Evaluation of NHS-Acetate and DEPC labelling for determination of solvent accessible amino acid residues in protein complexes
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Oryctolagus Cuniculus Cuniculus Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: 
Marie Alfes
LAB HEAD: Carla Schmidt
PROVIDER: PXD015940 | Pride | 2020-05-05
REPOSITORIES: Pride
Publications
Evaluation of NHS-Acetate and DEPC labelling for determination of solvent accessible amino acid residues in protein complexes.
Barth Marie M Bender Julian J Kundlacz Til T Schmidt Carla C
Journal of proteomics 20200427
The activity of most proteins and protein complexes relies on the formation of defined three-dimensional structures. The analysis of these arrangements is therefore key for understanding their function and regulation in the cell. Besides the traditional structural techniques, structural mass spectrometry delivers insights into the various aspects of protein structure, including stoichiometry, protein-ligand interactions and solvent accessibility. The latter is usually obtained from labelling exp ...[more]
Similar Datasets
Project description:Synaptic vesicles are storage organelles for neurotransmitters in the pre-synaptic cytosol. When an action potential arrives at the pre-synaptic terminal, they fuse with the pre-synaptic membrane and neurotransmitters are released. In this project, we set out to study the protein interactions formed in synaptic vesicle membranes. For this, we first assessed the proteome of five independent vesicle preparations. Using chemical cross-linking, we then tackled the interactions of the major protein components. To gain further insights, we combined this approach with a chemical labelling strategy. Specific protein interactions were then studied after (i) treating the vesicles with Botulinum neurotoxin B, (ii) binding the soluble SNARE complex, and (iii) fusion of the vesicles with empty liposomes. Our findings reveal stable interaction modules in synaptic vesicles.
2020-12-09 | PXD020859 | Pride
Project description:Identification of the major protease sites in MSLN and preparation of a monoclonal antibody, 15B6, that binds next to cell membrane at the protease sensitive region and inhibits MSLN shedding. Mab 15B6 makes very active CAR-T cells whose activity is not blocked by shed MSLN. The 15B6 CAR-T cells have greatly superior anti-tumor activity in mice than CAR-T cells targeting an epitope in shed MSLN.
2022-04-13 | PXD033137 | Pride
Project description:During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to successfully occur. This requires bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Besides being resistant to it, Salmonella can also successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. In order to gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit. For in vitro studies, bile was extracted from C57BL/6 mice and used immediately. An overnight culture of Salmonella enterica serovar Typhimurium SL1344 was used to inoculate 10 ?L of bile at an approximate density of 5x10^6 cells/mL. The experiment was performed in duplicate, and a total of 2 uninfected and 2 infected bile samples were studied. Samples were incubated for 24 hours at 37 oC with shaking. After incubation, samples were centrifuged to remove bacteria and the supernatant was saved for metabolomic analyses. For in vivo studies, C57BL/6 mice were infected with approximately 10^8 bacterial cells by oral gavage. Four groups of three to four mice each were either infected with Salmonella or kept uninfected (total of 11 mice per treatment group). Five days after infection, all mice were sacrificed and bile was collected. Samples were centrifuged and supernatants saved for metabolomic analyses. Samples were prepared by mixing equal volumes of bile from three to four mice, generating three samples per treatment (uninfected and infected), which were used in the subsequent steps. Seven microliters of each sample was evaporated and the residue resuspended in 50% acetonitrine. Extracts were infused into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140). To identify differences in metabolite composition between different groups of samples, we filtered the list of masses for metabolites which were present on one set of samples but not the other. Additionally, we calculated the ratios between averaged intensities of metabolites from each group of mice. To assign possible metabolite identities to the masses selected as described above, the monoisotopic neutral masses of interest were queried against the Human Metabolome Database (HMDB, http://www.hmdb.ca), with a tolerance of 0.001 Da.
2011-07-06 | E-GEOD-30404 | biostudies-arrayexpress
Project description:Agilent 4x44k Whole Mouse Genome v1 Microarrays were used to analyze aortic transcriptome of mice substituted with medroxyprogesterone acetate, norethisterone acetate or placebo after having been ovariectomized. The aim of this experiment was to detect genes regulated in the gestagen-treated groups as compared to placebo that might be involved in thrombotic events Aortic gene expression was analyzed in ApoE-deficient mice after ovariectomy and 90 days of hormone- or placebo-treatment (medroxyprogesterone acetate or norethisterone acetate) and feeding a high-fat Western-type diet.
2014-06-30 | E-GEOD-57972 | biostudies-arrayexpress
Project description:E. coli MG1655 was grown on M9 minimal medium supplemented with 15mM glucose and either 0, 10, 50 or 100 mM acetate. Transcriptomic analysis from exponential growing cells was performed using Agilent microarray A-MEXP-2232. This work was done with the help of students during practicals. Consequently, it is of special interest since we have four biological replicates done over four years and two technical replicates per biological replicate (n=8 per condition). We therefore thank all the INSA-Toulouse students that participated in this project: Leidy Caraballo, Xavier Caron, Pauline Chanut, Sarah Guiziou, Ngoc Thu Hang Pham, Diane Barbay, Céline Ben Hassen, Thomas Cerutti, Cécile Roland, Sarah Srour, Audrey Baylet, Mathilde Beraud, Claudie Bosc, Lilas Courtot, Violaine Dolfo, Anna Kaci, Manon Chevallot-Beroux, Sarah Colom, Fanny Leclerc, Zihan Liao, Grégoire Quinet and Mélina Vaurs.
2020-08-21 | E-MTAB-9086 | biostudies-arrayexpress
Project description:The interplay between pathogens and hosts has been studied for decades using targeted approaches such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods such as transcriptomics and proteomics have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomics study of Salmonella enterica serovar Typhimurium infection. We used Fourier Transform Ion Cyclotron Resonance Mass Spectrometry with Direct Infusion to reveal that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease, and by the host to counter infection. Female C57BL/6 mice were infected with Salmonella enterica serovar Typhimurium SL1344 cells by oral gavage. Feces and livers were collected and metabolites extracted using acetonitrile. For experiments with feces, samples were collected from 4 mice before and after infection. For liver experiments, 11 uninfected and 11 infected mice were used. Samples were combined into 3 groups of 3-4 mice each, resulting in the analysis of 3 group samples of uninfected and 3 of infected mice. Extracts were infused into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140 [PMID 19081807]). To identify differences in metabolite composition between uninfected and infected samples, we filtered the list of masses for metabolites which were present on one set of samples but not the other. Additionally, we calculated the ratios between averaged intensities of metabolites from uninfected and infected mice. To assign possible metabolite identities, monoisotopic neutral masses of interest were queried against MassTrix (http://masstrix.org). Masses were searched against the Mus musculus database within a mass error of 3 ppm. Data were analyzed by unpaired t tests with 95% confidence intervals.
2011-02-08 | E-GEOD-21720 | biostudies-arrayexpress
Project description:The importance of the mammalian intestinal microbiota to human health has been intensely studied over the past few years. It is now clear that the interactions between human hosts and their associated microbial communities need to be characterized in molecular detail if we are to truly understand human physiology. Additionally, the study of such host-microbe interactions is likely to provide us with new strategies to manipulate such complex systems to maintain or restore homeostasis in order to prevent or cure pathological states. We describe the use of high-throughput metabolomics to shed light on the interactions between the intestinal microbiota and the host. We show that treatment with the antibiotic streptomycin disrupts intestinal homeostasis and has a profound impact on the intestinal metabolome, affecting the levels of over 87% of all metabolites detected. Many metabolic pathways that are critical for host physiology were affected, including bile acid, eicosanoid and steroid hormone synthesis. Interestingly, many of these pathways are also affected by intestinal pathogens. Dissecting the effect of both beneficial and pathogenic bacteria on some of these pathways will be instrumental in understanding the interplay between the host, the resident microbiota and incoming pathogens and may aid in the design of new therapeutic strategies that target these interactions. Age-matched female C57BL/6 mice were used. Fresh feces were collected and stored at -80 oC. Mice were then treated with 20 mg of streptomycin through oral gavage and fresh feces were collected 24 hours after treatment and stored at -80 oC until used. To extract metabolites from feces, acetonitrile was added to samples (approximately 10-25 μL of acetonitrile per 1 mg of tissue), which were then homogenized. The samples were then cleared by centrifugation and the supernatant was collected and dried at room temperature using a centrifuge equipped with a vacuum pump. All extracts were kept at -80 oC until used. For metabolic profiling, the dried extracts from mouse feces were suspended in a 2:3 mixture of water and acetonitrile (10 μL per 1 mg of tissue), vortexed and cleared by centrifugation. Supernatants were collected and used as described below. Extracts were diluted 1:5 with 60% acetonitrile containing either 0.2% formic acid (for positive ion mode) or 0.5% ammonium hydroxide (for negative ion mode) and spiked with predefined amounts of the "ES tuning mix" solution as the internal standard for mass calibration. The solutions were then infused, using a syringe pump (KDS Scientific, Holliston, USA) at a flow rate of 2.5 µL per minute, into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer (Bruker Daltonics, Billerica, USA) equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Data were recorded in positive and negative ion modes with broadband detection and an FT acquisition size of 1024 kilobytes per second, within a range of m/z 150 to 1100. Under these settings, a mass resolution of ca. 100,000 (full width at half maximum, FWHM) at m/z 400 and a mass accuracy within 2 ppm or less for all detected components, following internal mass calibration, were observed. Other experimental parameters were: capillary electrospray voltage of 3600-3750 V, spray shield voltage of 3300-3450 V, source ion accumulation time of 0.1 s and collision cell ion accumulation time of 0.2 s. To increase detection sensitivity, survey scan mass spectra in positive and negative ion modes were acquired from the accumulation of 200 scans per spectrum, and duplicate acquisitions per sample were carried out to ensure data reproducibility. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140). To identify differences in metabolite composition between untreated and treated samples, we first filtered our list of masses for metabolites that were present on one set of samples (untreated or treated) but not the other. Additionally, we averaged the mass intensities of metabolites in each group and calculated the ratios between averaged intensities of metabolites from untreated and treated samples. To assign possible metabolite identities to the masses present in only one of the sample groups or showing at least a 2-fold change in intensities between the sample groups, the monoisotopic neutral masses of interest were queried against MassTrix (http://masstrix.org), a free-access software designed to incorporate masses into metabolic pathways. Masses were searched against the Mus musculus database within a mass error of 3 ppm. Data were analyzed by unpaired t tests with 95% confidence intervals.
2011-02-08 | E-GEOD-25687 | biostudies-arrayexpress
Project description:This experiment was performed to challenge E. coli response to acetate when its growth rate flux is reduced by 40% with 100 mM methyl α-D-glucopyranoside. Control without inhibition in the exact same conditions was already published on ArrayExpress database (accession number E-MTAB-9086) and could be used for comparison purpose. Here, E. coli BW25113 strain was grown in M9 medium complemented with 15 mM glucose, 100 mM αMG, and 0, 10, 50 or 100 mM sodium acetate. The cells were grown to mid-exponential phase in shake flasks at 37 °C and 200 rpm, in 50 mL of medium before proceeding to RNA extraction and subsequent microarray analysis. Three to four replicates (with two to three biological replicates) were analyzed for each condition. We thank all the INSA-Toulouse students that participated in this project: Lola Blayac, Romane Ducloux, Benjamin Jung, Oliver Larousse, Leyre Sarrias, Arno Bruel, Alexis Charbinat, Justine Dumas-Perdriau, Solène Frapard, Sophie Germain, Nicolas Papadopoulos, Marine Rodeghiero & Auriane Thomas.
2022-09-23 | E-MTAB-11717 | biostudies-arrayexpress
Project description:N-terminal proteoforms stem from the same gene but differ at their N-terminus, and most of these are found to be truncated, though some are N-terminally extended caused by ribosomes starting translation from codons in the annotated 5’UTR, and/or carry modified N-termini different from those of the canonical protein. Biological functions of N-terminal proteoforms are emerging, however, it remains unknown to what extend N-terminal proteoforms further expand the functional complexity. To address this in a more global manner, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. For this, we first generated an in-depth catalogue of N-terminal proteoforms in the cytosol of HEK293T cells. As the N-terminal region is the part that differs between the proteoforms, we performed N-terminal enrichment via COFRADIC on the cytosol of HEK293T cells. We combined three digestion enzymes to increase the depth of analysis. Data was searched twise: once with a regular UniProt database and a second time with a custom database (combining the sequences of UniProt proteins, UniProt isoforms and publicly available Ribo-seq data). Data was filtered and this resulted in a catalogue of 3,306 N-termini from which 20 pairs of canonical protein and N-terminal proteoform(s) were selected for interactome analysis. Our analysis of these pairs revealed that the overlap of the interactomes for both proteoforms is in general high, showing their functional relation. However, for all pairs tested we do report differences as well. We show that N-terminal proteoforms can be engaged in new/different interactions and as well can lose several interactions compared to the canonical protein.
2023-06-08 | PXD039392 | Pride
Project description:Ribosome profiling revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream open-reading frames (ORFs), long non-coding RNAs, ORFs in UTRs or ORFs in alternative reading frames. Ribo-seq but also bioinformatics-based prediction and RNA-sequencing reported translation of thousands of ORFs derived from non-translated regions (NTRs). Although such ORFs gained increased attention over the years, their actual coding potential remains debated as protein products of only a fraction of them were identified by mass spectrometry. Here, we introduced a new workflow to discover translation products of NTRs at a large-scale. We combined reducing sample complexity (by enriching N-terminal peptides of cytosolic proteins as such peptides are ideal proxies for translation) with and extend search space (combining the sequences of UniProt proteins, UniProt isoforms and publicly available Ribo-seq data) reasoning that this combination increased chances of identifying proteins from NTRs. Further, we introduced rigorous data analysis and results curation workflows to cope with the increased complexity of the search space and to mine identified peptides. This stringent filtering approach was found essential to retain confident translational evidence at the peptide level for NTRs. We show that theoretically our strategy facilitates the detection of translation events of transcripts from NTRs, but experimentally less than 1% of all identified peptides might originate from such translation events.
2022-07-12 | PXD030601 | Pride