Molecular Profiling of Axial Spondyloarthritis Patients Reveals an Association between Innate and Adaptive Cell Populations and Therapeutic Response to Tumor Necrosis Factor Inhibitors.

This study aims at identifying molecular biomarkers differentiating responders and non-responders to treatment with Tumor Necrosis Factor inhibitors (TNFi) among patients with axial spondyloarthritis (axSpA). Whole blood mRNA and plasma proteins were measured in a cohort of biologic-naïve axSpA patients (n = 35), pre and post (14 weeks) TNFi treatment with adalimumab. Differential expression analysis was used to identify the most enriched pathways and in predictive models to distinguish responses to TNFi. A treatment-associated signature suggests a reduction in inflammatory activity. We found transcripts and proteins robustly differentially expressed between baseline and week 14 in responders. C-reactive protein (CRP) and Haptoglobin (HP) proteins showed strong and early decrease in the plasma of axSpA patients, while a cluster of apolipoproteins (APOD, APOA2, APOA1) showed increased expression at week 14. Responders to TNFi treatment present higher levels of markers of innate immunity at baseline, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP, gender, and AFF3, the top differentially expressed gene at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC = 0.97). In conclusion, innate and adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axSpA patients. A model including clinical and gene expression variables should also be considered.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

Hyperglycemia triggers RyR2-dependent alterations of mitochondrial calcium homeostasis in response to cardiac ischemia-reperfusion: Key role of DRP1 activation.

Hyperglycemia increases the heart sensitivity to ischemia-reperfusion (IR), but the underlying cellular mechanisms remain unclear. Mitochondrial dynamics (the processes that govern mitochondrial morphology and their interactions with other organelles, such as the reticulum), has emerged as a key factor in the heart vulnerability to IR. However, it is unknown whether mitochondrial dynamics contributes to hyperglycemia deleterious effect during IR. We hypothesized that (i) the higher heart vulnerability to IR in hyperglycemic conditions could be explained by hyperglycemia effect on the complex interplay between mitochondrial dynamics, Ca2+ homeostasis, and reactive oxygen species (ROS) production; and (ii) the activation of DRP1, a key regulator of mitochondrial dynamics, could play a central role. Using transmission electron microscopy and proteomic analysis, we showed that the interactions between sarcoplasmic reticulum and mitochondria and mitochondrial fission were increased during IR in isolated rat hearts perfused with a hyperglycemic buffer compared with hearts perfused with a normoglycemic buffer. In isolated mitochondria and cardiomyocytes, hyperglycemia increased mitochondrial ROS production and Ca2+ uptake. This was associated with higher RyR2 instability. These results could contribute to explain the early mPTP activation in mitochondria from isolated hearts perfused with a hyperglycemic buffer and in hearts from streptozotocin-treated rats (to increase the blood glucose). DRP1 inhibition by Mdivi-1 during the hyperglycemic phase and before IR induction, normalized Ca2+ homeostasis, ROS production, mPTP activation, and reduced the heart sensitivity to IR in streptozotocin-treated rats. In conclusion, hyperglycemia-dependent DRP1 activation results in higher reticulum-mitochondria calcium exchange that contribute to the higher heart vulnerability to IR.

Flash MS/MS proteotyping allows identifying microbial isolates in 36 s of mass spectrometry signal.

Rapid identification of microorganisms is essential for medical diagnostics, sanitary controls, and food safety. High-throughput analytical platforms currently rely on whole-cell MALDI-TOF mass spectrometry to process hundreds of samples per day. Although this technology has become a reference method, it is unable to process most environmental isolates and opportunistic pathogens due to an incomplete experimental spectrum database. In most cases, its discriminating power is limited to the species taxonomical rank. By recording much more sequence information at the peptide level, proteotyping by tandem mass spectrometry is able to identify the taxonomic position of any microorganism in the tree of life and can be highly discriminating at the subspecies level. We propose here a methodology for ultra-fast identification of microorganisms by tandem mass spectrometry based on direct sample infusion and a highly sensitive procedure for data processing and taxonomic identification. Results obtained on reference strains and hitherto uncharacterized bacterial isolates show identification to species level in 36 s of tandem mass spectrometry signal, 102 s when including the injection procedure. Flash proteotyping is highly discriminating, as it can provide information down to strain level. The methodology enables high throughput identification of isolates, opening up new prospects, particularly in culturomics, and diagnostics.

Organ-oriented proteogenomics functional atlas of three aquatic invertebrate sentinel species.

Proteogenomic methodologies have enabled the identification of protein sequences in wild species without annotated genomes, shedding light on molecular mechanisms affected by pollution. However, proteomic resources for sentinel species are limited, and organ-level investigations are necessary to expand our understanding of their molecular biology. This study presents proteomic resources obtained from proteogenomic analyses of key organs (hepatopancreas, gills, hemolymph) from three established aquatic sentinel invertebrate species of interest in ecotoxicological/ecological research and environmental monitoring: Gammarus fossarum, Dreissena polymorpha, and Palaemon serratus. Proteogenomic analyses identified thousands of proteins for each species, with over 90% of them being annotated to putative function. Functional analysis validated the relevance of the proteomic atlases by revealing similarities in functional annotation of catalogues of proteins across analogous organs in the three species, while deep contrasts between functional profiles are delimited across different organs in the same organism. These organ-level proteomic atlases are crucial for future research on these sentinel animals, aiding in the evaluation of aquatic environmental risks and providing a valuable resource for ecotoxicological studies.

Bioaccumulation and molecular effects of carbamazepine and methylmercury co-exposure in males of Dreissena polymorpha.

Dreissena polymorpha is a bivalve promising for biomonitoring in freshwater ecosystems thanks to its abundance and high filtration activity allowing rapid uptake of toxicants and identification of their negative effects. Nonetheless, we still lack knowledge on its molecular responses to stress under realistic scenario, e.g. multi-contamination. Carbamazepine (CBZ) and Hg are ubiquitous pollutants sharing molecular toxicity pathways, e.g. oxidative stress. A previous study in zebra mussels showed their co-exposure to cause more alterations than single exposures, but molecular toxicity pathways remained unidentified. D. polymorpha was exposed 24 h (T24) and 72 h (T72) to CBZ (6.1 ± 0.1 µg L-1), MeHg (430 ± 10 ng L-1) and the co-exposure (6.1 ± 0.1 µg L-1CBZ and 500 ± 10 ng L-1 MeHg) at concentrations representative of polluted areas (~10× EQS). RedOx system at the gene and enzyme level, the proteome and the metabolome were compared. The co-exposure resulted in 108 differential abundant proteins (DAPs), as well as 9 and 10 modulated metabolites at T24 and T72, respectively. The co-exposure specifically modulated DAPs and metabolites involved in neurotransmission, e.g. dopaminergic synapse and GABA. CBZ specifically modulated 46 DAPs involved in calcium signaling pathways and 7 amino acids at T24. MeHg specifically modulated 55 DAPs involved in the cytoskeleton remodeling and hypoxia-induced factor 1 pathway, without altering the metabolome. Single and co-exposures commonly modulated proteins and metabolites involved in energy and amino acid metabolisms, response to stress and development. Concomitantly, lipid peroxidation and antioxidant activities were unchanged, supporting that D. polymorpha tolerated experimental conditions. The co-exposure was confirmed to cause more alterations than single exposures. This was attributed to the combined toxicity of CBZ and MeHg. Altogether, this study underlined the necessity to better characterize molecular toxicity pathways of multi-contamination that are not predictable on responses to single exposures, to better anticipate adverse effects in biota and improve risk assessment.

Insights into peculiar fungal LPMO family members holding a short C-terminal sequence reminiscent of phosphate binding motifs.

Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature. White-rot Polyporales, which are major fungal wood decayers, may possess up to 60 LPMO-encoding genes belonging to the auxiliary activities family 9 (AA9). Yet, the functional relevance of such multiplicity remains to be uncovered. Previous comparative transcriptomic studies of six Polyporales fungi grown on cellulosic substrates had shown the overexpression of numerous AA9-encoding genes, including some holding a C-terminal domain of unknown function ("X282"). Here, after carrying out structural predictions and phylogenetic analyses, we selected and characterized six AA9-X282s with different C-term modularities and atypical features hitherto unreported. Unexpectedly, after screening a large array of conditions, these AA9-X282s showed only weak binding properties to cellulose, and low to no cellulolytic oxidative activity. Strikingly, proteomic analysis revealed the presence of multiple phosphorylated residues at the surface of these AA9-X282s, including a conserved residue next to the copper site. Further analyses focusing on a 9 residues glycine-rich C-term extension suggested that it could hold phosphate-binding properties. Our results question the involvement of these AA9 proteins in the degradation of plant cell wall and open new avenues as to the divergence of function of some AA9 members.

Dodecyl creatine ester improves cognitive function and identifies key protein drivers including KIF1A and PLCB1 in a mouse model of creatine transporter deficiency.

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8-/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.

The plasma membrane-associated cation-binding protein PCaP1 of Arabidopsis thaliana is a uranyl-binding protein.

Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.

High-resolution shotgun proteomics reveals that increased air [CO2] amplifies the acclimation response of coffea species to drought regarding antioxidative, energy, sugar, and lipid dynamics.

As drought threatens crop productivity it is crucial to characterize the defense mechanisms against water deficit and unveil their interaction with the expected rise in the air [CO2]. For that, plants of Coffea canephora cv. Conilon Clone 153 (CL153) and C. arabica cv. Icatu grown under 380 (aCO2) or 700 µL L-1 (eCO2) were exposed to moderate (MWD) and severe (SWD) water deficits. Responses were characterized through the activity and/or abundance of a selected set of proteins associated with antioxidative (e.g., Violaxanthin de-epoxidase, Superoxide dismutase, Ascorbate peroxidases, Monodehydroascorbate reductase), energy/sugar (e.g., Ferredoxin-NADP reductase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, sucrose synthase, mannose-6-phosphate isomerase, Enolase), and lipid (Lineolate 13S-lipoxygenase) processes, as well as with other antioxidative (ascorbate) and protective (HSP70) molecules. MWD caused small changes in both genotypes regardless of [CO2] level while under the single imposition to SWD, only Icatu showed a global reinforcement of most studied proteins supporting its tolerance to drought. eCO2 alone did not promote remarkable changes but strengthened a robust multi-response under SWD, even supporting the reversion of impacts already observed by CL153 at aCO2. In the context of climate changes where water constraints and [CO2] levels are expected to increase, these results highlight why eCO2 might have an important role in improving drought tolerance in Coffea species.

Characterization of Soluble Cell-Free Coelomic Fluid Proteome from the Starfish Marthasterias glacialis.

Proteomics combined to advanced bioinformatics tools is acquiring a pivotal role in the comprehensive understanding of living organism's biology, in particular for non-model organisms, which includes most marine and aquatic invertebrates. Depicting of protein composition in a whole organ/organism followed by their assembling in functional protein association networks promotes the understanding of key biological processes. Here, we provide a detailed description of the extraction procedure of cell-free coelomic fluid soluble proteins and the characterization of the proteome of the starfish Marthasterias glacialis. Due to coelomic fluid richness in glycoproteins, which complicates protein identification, extracts of soluble proteins are deglycosylated prior to tandem mass spectrometry. This experimental approach is useful at improving knowledge on the coelomic fluid physiological role and deciphering its involvement in regeneration of starfish body parts when comparing different regeneration conditions.

Identification and Characterization of Marine Microorganisms by Tandem Mass Spectrometry Proteotyping.

The vast majority of marine microorganisms and their functions are yet to be explored. The considerable diversity they encompass is an endless source of knowledge and wealth that can be valued on an industrial scale, emphasizing the need to develop rapid and efficient identification and characterization techniques. In this study, we identified 26 microbial isolates from coastal water of the NW Mediterranean Sea, using phylopeptidomics, a cutting-edge tandem mass spectrometry proteotyping technique. Taxonomical identification at the species level was successfully conducted for all isolates. The presence of strains belonging to the newly described Balneolaeota phylum, yet uncharacterized at the proteomics scale, was noted. The very first proteomics-based investigation of a representative of the Balneolaeota phylum, Balneola vulgaris, is proposed, demonstrating the use of our proteotyping workflow for the rapid identification and in-depth molecular characterization, in a single MS/MS analytical run. Tandem mass spectrometry proteotyping is a valuable asset for culturomic programs as the methodology is able to quickly classify the most atypical isolates.

Infection of Human Endothelial Cells with Lassa Virus Induces Early but Transient Activation and Low Type I IFN Response Compared to the Closely-Related Nonpathogenic Mopeia Virus.

Lassa virus (LASV), an Old World arenavirus, is responsible for hemorrhagic fevers in western Africa. The privileged tropism of LASV for endothelial cells combined with a dysregulated inflammatory response are the main cause of the increase in vascular permeability observed during the disease. Mopeia virus (MOPV) is another arenavirus closely related to LASV but nonpathogenic for non-human primates (NHPs) and has never been described in humans. MOPV is more immunogenic than LASV in NHPs and in vitro in human immune cell models, with more intense type I IFN and adaptive cellular responses. Here, we compared the transcriptomic and proteomic responses of human umbilical vein endothelial cells (HUVECs) to infection with the two viruses to further decipher the mechanisms involved in their differences in immunogenicity and pathogenicity. Both viruses replicated durably and efficiently in HUVECs, but the responses they induced were strikingly different. Modest activation was observed at an early stage of LASV infection and then rapidly shut down. By contrast, MOPV induced a late but more intense response, characterized by the expression of genes and proteins mainly associated with the type I IFN response and antigen processing/presentation. Such a response is consistent with the higher immunogenicity of MOPV relative to LASV, whereas the lack of an innate response induced in HUVECs by LASV is consistent with its uncontrolled systemic dissemination through the vascular endothelium.

Antibiotic tolerance and degradation capacity of the organic pollutant-degrading bacterium Rhodococcus biphenylivorans TG9T.

Antibiotics are ubiquitous in soil due to natural ecological competition, as well as emerging contaminants due to anthropogenic inputs. Under environmental factors like antibiotic stress, some bacteria, including those that degrade environmental pollutants, can enter a dormant state as a survival strategy, thereby limiting their metabolic activity and function. Dormancy has a critical influence on the degradative activity of bacteria, dramatically decreasing the rate at which they transform organic pollutants. To better understand this phenomenon in environmental pollutant-degrading bacteria, we investigated dormancy transitions induced with norfloxacin in Rhodococcus biphenylivorans TG9T using next-generation proteomics, proteogenomics, and additional experiments. Our results suggest that exposure to norfloxacin inhibited DNA replication, which led to damage to the cell. Dormant cells then likely triggered DNA repair, particularly homologous recombination, for continued survival. The results also indicated that substrate transport (ATP-binding cassette transporter), ATP production, and the tricarboxylic acid (TCA) cycle were repressed during dormancy, and degradation of organic pollutants was down-regulated. Given the widespread phenomenon of dormancy among bacteria involved in pollutant removal systems, this study improves our understanding of possible implications of antibiotic survival strategies on biotransformation of mixtures containing antibiotics as well as other organics.

Increased protein S-nitrosylation in mitochondria: a key mechanism of exercise-induced cardioprotection.

Endothelial nitric oxide synthase (eNOS) activation in the heart plays a key role in exercise-induced cardioprotection during ischemia-reperfusion, but the underlying mechanisms remain unknown. We hypothesized that the cardioprotective effect of exercise training could be explained by the re-localization of eNOS-dependent nitric oxide (NO)/S-nitrosylation signaling to mitochondria. By comparing exercised (5 days/week for 5 weeks) and sedentary Wistar rats, we found that exercise training increased eNOS level and activation by phosphorylation (at serine 1177) in mitochondria, but not in the cytosolic subfraction of cardiomyocytes. Using confocal microscopy, we confirmed that NO production in mitochondria was increased in response to H2O2 exposure in cardiomyocytes from exercised but not sedentary rats. Moreover, by S-nitrosoproteomic analysis, we identified several key S-nitrosylated proteins involved in mitochondrial function and cardioprotection. In agreement, we also observed that the increase in Ca2+ retention capacity by mitochondria isolated from the heart of exercised rats was abolished by exposure to the NOS inhibitor L-NAME or to the reducing agent ascorbate, known to denitrosylate proteins. Pre-incubation with ascorbate or L-NAME also increased mitochondrial reactive oxygen species production in cardiomyocytes from exercised but not from sedentary animals. We confirmed these results using isolated hearts perfused with L-NAME before ischemia-reperfusion. Altogether, these results strongly support the hypothesis that exercise training increases eNOS/NO/S-nitrosylation signaling in mitochondria, which might represent a key mechanism of exercise-induced cardioprotection.

Redox proteomic study of Bacillus cereus thiol proteome during fermentative anaerobic growth.

BACKGROUND: Bacillus cereus is a notorious foodborne pathogen, which can grow under anoxic conditions. Anoxic growth is supported by endogenous redox metabolism, for which the thiol redox proteome serves as an interface. Here, we studied the cysteine (Cys) proteome dynamics of B. cereus ATCC 14579 cells grown under fermentative anoxic conditions. We used a quantitative thiol trapping method combined with proteomics profiling. RESULTS: In total, we identified 153 reactive Cys residues in 117 proteins participating in various cellular processes and metabolic pathways, including translation, carbohydrate metabolism, and stress response. Of these reactive Cys, 72 were detected as reduced Cys. The B. cereus Cys proteome evolved during growth both in terms of the number of reduced Cys and the Cys-containing proteins identified, reflecting its growth-phase-dependence. Interestingly, the reduced status of the B. cereus thiol proteome increased during growth, concomitantly to the decrease of extracellular oxidoreduction potential. CONCLUSIONS: Taken together, our data show that the B. cereus Cys proteome during unstressed fermentative anaerobic growth is a dynamic entity and provide an important foundation for future redox proteomic studies in B. cereus and other organisms.

Heterogeneity of SARS-CoV-2 virus produced in cell culture revealed by shotgun proteomics and supported by genome sequencing.

COVID-19 is the most disturbing pandemic of the past hundred years. Its causative agent, the SARS-CoV-2 virus, has been the subject of an unprecedented investigation to characterize its molecular structure and intimate functioning. While markers for its detection have been proposed and several diagnostic methodologies developed, its propensity to evolve and evade diagnostic tools and the immune response is of great concern. The recent spread of new variants with increased infectivity requires even more attention. Here, we document how shotgun proteomics can be useful for rapidly monitoring the evolution of the SARS-CoV-2 virus. We evaluated the heterogeneity of purified SARS-CoV-2 virus obtained after culturing in the Vero E6 cell line. We found that cell culture induces significant changes that are translated at the protein level, such changes being detectable by tandem mass spectrometry. Production of viral particles requires careful quality control which can be easily performed by shotgun proteomics. Although considered relatively stable so far, the SARS-CoV-2 genome turns out to be prone to frequent variations. Therefore, the sequencing of SARS-CoV-2 variants from patients reporting only the consensus genome after its amplification would deserve more attention and could benefit from more in-depth analysis of low level but crystal-clear signals, as well as complementary and rapid analysis by shotgun proteomics.

Lysine-specific acetylated proteome from the archaeon Thermococcus gammatolerans reveals the presence of acetylated histones.

Thermococcus gammatolerans EJ3 is an extremophile archaeon which was revealed as one of the most radioresistant organisms known on Earth, withstanding up to 30 kGy gamma-ray radiations. While its theoretical proteome is rather small, T. gammatolerans may enhance its toolbox by post-translational modification of its proteins. Here, we explored its extent of Nε-acetylation of lysines. For this, we immunopurified with two acetylated-lysine antibodies the acetylated peptides resulting from a proteolysis of soluble proteins with trypsin. The comparison of acetylated proteomes of two archaea highlights some common acetylation patterns but only 4 out of 26 orthologous proteins found to be acetylated in both species, are acetylated on the same lysine site. We evidenced that histone B is acetylated in T. gammatolerans at least at two different sites (K27 and K36), and a peptide common at the C-terminus of histones A and B is also acetylated. We verified that acetylation of histones is a common trait among Thermococcales after recording data on Thermococcus kodakaraensis histones and identifying three acetylated sites. This discovery reinforces the strong evolutionary link between Archaea and Eukaryotes and should be an incentive for further investigation on the extent and role of acetylation of histones in Archaea. SIGNIFICANCE: Acetylation is an important post-translational modification of proteins that has been extensively described in Eukaryotes, and more recently in Bacteria. Here, we report for the first time ever that histones in Archaea are also modified by acetylation after a systematic survey of acetylated peptides in Thermococcus gammatolerans. Structural models of histones A and B indicates that acetylation of the identified modified residues may play an important role in histone assembly and/or interaction with DNA. The in-depth protein acetylome landscape in T. gammatolerans includes at least 181 unique protein sequences, some of them being modified on numerous residues. Proteins involved in metabolic processes, information storage and processing mechanisms are over-represented categories in this dataset, highlighting the ancient role of this protein post-translational modification in primitive cells.

Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it can optimize whole viral particle antigen production for vaccines.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.

High-throughput proteotyping of bacterial isolates by double barrel chromatography-tandem mass spectrometry based on microplate paramagnetic beads and phylopeptidomics.

Tandem mass spectrometry-based proteotyping of microorganisms presents several advantages over whole-cell MALDI-TOF mass spectrometry: because a larger number of signals are recorded with better accuracy and precision, the approach allows for the identification of microorganisms at more resolved taxonomic levels, and can easily manage complex samples. Additionally, the use of SP3 paramagnetic beads for cell lysis and protein cleanup simplifies sample preparation for proteotyping. Based on these features, we have developed and tested a 96-well plate platform for high-throughput proteotyping of a large variety of bacteria. We evaluated the performance of the platform in terms of bacterial load and found no cross-contamination between wells. Likewise, phylopeptidomics analysis revealed no alteration in the relative quantifications of microorganisms. Finally, we applied this new format for rapid proteotyping of a large set of dental isolates using double-barrel chromatography coupled to tandem mass spectrometry, which maximizes the number of spectra per unit of time. The procedure allowed us to establish whether these isolates were pure strains or mixtures of strains and to identify the microorganisms at the most resolved taxonomic level. SIGNIFICANCE: The rapid and accurate identification of microorganisms is a clinical priority in medical diagnostics; however, specimens containing mixtures of microorganisms are difficult to analyze and the procedure is time-consuming. Tandem mass spectrometry proteotyping allows the fast identification of complex mixtures of microorganisms, known or unknown, and can also establish the biomass ratio of each component. We describe here a new workflow for preparing microbial samples in a 96-well-plate format for tandem mass spectrometry proteotyping and document its advantages and limitations. We demonstrate that this new format coupled to a highly efficient double-barrel LC-MS/MS system allows proteotyping of 96 isolates in 55 h.