[Show abstract][Hide abstract] ABSTRACT: We evaluate the potential of native mass spectrometry (MS) and ion mobility (IM-MS) for the screening of protein : ligand complexes when very subtle conformational changes are involved. As a proof of concept, we investigate the interactions between a peptide deformylase (PDF1B), a promising target for the development of new antibiotics, and three of its specific inhibitors that bind in different modes. First, real-time native MS reveals two types of ligands, both interacting in a 1 : 1 stoichiometry with PDF1B but with different affinities and gas phase stabilities. Conformational IM-MS screening then highlights two very close but significantly distinct ligand-induced conformations with collision cross sections that differ by less than 1%. Real-time IM-MS is used to monitor not only the dynamics of ligand binding to apoPDF1B but also the switching between holo conformations. This study provides additional evidence that the most potent ligands inhibit peptide deformylases through a slow-tight binding mechanism, in agreement with previous structural and enzymology studies. Furthermore, this approach, wherein the characteristics obtained by native MS are combined with IM-MS conformational screening, prove valuable in characterizing extremely subtle dynamic conformational changes induced when ligands bind to protein assemblies. We discuss the promise and limitations of IM-MS in the context of detection of very small conformational changes induced upon ligand binding.
The Analyst 09/2015; 140(21). DOI:10.1039/c5an01311a · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Significance:
Our paper tackles the open question of the cost of mounting an innate immune response. Evolutionary biologists are familiar since a long time with the concept of trade-offs among key traits of an organism, trade-offs that shape life history trajectories of species and individuals, ultimately in terms of reproduction and survival. On the other hand, medicine and molecular biologists study the intimate mechanisms of immune senescence and underline that oxidative imbalance is probably playing a key role in the progressive loss of immune function with age. This paper merges the two fields by exploring the nature of the cellular pathways that are mainly affected by age when the innate immunity is triggered. To this purpose, a proteomic approach was used to explore liver protein profiles and provide for the first time convincing data supporting the idea that oxidative stress constitutes a cost of innate immune response in old mice, possibly contributing to senescence. Proteomics-derived hypotheses were furthermore validated using biochemical assays. This paper therefore illustrates the added value of using proteomics to answer evolutionary biology questions, and opens a promising way to study the inter-specific variability in the rates of immune-senescence.
Journal of proteomics 09/2015; DOI:10.1016/j.jprot.2015.09.008 · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)-an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol-thiol heteroconjugates. Namely, an antibody-drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we applied the combination of one-dimensional gel electrophoresis, immunoblot and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to identify potential immunogenic proteins of Toxoplasma gondii tachyzoites that can be used for the development of reliable assays in the serodiagnosis of acquired toxoplasmosis in immunocompetent subjects. For this purpose, we developed an immunoblot using soluble and membrane extracts of GT1 Toxoplasma gondii tachyzoites and tested 194 positive and 100 negative sera obtained from pregnant women.
Five bands of soluble antigens (98 kDa, 36 kDa, 33 kDa, 32 kDa and 21 kDa) and 4 bands of membrane antigens (41 kDa, 35 kDa, 32 kDa and 30 kDa) were selected as the most valuable in terms of sensitivity and specificity.
Among these bands, only 2 bands of soluble antigen (33 kDa and 32 kDa) and 2 bands of membrane antigen (32 kDa and 30 kDa) showed a specificity ≥ 90%.
After mass spectrometry and bioinformatics analysis, 7 proteins were identified as potential markers for
serodiagnosis of toxoplasmosis. These proteins are: SRS34A, GRA7, GRA1, DG32, MIC5, ROP5 and Toxofilin.
These proteins showed a 86% to 100% homology with proteins of both VEG and ME49 strains of T. gondii and a 58% to 87% homology with Hammondia hammondi; and can be considered as attractive candidates for the development of an immunochromatography test that can be used for the rapid diagnosis of toxoplasmosis and as a confirmatory test when routine techniques give equivocal results.
Journal of Bacteriology & Parasitology 08/2015; 6(4):1-10.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we applied the combination of one-dimensional gel electrophoresis, immunoblot and nano liquidchromatography-tandem mass spectrometry (nanoLC-MS/MS) to identify potential immunogenic proteins of Toxoplasma gondii tachyzoites that can be used for the development of reliable assays in the serodiagnosis of acquired toxoplasmosis in immunocompetent subjects. For this purpose, we developed an immunoblot using soluble and membrane extracts of GT1 Toxoplasma gondii tachyzoites and tested 194 positive and 100 negative sera obtained from pregnant women.
Five bands of soluble antigens (98 kDa, 36 kDa, 33 kDa, 32 kDa and 21 kDa) and 4 bands of membrane antigens (41 kDa, 35 kDa, 32 kDa and 30 kDa) were selected as the most valuable in terms of sensitivity and specificity. Among these bands, only 2 bands of soluble antigen (33 kDa and 32 kDa) and 2 bands of membrane antigen (32 kDa and 30 kDa) showed a specificity ≥ 90%.
After mass spectrometry and bioinformatics analysis, 7 proteins were identified as potential markers for
serodiagnosis of toxoplasmosis. These proteins are: SRS34A, GRA7, GRA1, DG32, MIC5, ROP5 and Toxofilin. These proteins showed a 86% to 100% homology with proteins of both VEG and ME49 strains of T. gondii and a 58% to 87% homology with Hammondia hammondi; and can be considered as attractive candidates for the development of an immunochromatography test that can be used for the rapid diagnosis of toxoplasmosis and as a confirmatory test when routine techniques give equivocal results.
[Show abstract][Hide abstract] ABSTRACT: Microorganisms, such as bacteria, are one of the first targets of nanoparticles in the environment. In this study, we tested the effect of two nanoparticles, ZnO and TiO2, with the salt ZnSO4 as the control, on the Gram-positive bacterium Bacillus subtilis by 2D gel electrophoresis-based proteomics. Despite a significant effect on viability (LD50), TiO2 NPs had no detectable effect on the proteomic pattern, while ZnO NPs and ZnSO4 significantly modified B. subtilis metabolism. These results allowed us to conclude that the effects of ZnO observed in this work were mainly attributable to Zn dissolution in the culture media. Proteomic analysis highlighted twelve modulated proteins related to central metabolism: MetE and MccB (cysteine metabolism), OdhA, AspB, IolD, AnsB, PdhB and YtsJ (Krebs cycle) and XylA, YqjI, Drm and Tal (pentose phosphate pathway). Biochemical assays, such as free sulfhydryl, CoA-SH and malate dehydrogenase assays corroborated the observed central metabolism reorientation and showed that Zn stress induced oxidative stress, probably as a consequence of thiol chelation stress by Zn ions. The other patterns affected by ZnO and ZnSO4 were the stringent response and the general stress response. Nine proteins involved in or controlled by the stringent response showed a modified expression profile in the presence of ZnO NPs or ZnSO4: YwaC, SigH, YtxH, YtzB, TufA, RplJ, RpsB, PdhB and Mbl. An increase in the ppGpp concentration confirmed the involvement of the stringent response during a Zn stress. All these metabolic reorientations in response to Zn stress were probably the result of complex regulatory mechanisms including at least the stringent response via YwaC.
Journal of proteomics 07/2015; DOI:10.1016/j.jprot.2015.07.018 · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the framework of the C-HPP, our Franco-Swiss consortium has adopted chromosomes 2 and 14, coding for a total of 382 missing proteins (proteins for which evidence is lacking at protein level). Over the last four years, the French proteomics infrastructure has collected high quality datasets from 40 human samples, including a series of rarely studied cell lines, tissue types and sample preparations. Here, we described a step-by-step strategy based on the use of bioinformatics screening and subsequent mass spectrometry (MS)-based validation to identify what were up to now missing proteins in these datasets. Screening database search results (85,326 .dat files) identified 58 of the missing proteins (36 on chromosome 2 and 22 on chromosome 14) by 83 unique peptides following the latest release of neXtProt (2014-10). PSMs corresponding to these peptides were thoroughly examined by applying two different MS-based criteria: peptide-level False Discovery Rate (FDR) calculation and expert PSM quality assessment. Synthetic peptides were then produced and used to generate reference MS/MS spectra. A spectral similarity score was then calculated for each pair of reference-endogenous spectra and used as a third criterion for missing protein validation. Finally, LC-SRM assays were developed to target proteotypic peptides from 4 of the missing proteins detected in tissue/cell samples which were still available and for which sample preparation could be reproduced. These LC-SRM assays unambiguously detected the endogenous unique peptide for 3 of the proteins. For 2 of these, identification was confirmed by additional proteotypic peptides. We concluded that of the initial set of 58 proteins detected by the bioinformatics screen, the consecutive MS-based validation criteria led to propose the identification of 13 of these proteins (8 on chromosome 2 and 5 on chromosome 14) that passed at least two of the three MS-based criteria. Thus, a rigorous step-by-step approach combining bioinformatics screening and MS-based validation assays is particularly suitable to obtain protein-level evidence for proteins previously considered as missing. All MS/MS data have been deposited in ProteomeXchange under identifier PXD002131.
Journal of Proteome Research 07/2015; 14(9). DOI:10.1021/pr5010345 · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study aimed to identify a protein marker that can differentiate between fresh skinless and frozen-thawed sea bass (Dicentrarchus labrax) fillets using the two-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Distinct gel patterns, due to proteins with low molecular weight and low isoelectric points, distinguished fresh fillets from frozen-thawed ones. Frozen-thawed fillets showed two specific protein spots as early as the first day of the study. However, these spots were not observed in fresh fillets until at least 13 days of storage between 0 and 4°C, fillets were judged, beyond this period, fish were unfit for human consumption as revealed by complementary studies on fish spoilage indicators namely total volatile basic nitrogen and biogenic amines. Mass spectrometry identified the specific proteins as parvalbumin isoforms. Parvalbumins may thus be useful markers of differentiation between fresh and frozen-thawed sea bass fillets.
[Show abstract][Hide abstract] ABSTRACT: The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenyl phosphonium (TMPP) labeling approaches. Owing to the extent of protein import and cleavage for mitochondrial proteins, determining the new N-termini generated after translocation/processing events for mitochondrial proteins is crucial to understand the transformation of precursors to mature proteins. The dN-TOP strategy based on a double light/heavy TMPP labeling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis. A total of 2714 proteins were identified and 897 N-terminal peptides were characterized (424 N-α-acetylated and 473 TMPP-labelled peptides). These results allowed the precise identification of the N-terminus of 693 unique proteins corresponding to 26% of all identified proteins. Overall, 120 already annotated processing cleavage sites were confirmed while 302 new cleavage sites were characterized. The accumulation of experimental evidence of mature N-termini should allow increasing the knowledge of processing mechanisms and consequently also enhance cleavage sites prediction algorithms. Complete datasets have been deposited to the ProteomeXchange Consortium with identifiers PXD001521, PXD001522 and PXD001523. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.
PLoS ONE 04/2015; 10(4):e0124496. DOI:10.1371/journal.pone.0124496 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The bioproduction of recombinant monoclonal antibodies (mAbs) results in complex mixtures of a main isoform and numerous macro and micro-variants. MAbs present therefore different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities are truncation and fragmentation of the primary amino-acid sequence resulting in shorter or cleaved polypeptide chains. Another reported heterogeneity comes from the incomplete processing of the signal peptide resulting in N-terminal elongated polypeptide chains. Here we present a novel in-gel protein N-terminal chemical derivatization method with trimethoxyphenyl phosphonium (TMPP) that enhances the mass spectrometry detection of N-terminal positions of proteins and allows their unambiguous assignment without altering the identification of internal digestion peptides. This method requires only one additional step to a classical peptide mapping workflow. This in-gel N-TOP (N-Terminal Oriented Proteomics) strategy was applied successfully to detect and characterize the N-terminal sequence heterogeneities of several monoclonal antibodies among which minor unexpected proteoforms.
[Show abstract][Hide abstract] ABSTRACT: Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are 5-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native mass spectrometry (MS) to build a 2D connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Amine-to-thiol coupling is the most common route for the preparation of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. A series of antibody-dye conjugates have been prepared using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.
[Show abstract][Hide abstract] ABSTRACT: To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB).
TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status.
In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (ie, capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range.
Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB.
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Gut 11/2014; DOI:10.1136/gutjnl-2014-308041 · 14.66 Impact Factor