposure assessment and provide a quantitative assessment tool for potential dermal exposure. When used in conjunction with a thorough occupational hygiene program that includes hazard communication, engineering controls, and personal protective equipment, skin exposure and consequent sensitization risks in the workplace can be minimized., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10% additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hua et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

Protein aggregation is now recognized as a generic and significant component of the protein energy landscape. Occurring through a complex and dynamic pathway of structural interconversion, the assembly of misfolded proteins to form soluble oligomers and insoluble aggregates remains a challenging topic of study, both in vitro and in vivo. Since the etiology of numerous human diseases has been associated with protein aggregation, and it has become a field of increasing importance in the biopharmaceutical industry, the biophysical characterization of protein misfolded states and their aggregation mechanisms continues to receive increased attention. Mass spectrometry (MS) has firmly established itself as a powerful analytical tool capable of both detection and characterization of proteins at all levels of structure. Given inherent advantages of biological MS, including high sensitivity, rapid timescales of analysis, and the ability to distinguish individual components from complex mixtures with unrivalled specificity, it has found widespread use in the study of protein aggregation, importantly, where traditional structural biology approaches are often not amenable. The present review aims to provide a brief overview of selected MS-based approaches that can provide a range of biophysical descriptors associated with protein conformation and the aggregation pathway. Recent examples highlight where this technology has provided unique structural and mechanistic understanding of protein aggregation., (© 2023 The Author(s).)

Polyamines and polyamine-containing metabolites are involved in many cellular processes related to bacterial cell growth and survival. In Escherichia coli , the bifunctional enzyme glutathionylspermidine synthetase/amidase (GspSA) controls the production of glutathionylspermidine, which has a protective role against oxidative stress. E. coli also encodes two enzymes with homology to the synthetase domain of GspSA, YgiC, and YjfC; however, these do not catalyze the formation of glutathionylspermidine, and their catalytic function remained unknown. Here, we detail the structural and functional characterization of YgiC and YjfC. Using X-ray crystallography, the high-resolution crystal structures of YgiC and YjfC were obtained. This revealed that YgiC and YjfC possess multiple substitutions in key residues required for binding of glutathione in GspSA. Despite this difference, these enzymes share a similar active site structure to GspSA, suggesting that they catalyze the formation of an alternate peptide─spermidine conjugate. As the physiological substrates of YgiC and YjfC are unknown, this was probed using the peptide triglycine as a model substrate. A combination of enzyme activity assays and mass spectrometry revealed that YgiC and YjfC can function as peptide-spermidine ligases, forming a triglycine-spermidine conjugate. For both enzymes, conjugate formation was only observed in the presence of spermidine, but not other common polyamines, supporting that spermidine or a spermidine derivative is the physiological substrate. Importantly, since YgiC and YjfC are widely distributed in Gram-negative bacterial species, this suggests that these enzymes function in a conserved cellular process, representing a currently unknown aspect of bacterial polyamine metabolism.

Anthocyanins are a subclass of plant-derived flavonoids that demonstrate immense structural heterogeneity which is challenging to capture in complex extracts by traditional liquid chromatography-mass spectrometry (MS)-based approaches. Here, we investigate direct injection ion mobility-MS as a rapid analytical tool to characterize anthocyanin structural features in red cabbage ( Brassica oleracea ) extracts. Within a 1.5 min sample run time, we observe localization of structurally similar anthocyanins and their isobars into discrete drift time regions based upon their degree of chemical modifications. Furthermore, drift time-aligned fragmentation enables simultaneous collection of MS, MS/MS, and collisional cross-section data for individual anthocyanin species down to a low picomole scale to generate structural identifiers for rapid identity confirmation. We finally identify anthocyanins in three other Brassica oleracea extracts based on red cabbage anthocyanin identifiers to demonstrate our high-throughput approach. Direct injection ion mobility-MS therefore provides wholistic structural information on structurally similar, and even isobaric, anthocyanins in complex plant extracts, which can inform the nutritional value of a plant and bolster drug discovery pipelines., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Alzheimer's disease (AD) is an insidious disease. Its distinctive pathology forms over a considerable length of time without symptoms. There is a need to detect this disease, before even subtle changes occur in cognition. Hallmark AD biomarkers, tau and amyloid-β, have shown promising results in CSF and blood. However, detecting early changes in these biomarkers and others will involve screening a wide group of healthy, asymptomatic individuals. Saliva is a feasible alternative. Sample collection is economical, non-invasive and saliva is an abundant source of proteins including tau and amyloid-β. This work sought to extend an earlier promising untargeted mass spectrometry study in saliva from individuals with mild cognitive impairment (MCI) or AD with age- and gender-matched cognitively normal from the South Australian Neurodegenerative Disease cohort. Five proteins, with key roles in inflammation, were chosen from this study and measured by ELISA from individuals with AD ( n = 16), MCI ( n = 15) and cognitively normal ( n = 29). The concentrations of Cystatin-C, Interleukin-1 receptor antagonist, Stratifin, Matrix metalloproteinase 9 and Haptoglobin proteins had altered abundance in saliva from AD and MCI, consistent with the earlier study. Receiver operating characteristic analysis showed that combinations of these proteins demonstrated excellent diagnostic accuracy for distinguishing both MCI (area under curve = 0.97) and AD (area under curve = 0.97) from cognitively normal. These results provide evidence for saliva being a valuable source of biomarkers for early detection of cognitive impairment in individuals on the AD continuum and potentially other neurodegenerative diseases., Competing Interests: Author RR declared that he was a shareholder of Biosensis Pty Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McNicholas, François, Liu, Doecke, Hecker, Faunt, Maddison, Johns, Pukala, Rush and Leifert.)

Enzymes involved in Staphylococcus aureus amino acid metabolism have recently gained traction as promising targets for the development of new antibiotics, however, not all aspects of this process are understood. The ATP-grasp superfamily includes enzymes that predominantly catalyze the ATP-dependent ligation of various carboxylate and amine substrates. One subset, ʟ-amino acid ligases (LALs), primarily catalyze the formation of dipeptide products in Gram-positive bacteria, however, their involvement in S. aureus amino acid metabolism has not been investigated. Here, we present the characterization of the putative ATP-grasp enzyme (SAOUHSC_02373) from S. aureus NCTC 8325 and its identification as a novel LAL. First, we interrogated the activity of SAOUHSC_02373 against a panel of ʟ-amino acid substrates. As a result, we identified SAOUHSC_02373 as an LAL with high selectivity for ʟ-aspartate and ʟ-methionine substrates, specifically forming an ʟ-aspartyl-ʟ-methionine dipeptide. Thus, we propose that SAOUHSC_02373 be assigned as ʟ-aspartate-ʟ-methionine ligase (LdmS). To further understand this unique activity, we investigated the mechanism of LdmS by X-ray crystallography, molecular modeling, and site-directed mutagenesis. Our results suggest that LdmS shares a similar mechanism to other ATP-grasp enzymes but possesses a distinctive active site architecture that confers selectivity for the ʟ-Asp and ʟ-Met substrates. Phylogenetic analysis revealed LdmS homologs are highly conserved in Staphylococcus and closely related Gram-positive Firmicutes. Subsequent genetic analysis upstream of the ldmS operon revealed several trans-acting regulatory elements associated with control of Met and Cys metabolism. Together, these findings support a role for LdmS in Staphylococcal sulfur amino acid metabolism., Competing Interests: Conflict of interest J. L. P. and A. J. H. are recipients of an Australian Government Research Training Program stipend scholarship. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The deposition of α-synuclein (αS) aggregates in the gut and the brain is ever present in cases of Parkinson's disease. While the central non-amyloidogenic-component (NAC) region of αS plays a critical role in fibrilization, recent studies have identified a specific sequence from within the N-terminal region (NTR, residues 36-42) as a key modulator of αS fibrilization. Due to the lack of effective therapeutics which specifically target αS aggregates, we have developed a strategy to prevent the aggregation and subsequent toxicity attributed to αS fibrilization utilizing NTR targeting peptides. In this study, L- and D-isoforms of a hexa- (VAQKTV-Aib, 77-82 NAC) and heptapeptide (GVLYVGS-Aib, 36-42 NTR) containing a self-recognition component unique to αS, as well as a C-terminal disruption element, were synthesized to target primary sequence regions of αS that modulate fibrilization. The D-peptide that targets the NTR (NTR-TP-D) was shown by ThT fluorescence assays and TEM to be the most effective at preventing fibril formation and elongation, as well as increasing the abundance of soluble monomeric αS. In addition, NTR-TP-D alters the conformation of destabilised monomers into a less aggregation-prone state and reduces the hydrophobicity of αS fibrils via fibril remodelling. Furthermore, both NTR-TP isoforms alleviate the cytotoxic effects of αS aggregates in both Neuro-2a and Caco-2 cells. Together, this study highlights how targeting the NTR of αS using D-isoform peptide inhibitors may effectively combat the deleterious effects of αS fibrilization and paves the way for future drug design to utilise such an approach to treat Parkinson's disease., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mucin 1 is a transmembrane glycoprotein which overexpresses cancer-specific epitopes (MUC1-CE) on pancreatic ductal adenocarcinoma (PDAC) cells. As PDAC is a low survival and highly aggressive malignancy, developing radioimmunoconjugates capable of targeting MUC1-CE could lead to improvements in PDAC outcomes. The aim of this study was to develop and perform preliminary testing of diagnostic and therapeutic radioimmunoconjugates for PDAC using an anti-MUC1 antibody, C595. Firstly, p-SCN-Bn-DOTA was conjugated to the C595 antibody to form a DOTA-C595 immunoconjugate. The stability and binding affinity of the DOTA-C595 conjugate was evaluated using mass spectrometry and ELISA. DOTA-C595 was radiolabelled to Copper-64, Lutetium-177, Gallium-68 and Technetium-99m to form novel radioimmunoconjugates. Cell binding assays were performed in PANC-1 (strong MUC1-CE expression) and AsPC-1 (weak MUC1-CE expression) cell lines using 64 Cu-DOTA-C595 and 177 Lu-DOTA-C595. An optimal molar ratio of 4:1 DOTA groups per C595 molecule was obtained from the conjugation process. DOTA-C595 labelled to Copper-64, Lutetium-177, and Technetium-99m with high efficiency, although the Gallium-68 labelling was low. 177 Lu-DOTA-C595 demonstrated high cellular binding to the PANC-1 cell lines which was significantly greater than AsPC-1 binding at concentrations exceeding 100 nM ( p < 0.05). 64 Cu-DOTA-C595 showed similar binding to the PANC-1 and AsPC-1 cells with no significant differences observed between cell lines ( p > 0.05). The high cellular binding of 177 Lu-DOTA-C595 to MUC1-CE positive cell lines suggests promise as a therapeutic radioimmunoconjugate against PDAC while further work is required to harness the potential of 64 Cu-DOTA-C595 as a diagnostic radioimmunoconjugate.

Magenta lilly pilly ( Syzygium paniculatum ) is an Australian native tree that produces berry fruits that are rich in phytochemicals reportedly beneficial to human health. Here we explored the biological activities of polyphenol-enriched extracts from the magenta lilly pilly fruit, benchmarking it against traditional sources including purple sweet potato and blackberry. We show that the extracts exert potent antioxidant and neuroprotective properties as well as antimicrobial activity against Staphylococcus aureus . The phenolic composition of lilly pilly was investigated using liquid chromatography coupled to mass spectrometry (HPLC-DAD-MS), revealing anthocyanins to be the primary component in high abundance compared to traditional anthocyanin-containing plants. Three anthocyanins from lilly pilly, along with their glycosylation patterns and stability, were characterised. Altogether, our results demonstrate the potential to exploit magenta lilly pilly fruits as a high-yielding source of phenolics with beneficial biological properties of potential interest for multiple downstream applications.

The metabolic enzyme, enolase, plays a crucial role in the cytoplasm where it maintains cellular energy production within the process of glycolysis. The main role of enolase in glycolysis is to convert 2-phosphoglycerate to phosphoenolpyruvate; however, enolase can fulfill roles that deviate from this function. In pathogenic bacteria and fungi, enolase is also located on the cell surface where it functions as a virulence factor. Surface-expressed enolase is a receptor for human plasma proteins, including plasminogen, and this interaction facilitates nutrient acquisition and tissue invasion. A novel approach to developing antifungal drugs is to inhibit the formation of this complex. To better understand the structure of enolase and the interactions that may govern complex formation, we have solved the first X-ray crystal structure of enolase from Aspergillus fumigatus (2.0 Å) and have shown that it preferentially adopts a dimeric quaternary structure using native mass spectrometry. Two additional X-ray crystal structures of A. fumigatus enolase bound to the endogenous substrate 2-phosphoglycerate and product phosphoenolpyruvate were determined and kinetic characterization was carried out to better understand the details of its canonical function. From these data, we have produced a model of the A. fumigatus enolase and human plasminogen complex to provide structural insights into the mechanisms of virulence and aid future development of small molecules or peptidomimetics for antifungal drug design., (© 2022 Wiley Periodicals LLC.)

The Australasian region is home to the most diverse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared to elapids on other continents; however, their venom compositions are poorly known. The Australian elapid Hoplocephalus stephensii (Stephen's banded snake) is an arboreal snake with a unique morphology. Human envenoming results in venom-induced consumption coagulopathy, without neurotoxicity. Using transcriptomics and a multi-step fractionation method involving reverse-phase high-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and bottom-up proteomics, we characterized the venom proteome of H. stephensii. 92% of the total protein component of the venom by weight was characterized, and included all dominant protein families and 4 secondary protein families. Eighteen toxins made up 76% of the venom, four previously characterized and 14 new toxins. The four dominant protein families made up 77% of the venom, including snake venom metalloprotease (SVMP; 36.7%; three identified toxins), phospholipase A 2 (PLA 2 ; 24.0%; five identified toxins), three-finger toxin (3FTx; 10.2%; two toxins) and snake venom serine protease (SVSP; 5.9%; one toxin; Hopsarin). Secondary protein families included L-amino acid oxidase (LAAO; 10.8%; one toxin), natriuretic peptide (NP; 0.8%; two toxins), cysteine-rich secretory protein (CRiSP; 1.7%; two toxins), c-type lectin (CTL; 1.1%; one toxin), and one minor protein family, nerve growth factor (NGF; 0.8%; one toxin). The venom composition of H. stephensii differs to other elapids, with a large proportion of SVMP and LAAO, and a relatively small amount of 3FTx. H. stephensii venom appeared to have less toxin diversity than other elapids, with only 18 toxins making up three-quarters of the venom.

Purine biosynthesis is a fundamental cellular process that sustains life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As an integral determinant of fungal survival and virulence, the enzymes in this metabolic pathway have been pursued as potential antifungal targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in the clinically prominent fungal pathogens Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. However, a lack of structural information on GMP synthase has hindered drug-design efforts. Here, the first structure of a GMP synthase of fungal origin, that from A. fumigatus (at 2.3 Å resolution), is presented. Structural analysis of GMP synthase shows a distinct absence of the D1 dimerization domain that is present in the human homologue. Interestingly, A. fumigatus GMP synthase adopts a dimeric state, as determined by native mass spectrometry and gel-filtration chromatography, in contrast to the monomeric human homologue. Analysis of the substrate-binding pockets of A. fumigatus GMP synthase reveals key differences in the ATP- and XMP-binding sites that can be exploited for species-specific inhibitor drug design. Furthermore, the inhibitory activities of the glutamine analogues acivicin (IC 50 = 16.6 ± 2.4 µM) and 6-diazo-5-oxo-L-norleucine (IC 50 = 29.6 ± 5.6 µM) against A. fumigatus GMP synthase are demonstrated. Together, these data provide crucial structural information required for specifically targeting A. fumigatus GMP synthase for future antifungal drug-discovery endeavours.

Understanding snake venom proteomes is becoming increasingly important to understand snake venom biology, evolution and especially clinical effects of venoms and approaches to antivenom development. To explore the current state of snake venom proteomics and transcriptomics we investigated venom proteomic methods, associations between methodological and biological variability and the diversity and abundance of protein families. We reviewed available studies on snake venom proteomes from September 2017 to April 2021. This included 81 studies characterising venom proteomes of 79 snake species, providing data on relative toxin abundance for 70 species and toxin diversity (number of different toxins) for 37 species. Methodologies utilised in these studies were summarised and compared. Several comparative studies showed that preliminary decomplexation of crude venom by chromatography leads to increased protein identification, as does the use of transcriptomics. Combining different methodological strategies in venomic approaches appears to maximize proteome coverage. 48% of studies used the RP-HPLC →1D SDS-PAGE → in-gel trypsin digestion → ESI -LC-MS/MS pathway. Protein quantification by MS1-based spectral intensity was used twice as commonly as MS2-based spectral counting (33-15 studies). Total toxin diversity was 25-225 toxins/species, with a median of 48. The relative mean abundance of the four dominant protein families was for elapids; 3FTx-52%, PLA 2 -27%, SVMP-2.8%, and SVSP-0.1%, and for vipers: 3FTx-0.5%, PLA 2 -24%, SVMP-27%, and SVSP-12%. Viper venoms were compositionally more complex than elapid venoms in terms of number of protein families making up most of the venom, in contrast, elapid venoms were made up of fewer, but more toxin diverse, protein families. No relationship was observed between relative toxin diversity and abundance. For equivalent comparisons to be made between studies, there is a need to clarify the differences between methodological approaches and for acceptance of a standardised protein classification, nomenclature and reporting procedure. Correctly measuring and comparing toxin diversity and abundance is essential for understanding biological, clinical and evolutionary implications of snake venom composition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tasoulis, Pukala and Isbister.)

Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-β 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development., (© 2021 Federation of European Biochemical Societies.)

A three-step strategy is introduced to develop inherent iminodiacetic (IDA)-functionalized nanopolymer. SEM micrographs show homogenous spherical beads with a particle size of 500 nm. Further modification to COOH-functionalized 1,2-epoxy-5-hexene/DVB mesoporous nanopolymer enriches glycopeptides via hydrophilic interactions followed by their MS determination. Significantly high BET surface area 433.4336 m 2  g -1 contributes to the improved surface hydrophilicity which is also shown by high concentration of ionizable carboxylic acids, 14.59 ± 0.25 mmol g -1 . Measured surface area is the highest among DVB-based polymers and in general much higher in comparison to the previously reported BET surface areas of co-polymers, terpolymers, MOFs, and graphene-based composites. Thirty-one, 19, and 16 N-glycopeptides are enriched/identified by nanopolymer beads from tryptic digests of immunoglobulin G, horseradish peroxidase, and chicken avidin, respectively, without additional desalting steps. Material exhibits high selectivity (1:400 IgG:BSA), sensitivity (down to 0.1 fmol), regeneration ability up to three cycles, and batch-to-batch reproducibility (RSD > 1%). Furthermore, from 1 μL of digested human serum, 343 N-glycopeptide characteristics of 134 glycoproteins including 30 FDA-approved serum biomarkers are identified via nano-LC-MS/MS. The developed strategy to self-generate IDA on polymeric surface with improved surface area, porosity, and ordered morphology is insignia of its potential as chromatographic tool contributing to future developments in large-scale biomedical glycoproteomics studies., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Diels-Alder chemistry is a well-explored avenue for the synthesis of bioactive materials; however, its potential applications have recently expanded following the development of reactions that can be performed in buffered aqueous environments at low temperatures, including fulvene-maleimide [4 + 2] cycloadditions. In this study, we synthesized two novel amine-reactive fulvene linkers to demonstrate the application of this chemistry for generating mass spectrometry-cleavable labels ("mass tags"), which can be used for the labeling and detection of proteins. Successful conjugation of these linkers to maleimide-labeled peptides was observed at low temperatures in phosphate-buffered saline, allowing the non-destructive modification of proteins with such mass tags. The labile nature of fulvene-maleimide adducts in the gas phase also makes them suitable for both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometric analysis. Unlike previous examples of MALDI mass tags, we show that fulvene-maleimide cycloaddition adducts fragment predictably upon gas-phase activation without the need for bulky photocleavable groups. Further exploration of this chemistry could therefore lead to new approaches for mass spectrometry-based bioassays.

Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s -1 ), specificity constant (626 ± 110.0 mm -1 ·s -1 ) and binding free energy change (-37.0 ± 3.5 kcal·mol -1 ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s -1 ) and lowest specificity constant (40.2 ± 4.4 mm -1 ·s -1 ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV)., (© 2020 Federation of European Biochemical Societies.)

The association between protein aggregation and neurodegenerative diseases such as Parkinson's disease continues to be well interrogated but poorly elucidated at a mechanistic level. Nevertheless, the formation of amyloid fibrils from the destabilization and misfolding of native proteins is a molecular hallmark of disease. Consequently, there is ongoing demand for the identification and development of small molecules which prevent fibril formation. This study comprehensively assesses the inhibitory properties of two small molecules, the lignan polyphenol honokiol and the flavonoid 2',3',4'-trihydroxyflavone, in preventing α-synuclein fibrilization. The data shows that honokiol does not prevent α-synuclein fibril elongation, while 2',3',4'-trihydroxyflavone is effective at inhibiting fibril elongation and induces oligomer formation (for both wild-type α-synuclein and the disease-associated A53T mutation). Moreover, the exposed hydrophobicity of α-synuclein fibrils is reduced in the presence of 2',3',4'-trihydroxyflavone, whereas the addition of honokiol did not reduce the hydrophobicity of fibrils. In addition, ion mobility-mass spectrometry revealed that the conformation of α-synuclein wild-type and A53T monomers after disassembly is restored to a nonaggregation-prone state upon 2',3',4'-trihydroxyflavone treatment. Collectively, this study shows that the mechanisms by which these polyphenols and flavonoids prevent fibril formation are distinct by their interactions at various phases of the fibril-forming pathway. Furthermore, this study highlights how thorough biophysical interrogation of the interaction is required for understanding the ability of inhibitors to prevent protein aggregation associated with disease.

Developments in immunoassays and mass spectrometry have independently influenced diagnostic technology. However, both techniques possess unique strengths and limitations, which define their ability to meet evolving requirements for faster, more affordable and more accurate clinical tests. In response, hybrid techniques, which combine the accessibility and ease-of-use of immunoassays with the sensitivity, high throughput and multiplexing capabilities of mass spectrometry are continually being explored. Developments in antibody conjugation methodology have expanded the role of these biomolecules to applications outside of conventional colorimetric assays and histology. Furthermore, the range of different mass spectrometry ionisation and analysis technologies has enabled its successful adaptation as a detection method for numerous clinically relevant immunological assays. Several recent examples of combined mass spectrometry-immunoassay techniques demonstrate the potential of these methods as improved diagnostic tests for several important human diseases. The present challenges are to continue technological advancements in mass spectrometry instrumentation and develop improved bioconjugation methods, which can overcome their existing limitations and demonstrate the clinical significance of these hybrid approaches., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Elsevier B.V. All rights reserved.)

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a 'substrate validation' activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brown seaweed (Phaeophyceae) polyphenolics such as phlorotannins are ascribed various biological activities, including neuroprotection. Of these seaweeds, Ecklonia radiata (E. radiata) is found abundantly along South Australian coastal regions; however it has not been explored for various biological activities relative to any component phlorotannins previously ascribed neuroprotective capacity. In the present study, we evaluated neuroprotective activity against the neurotoxic amyloid β protein (Aβ1-42) of an ethanol extract of E. radiata compared with various additional solvent-solubilised fractions in a neuronal PC-12 cell line. The ethyl acetate fraction comprising 62% phlorotannins demonstrated the most efficacious neuroprotective activity, inhibiting neurotoxicity at all Aβ1-42 concentrations. In addition, this fraction demonstrated a significant reduction in Aβ aggregate density, but did not alter overall aggregate morphology. Centrifugal partitioning chromatography was used to isolate the major component, eckol, in high yield and liquid chromatography-mass spectrometry was used to characterize the major components of the ethyl acetate fraction. Our results demonstrate that the prevalence of eckol-type phlorotannins are associated with neuroprotective bioactivity of E. radiata, suggestive of potential nutraceutical and biopharmaceutical uses of this brown seaweed phlorotannin in dementia.

Amyloid beta peptide (Aβ42) aggregation in the brain is thought to be responsible for the onset of Alzheimer's disease, an insidious condition without an effective treatment or cure. Hence, a strategy to prevent aggregation and subsequent toxicity is crucial. Bio-inspired peptide-based molecules are ideal candidates for the inhibition of Aβ42 aggregation, and are currently deemed to be a promising option for drug design. In this study, a hexapeptide containing a self-recognition component unique to Aβ42 was designed to mimic the β-strand hydrophobic core region of the Aβ peptide. The peptide is comprised exclusively of D-amino acids to enhance specificity towards Aβ42, in conjunction with a C-terminal disruption element to block the recruitment of Aβ42 monomers on to fibrils. The peptide was rationally designed to exploit the synergy between the recognition and disruption components, and incorporates features such as hydrophobicity, β-sheet propensity, and charge, that all play a critical role in the aggregation process. Fluorescence assays, native ion-mobility mass spectrometry (IM-MS) and cell viability assays were used to demonstrate that the peptide interacts with Aβ42 monomers and oligomers with high specificity, leading to almost complete inhibition of fibril formation, with essentially no cytotoxic effects. These data define the peptide-based inhibitor as a potentially potent anti-amyloid drug candidate for this hitherto incurable disease., (© 2020 The Author(s).)

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus ( S. aureus ) . In S. aureus , BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro , as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10 -9 ) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus ., Competing Interests: The authors declare no competing financial interest.

Snake venoms contain complex mixtures of proteins vital for the survival of venomous snakes. Aligned with their diverse pharmacological activities, the protein compositions of snake venoms are highly variable, and efforts to characterise the primary structures of such proteins are ongoing. Additionally, a significant knowledge gap exists in terms of the higher-order protein structures which modulate venom potency, posing a challenge for successful therapeutic applications. Here we use a multifaceted mass spectrometry approach to characterise proteins from venoms of Collett's snake Pseudechis colletti and the puff adder Bitis arietans. Following chromatographic fractionation and bottom-up proteomics analysis, native mass spectrometry identified, among other components, a non-covalent l-amino acid oxidase dimer in the P. colletti venom and a C-type lectin tetramer in the B. arietans venom. Furthermore, a covalently-linked phospholipase A 2 (PLA 2 ) dimer was identified in P. colletti venom, from which the PLA 2 species were shown to adopt compact geometries using ion mobility measurements. Interestingly, we show that the dimeric PLA 2 possesses greater bioactivity than the monomeric PLA 2 s. This work contributes to ongoing efforts cataloguing components of snake venoms, and notably, emphasises the importance of understanding higher-order venom protein interactions and the utility of a combined mass spectrometric approach for this task. SIGNIFICANCE: The protein constituents of snake venoms represent a sophisticated cocktail of biologically active molecules ideally suited for further exploration in drug design and development. Despite ongoing efforts to characterise the diverse protein components of such venoms there is still much work required in this area, particularly in moving from simply describing the protein primary sequence to providing an understanding of quaternary structure. The combined proteomic and native mass spectrometry workflow utilised here gives new insights into higher order protein structures in selected snake venoms, and can underpin further investigation into the protein interactions which govern snake venom specificity and potency., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's diseases. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion mobility-mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions. The molecular chaperone β-casein (β-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between β-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion mobility-mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of β-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein-chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms., (© 2020 The Author(s).)

Enrichment strategies are designed for the pretreatment of low-abundance glycans and glycopeptides prior to mass spectrometric (MS) analysis. Here, a tip-based strategy is being reported for the enrichment of glycopeptides and glycans using a piperazine modified polymeric monolithic tip. The tip is fabricated using the free radical polymerization. Fast separation (2 min) is achieved under optimized conditions with 20 cycles per step of loading, incubation, washing, and elution followed by MALDI-MS analysis. A total of 25, 22, and 34 glycopeptides covering all glycosylation sites are enriched by the modified tips from tryptic digests of horse radish peroxidase, chicken avidin, and human immunoglobulin G, respectively. Piperazine exhibits high selectivity 1:400 horse radish peroxidase/bovine serum albumin, sensitivity to 100 attomoles, recovery 89.51%, and batch to batch reproducibility (RSD > 1) in glycopeptides enrichment. Piperazine tips also enrich glycans from ovalbumin and human immunoglobulin G. High selectivity (1:1200, ovalbumin/BSA) and detection limit of 100 attomole is attained for glycans and furthermore 58 glycans are enriched from human serum. Thus, piperazine tips can be used as an enrichment tool for swift, cost-effective routine analysis of biological samples for separation of glycopeptides and glycans.

Norbornene derivatives were validated as probes for cysteine sulfenic acid on proteins and in live cells. Trapping sulfenic acids with norbornene probes is highly selective and revealed a different reactivity profile than the traditional dimedone reagent. The norbornene probe also revealed a superior chemoselectivity when compared to a commonly used dimedone probe. Together, these results advance the study of cysteine oxidation in biological systems.

An adequate supply of biotin is vital for the survival and pathogenesis of Staphylococcus aureus. The key protein responsible for maintaining biotin homeostasis in bacteria is the biotin retention protein A (BirA, also known as biotin protein ligase). BirA is a bi-functional protein that serves both as a ligase to catalyse the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates biotin biosynthesis, biotin transport and fatty acid elongation. The mechanism of BirA regulated transcription has been extensively characterized in Escherichia coli, but less so in other bacteria. Biotin-induced homodimerization of E. coli BirA (EcBirA) is a necessary prerequisite for stable DNA binding and transcriptional repression. Here, we employ a combination of native mass spectrometry, in vivo gene expression assays, site-directed mutagenesis and electrophoretic mobility shift assays to elucidate the DNA binding pathway for S. aureus BirA (SaBirA). We identify a mechanism that differs from that of EcBirA, wherein SaBirA is competent to bind DNA as a monomer both in the presence and absence of biotin and/or MgATP, allowing homodimerization on the DNA. Bioinformatic analysis demonstrated the SaBirA sequence used here is highly conserved amongst other S. aureus strains, implying this DNA-binding mechanism is widely employed.

Determination of collisional cross sections (CCS) by travelling wave ion mobility mass spectrometry (TWIM-MS) requires calibration against standards for which the CCS has been measured previously by drift tube ion mobility mass spectrometry (DTIM-MS). The different extents of collisional activation in TWIM-MS and DTIM-MS can give rise to discrepancies in the CCS of calibrants across the two platforms. Furthermore, the conditions required to ionize and transmit large, folded proteins and assemblies may variably affect the structure of the calibrants and analytes. Stable hetero-oligomeric phospholipase A 2 (PDx) and its subunits were characterized as calibrants for TWIM-MS. Conditions for acquisition of native-like TWIM (Synapt G1 HDMS) and DTIM (Agilent 6560 IM-Q-TOF) mass spectra were optimized to ensure the spectra exhibited similar charge state distributions. CCS measurements (DTIM-MS) for ubiquitin, cytochrome c, holo-myoglobin, serum albumin and glutamate dehydrogenase were in good agreement with other recent results determined using this and other DTIM-MS instruments. PDx and its β and γ subunits were stable across a wide range of cone and trap voltages in TWIM-MS and were stable in the presence of organic solvents. The CCS of PDx and its subunits were determined by DTIM-MS and were used as calibrants in determination of CCS of native-like cytochrome c, holo-myoglobin, carbonic anhydrase, serum albumin and haemoglobin in TWIM-MS. The CCS values were in good agreement with those measured by DTIM-MS where available. These experiments demonstrate conditions for analysis of native-like proteins using a commercially available DTIM-MS instrument, characterize robust calibrants for TWIM-MS, and present CCS values determined by DTIM-MS and TWIM-MS for native proteins to add to the current literature database. Graphical Abstract ᅟ.

Proteinopathies including cataracts and neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, are characterized by a series of aberrant protein folding events, resulting in amorphous aggregate or amyloid fibril formation. In the latter case, research has heavily focused on the development of small-molecule inhibitors with limited success during clinical trials. However, very few studies have focused on utilizing exogenous proteins as potential aggregation inhibitors. C-Phycocyanin, derived from Spirulina sp., has been known to exert anti-inflammatory properties; however, the ability of C-phycocyanin to inhibit protein aggregation has yet to be investigated. We have demonstrated that C-phycocyanin is an effective inhibitor of A53Tα-synuclein at extremely low substoichiometric ratios (200-fold excess of α-synuclein) and Aβ40/42 fibril formation. However, C-phycocyanin is relatively ineffective in inhibiting the reduction-induced amorphous aggregation of ADH and heat-induced aggregation of catalase. In addition, 2D NMR, ion mobility-mass spectrometry, and analytical-SEC demonstrate that the interaction between C-phycocyanin and α-synuclein is through nonstable interactions, indicating that transient interactions are likely to be responsible for preventing fibril formation. Overall, this work highlights how biomolecules from natural sources could be used to aid in the development of therapeutics to combat protein misfolding diseases.