Serum proteome signatures associated with ileal and colonic ulcers in Crohn's disease.

At a clinical level, ileal and colonic Crohn's disease (CD) are considered as separate entities. These subphenotypes need to be better supported by biological data to develop personalised medicine in CD. To this end, we combined different technologies (proximity extension assay, selected reaction monitoring, and high-sensitivity turbidimetric immunoassay (hsCRP)) to measure 207 immune-related serum proteins in CD patients presenting no endoscopic lesions (endoscopic remission) (n = 23), isolated ileal ulcers (n = 17), or isolated colonic ulcers (n = 16). We showed that isolated ileal ulcers and isolated colonic ulcers were specifically associated with 6 and 18 serum proteins, respectively: (high level: JUN, CNTNAP2; low level: FCRL6, LTA, CLEC4A, NTF4); (high level: hsCRP, IL6, APCS, CFB, MBL2, IL7, IL17A, CCL19, CXCL10, CSF3, IL10, CLEC4G, MMP12, VEGFA; low level: CLEC3B, GSN, TNFSF12, TPSAB1). Isolated ileal ulcers and isolated colonic ulcers were detected by hsCRP with an area under the receiver operating characteristics curve of 0.64 (p-value = 0.07) and 0.77 (p-value = 0.001), respectively. We highlighted distinct serum proteome profiles associated with ileal and colonic ulcers in CD, this finding might support the development of therapeutics and biomarkers tailored to disease location. SIGNIFICANCE: Although ileal and colonic Crohn's disease present important clinical differences (eg, progression, response to treatment and reliability of biomarkers), these two entities are managed with the same therapeutic strategy. The biological specificities of ileal and colonic Crohn's disease need to be better characterised to develop more personalised approaches. The present study used robust technologies (selected reaction monitoring, proximity extension assays and turbidimetric immunoassay) to quantify precisely 207 serum immune-related proteins in three groups of Crohn's disease patients presenting: 1) no endoscopic lesions (endoscopic remission) (n = 23); 2) isolated ileal ulcers (n = 17); 3) isolated colonic ulcers (n = 16). We found distinct serum proteome signatures associated with ileal and colonic ulcers. Our findings could foster the development of biomarkers and treatments tailored to Crohn's disease location.

Investigation of Structure-Stabilizing Elements in Proteins by Ion Mobility Mass Spectrometry and Collision-Induced Unfolding.

A recently developed proteolytic reactor, designed for protein structural investigation, was coupled to ion mobility mass spectrometry to monitor collisional cross section (CCS) evolution of model proteins undergoing trypsin-mediated mono enzymatic digestion. As peptides are released during digestion, the CCS of the remaining protein structure may deviate from the classical 2/3 power of the CCS-mass relationship for spherical structures. The classical relationship between CCS and mass (CCS = A × M2/3) for spherical structures, assuming a globular shape in the gas phase, may deviate as stabilizing elements are lost during digestion. In addition, collision-induced unfolding (CIU) experiments on partially digested proteins provided insights into the CCS resilience in the gas phase to ion activation, potentially due to the presence of stabilizing elements. The study initially investigated a model peptide ModBea (3 kDa), assessing the impact of disulfide bridges on CCS resilience in both reduced and oxidized forms. Subsequently, ß-lactoglobulin (2 disulfide bridges), calmodulin (Ca2+ coordination cation), and cytochrome c (heme) were selected to investigate the influence of common structuring elements on CCS resilience. CIU experiments probed the unfolding process, evaluating the effect of losing specific peptides on the energy landscapes of partially digested proteins. Comparisons of the TWCCSN2→He to trend curves describing the CCS/mass relationship revealed that proteins with structure-stabilizing elements consistently exhibit TWCCSN2→He and greater resilience toward CIU compared to proteins lacking these elements. The integration of online digestion, ion mobility, and CIU provides a valuable tool for identifying structuring elements in biopolymers in the gas phase.

An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing.

CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.

Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal.

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.

Oncogenic CALR mutant C-terminus mediates dual binding to the thrombopoietin receptor triggering complex dimerization and activation.

Calreticulin (CALR) frameshift mutations represent the second cause of myeloproliferative neoplasms (MPN). In healthy cells, CALR transiently and non-specifically interacts with immature N-glycosylated proteins through its N-terminal domain. Conversely, CALR frameshift mutants turn into rogue cytokines by stably and specifically interacting with the Thrombopoietin Receptor (TpoR), inducing its constitutive activation. Here, we identify the basis of the acquired specificity of CALR mutants for TpoR and define the mechanisms by which complex formation triggers TpoR dimerization and activation. Our work reveals that CALR mutant C-terminus unmasks CALR N-terminal domain, rendering it more accessible to bind immature N-glycans on TpoR. We further find that the basic mutant C-terminus is partially α-helical and define how its α-helical segment concomitantly binds acidic patches of TpoR extracellular domain and induces dimerization of both CALR mutant and TpoR. Finally, we propose a model of the tetrameric TpoR-CALR mutant complex and identify potentially targetable sites.

The structure of DarB in complex with RelNTD reveals nonribosomal activation of Rel stringent factors.

Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP-binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB2:RelNTD2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.

Lunaemycins, New Cyclic Hexapeptide Antibiotics from the Cave Moonmilk-Dweller Streptomyces lunaelactis MM109T.

Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S. lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S. lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and 1H/13C NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.

Sweat Proteomics in Cystic Fibrosis: Discovering Companion Biomarkers for Precision Medicine and Therapeutic Development.

In clinical routine, the diagnosis of cystic fibrosis (CF) is still challenging regardless of international consensus on diagnosis guidelines and tests. For decades, the classical Gibson and Cooke test measuring sweat chloride concentration has been a keystone, yet, it may provide normal or equivocal results. As of now, despite the combination of sweat testing, CFTR genotyping, and CFTR functional testing, a small fraction (1-2%) of inconclusive diagnoses are reported and justifies the search for new CF biomarkers. More importantly, in the context of precision medicine, with a view to early diagnosis, better prognosis, appropriate clinical follow-up, and new therapeutic development, discovering companion biomarkers of CF severity and phenotypic rescue are of utmost interest. To date, previous sweat proteomic studies have already documented disease-specific variations of sweat proteins (e.g., in schizophrenia and tuberculosis). In the current study, sweat samples from 28 healthy control subjects and 14 patients with CF were analyzed by nanoUHPLC-Q-Orbitrap-based shotgun proteomics, to look for CF-associated changes in sweat protein composition and abundance. A total of 1057 proteins were identified and quantified at an individual level, by a shotgun label-free approach. Notwithstanding similar proteome composition, enrichment, and functional annotations, control and CF samples featured distinct quantitative proteome profiles significantly correlated with CF, accounting for the respective inter-individual variabilities of control and CF sweat. All in all: (i) 402 sweat proteins were differentially abundant between controls and patients with CF, (ii) 68 proteins varied in abundance between F508del homozygous patients and patients with another genotype, (iii) 71 proteins were differentially abundant according to the pancreatic function, and iv) 54 proteins changed in abundance depending on the lung function. The functional annotation of pathophysiological biomarkers highlighted eccrine gland cell perturbations in: (i) protein biosynthesis and trafficking, (ii) CFTR proteostasis and membrane stability, and (iii) cell-cell adherence, membrane integrity, and cytoskeleton crosstalk. Cytoskeleton-related biomarkers were of utmost interest because of the consistency between variations observed here in CF sweat and variations previously documented in other CF tissues. From a clinical stance, nine candidate biomarkers of CF diagnosis (CUTA, ARG1, EZR, AGA, FLNA, MAN1A1, MIA3, LFNG, SIAE) and seven candidate biomarkers of CF severity (ARG1, GPT, MDH2, EML4 (F508del homozygous), MGAT1 (pancreatic insufficiency), IGJ, TOLLIP (lung function impairment)) were deemed suitable for further verification.

Structure of New Ferroverdins Recruiting Unconventional Ferrous Iron Chelating Agents.

Ferroverdins are ferrous iron (Fe2+)-nitrosophenolato complexes produced by a few Streptomyces species as a response to iron overload. Previously, three ferroverdins were identified: ferroverdin A, in which three molecules of p-vinylphenyl-3-nitroso-4-hydroxybenzoate (p-vinylphenyl-3,4-NHBA) are recruited to bind Fe2+, and Ferroverdin B and Ferroverdin C, in which one molecule of p-vinylphenyl-3,4-NHBA is substituted by hydroxy-p-vinylphenyl-3,4-NHBA, and by carboxy-p-vinylphenyl-3,4-NHBA, respectively. These molecules, especially ferroverdin B, are potent inhibitors of the human cholesteryl ester transfer protein (CETP) and therefore candidate hits for the development of drugs that increase the serum concentration of high-density lipoprotein cholesterol, thereby diminishing the risk of atherosclerotic cardiovascular disease. In this work, we used high-resolution mass spectrometry combined with tandem mass spectrometry to identify 43 novel ferroverdins from the cytosol of two Streptomyces lunaelactis species. For 13 of them (designated ferroverdins C2, C3, D, D2, D3, E, F, G, H, CD, DE, DF, and DG), we could elucidate their structure, and for the other 17 new ferroverdins, ambiguity remains for one of the three ligands. p-formylphenyl-3,4-NHBA, p-benzoic acid-3,4-NHBA, 3,4-NHBA, p-phenylpropionate-3,4-NHBA, and p-phenyacetate-3,4-NHBA were identified as new alternative chelators for Fe2+-binding, and two compounds (C3 and D3) are the first reported ferroverdins that do not recruit p-vinylphenyl-3,4-NHBA. Our work thus uncovered putative novel CETP inhibitors or ferroverdins with novel bioactivities.

Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization.

Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog "in cascade", between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.

Oligodendrocyte precursors guide interneuron migration by unidirectional contact repulsion.

In the forebrain, ventrally derived oligodendrocyte precursor cells (vOPCs) travel tangentially toward the cortex together with cortical interneurons. Here, we tested in the mouse whether these populations interact during embryogenesis while migrating. By coupling histological analysis of genetic models with live imaging, we show that although they are both attracted by the chemokine Cxcl12, vOPCs and cortical interneurons occupy mutually exclusive forebrain territories enriched in this chemokine. Moreover, first-wave vOPC depletion selectively disrupts the migration and distribution of cortical interneurons. At the cellular level, we found that by promoting unidirectional contact repulsion, first-wave vOPCs steered the migration of cortical interneurons away from the blood vessels to which they were both attracted, thereby allowing interneurons to reach their proper cortical territories.

HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources.

Despite the high prevalence of both cervico-vaginal human papillomavirus (HPV) infection and bacterial vaginosis (BV) worldwide, their causal relationship remains unclear. While BV has been presumed to be a risk factor for HPV acquisition and related carcinogenesis for a long time, here, supported by both a large retrospective follow-up study (n = 6,085) and extensive in vivo data using the K14-HPV16 transgenic mouse model, we report a novel blueprint in which the opposite association also exists. Mechanistically, by interacting with several core members (NEMO, CK1 and ß-TrCP) of both NF-κB and Wnt/ß-catenin signaling pathways, we show that HPV E7 oncoprotein greatly inhibits host defense peptide expression. Physiologically secreted by the squamous mucosa lining the lower female genital tract, we demonstrate that some of these latter are fundamental factors governing host-microbial interactions. More specifically, several innate molecules down-regulated in case of HPV infection are hydrolyzed, internalized and used by the predominant Lactobacillus species as amino acid source sustaining their growth/survival. Collectively, this study reveals a new viral immune evasion strategy which, by its persistent/negative impact on lactic acid bacteria, ultimately causes the dysbiosis of vaginal microbiota.

Label-Free Higher Order Structure and Dynamic Investigation Method of Proteins in Solution Using an Enzymatic Reactor Coupled to Electrospray High-Resolution Mass Spectrometry Detection.

For decades, structural analysis of proteins have received considerable attention, from their sequencing to the determination of their 3D structures either in the free state (e.g., no host-guest system, apoproteins) or (non)covalently bound complexes. The elucidation of the 3D structures and the mapping of intra- and intermolecular interactions are valuable sources of information to understand the physicochemical properties of such systems. X-ray crystallography and nuclear magnetic resonance are methods of choice for obtaining structures at the atomic level. Nonetheless, they still present drawbacks which limit their use to highly purified systems in a relatively high amount. On the contrary, mass spectrometry (MS) has become a powerful tool thanks to its selectivity, sensitivity, and the development of structural methods both at the global shape and the residue level. The combination of several MS-based methods is mandatory to fully assign a putative structure in combination with computational chemistry and bioinformatics. In that context, we propose a strategy which complements the existing methods of structural studies (e.g., circular dichroism, hydrogen/deuterium exchange and cross-links experiments, nuclear magnetic resonance). The workflow is based on the collection of structural information on proteins from the apparition rates and the time of appearance of released peptides generated by a protease in controlled experimental conditions with online detection by electrospray high-resolution mass spectrometry. Nondenaturing, partially or fully denatured proteins were digested by the enzymatic reactor, i.e., ß-lactoglobulin, cytochrome c, and ß-casein. The collected data are interpreted with regard to the kinetic schemes with time-dependent rates of the enzymatic digestion established beforehand, considering kinetics parameters in the Michaelis-Menten formalism including kcat (the turnover number), k1 (formation of the enzyme-substrate complex), k-1 (dissociation of the enzyme-substrate complex), koff (local refolding of the protein around the cleavage site), and kon (local unfolding of the protein around the cleavage site). Solvent-accessible surface analysis through digestion kinetics was also investigated. The initial apparition rates of released peptides varied according to the protein state (folded vs denatured) and informs the koff/kon ratio around the cleavage site. On the other hand, the time of appearance of a given peptide is related to its solvent accessibility and to the resilience of the residual protein structure in solution. Temperature-dependent digestion experiments allowed estimation of the type of secondary structures around the cleavage site.

Are Physicochemical Properties Shaping the Allergenic Potency of Plant Allergens?

This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing.

Are Physicochemical Properties Shaping the Allergenic Potency of Animal Allergens?

Key determinants for the development of an allergic response to an otherwise 'harmless' food protein involve different factors like the predisposition of the individual, the timing, the dose, the route of exposure, the intrinsic properties of the allergen, the food matrix (e.g. lipids) and the allergen modification by food processing. Various physicochemical parameters can have an impact on the allergenicity of animal proteins. Following our previous review on how physicochemical parameters shape plant protein allergenicity, the same analysis was proceeded here for animal allergens. We found that each parameter can have variable effects, ranging on an axis from allergenicity enhancement to resolution, depending on its nature and the allergen. While glycosylation and phosphorylation are common, both are not universal traits of animal allergens. High molecular structures can favour allergenicity, but structural loss and uncovering hidden epitopes can also have a similar impact. We discovered that there are important knowledge gaps in regard to physicochemical parameters shaping protein allergenicity both from animal and plant origin, mainly because the comparability of the data is poor. Future biomolecular studies of exhaustive, standardised design together with strong validation part in the clinical context, together with data integration model systems will be needed to unravel causal relationships between physicochemical properties and the basis of protein allergenicity.

New applications of advanced instrumental techniques for the characterization of food allergenic proteins.

Current approaches based on electrophoretic, chromatographic or immunochemical principles have allowed characterizing multiple allergens, mapping their epitopes, studying their mechanisms of action, developing detection and diagnostic methods and therapeutic strategies for the food and pharmaceutical industry. However, some of the common structural features related to the allergenic potential of food proteins remain unknown, or the pathological mechanism of food allergy is not yet fully understood. In addition, it is also necessary to evaluate new allergens from novel protein sources that may pose a new risk for consumers. Technological development has allowed the expansion of advanced technologies for which their whole potential has not been entirely exploited and could provide novel contributions to still unexplored molecular traits underlying both the structure of food allergens and the mechanisms through which they sensitize or elicit adverse responses in human subjects, as well as improving analytical techniques for their detection. This review presents cutting-edge instrumental techniques recently applied when studying structural and functional aspects of proteins, mechanism of action and interaction between biomolecules. We also exemplify their role in the food allergy research and discuss their new possible applications in several areas of the food allergy field.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

SCOPE: Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. METHODS AND RESULTS: The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. CONCLUSION: Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

Characterization of the Human Eccrine Sweat Proteome-A Focus on the Biological Variability of Individual Sweat Protein Profiles.

The potential of eccrine sweat as a bio-fluid of interest for diagnosis and personalized therapy has not yet been fully evaluated, due to the lack of in-depth sweat characterization studies. Thanks to recent developments in omics, together with the availability of accredited sweat collection methods, the analysis of human sweat may now be envisioned as a standardized, non-invasive test for individualized monitoring and personalized medicine. Here, we characterized individual sweat samples, collected from 28 healthy adult volunteers under the most standardized sampling methodology, by applying optimized shotgun proteomics. The thorough characterization of the sweat proteome allowed the identification of 983 unique proteins from which 344 were identified across all samples. Annotation-wise, the study of the sweat proteome unveiled the over-representation of newly addressed actin dynamics, oxidative stress and proteasome-related functions, in addition to well-described proteolysis and anti-microbial immunity. The sweat proteome composition correlated with the inter-individual variability of sweat secretion parameters. In addition, both gender-exclusive proteins and gender-specific protein abundances were highlighted, despite the high similarity between human female and male sweat proteomes. In conclusion, standardized sample collection coupled with optimized shotgun proteomics significantly improved the depth of sweat proteome coverage, far beyond previous similar studies. The identified proteins were involved in many diverse biological processes and molecular functions, indicating the potential of this bio-fluid as a valuable biological matrix for further studies. Addressing sweat variability, our results prove the proteomic profiling of sweat to be a promising bio-fluid analysis for individualized, non-invasive monitoring and personalized medicine.

Up-Regulated Salivary Proteins of Brown Marmorated Stink Bug Halyomorpha halys on Plant Growth-Promoting Rhizobacteria-Treated Plants.

Plant Growth-Promoting Rhizobacteria (PGPR) induce systemic resistance (SR) in plants, decreasing the development of phytopathogens. The FZB42 strain of Bacillus velezensis is known to induce an SR against pathogens in various plant species. Previous studies suggested that it could also influence the interactions between plants and associated pests. However, insects have developed several strategies to counteract plant defenses, including salivary proteins that allow the insect escaping detection, manipulating defensive pathways to its advantage, deactivating early signaling processes, or detoxifying secondary metabolites. Because Brown Marmorated Stink Bug (BMSB) Halyomorpha halys is highly invasive and polyphagous, we hypothesized that it could detect the PGPR-induced systemic defenses in the plant, and efficiently adapt its salivary compounds to counteract them. Therefore, we inoculated a beneficial rhizobacterium on Vicia faba roots and soil, previous to plant infestation with BMSB. Salivary gland proteome of BMSB was analyzed by LC-MS/MS and a label-free quantitative proteomic method. Among the differentially expressed proteins, most were up-regulated in salivary glands of insects exposed to PGPR-treated plants for 24 h. We could confirm that BMSB was confronted with a stress during feeding on PGPR-treated plants. The to-be-confirmed defensive state of the plant would have been rapidly detected by the invasive H. halys pest, which consequently modified its salivary proteins. Among the up-regulated proteins, many could be associated with a role in plant defense counteraction, and more especially in allelochemicals detoxification or sequestration.

Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process.

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.