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.

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.

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.

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.

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.

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.

New Proteins Contributing to Immune Cell Infiltration and Pannus Formation of Synovial Membrane from Arthritis Diseases.

An inflamed synovial membrane plays a major role in joint destruction and is characterized by immune cells infiltration and fibroblast proliferation. This proteomic study considers the inflammatory process at the molecular level by analyzing synovial biopsies presenting a histological inflammatory continuum throughout different arthritis joint diseases. Knee synovial biopsies were obtained from osteoarthritis (OA; n = 9), chronic pyrophosphate arthropathy (CPPA; n = 7) or rheumatoid arthritis (RA; n = 8) patients. The histological inflammatory score was determined using a semi-quantitative scale based on synovial hyperplasia, lymphocytes, plasmocytes, neutrophils and macrophages infiltration. Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS/MS). Differentially expressed proteins were confirmed by immunohistochemistry. Out of the 1871 proteins identified and quantified by LC-MS/MS, 10 proteins (LAP3, MANF, LCP1, CTSZ, PTPRC, DNAJB11, EML4, SCARA5, EIF3K, C1orf123) were differentially expressed in the synovial membrane of at least one of the three disease groups (RA, OA and CPPA). Significant increased expression of the seven first proteins was detected in RA and correlated to the histological inflammatory score. Proteomics is therefore a powerful tool that provides a molecular pattern to the classical histology usually applied for synovitis characterization. Except for LCP1, CTSZ and PTPRC, all proteins have never been described in human synovitis.

Discovery of biomarker candidates associated with the risk of short-term and mid/long-term relapse after infliximab withdrawal in Crohn's patients: a proteomics-based study.

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse were searched in the baseline serum of CD patients stopping infliximab when they were under combined therapy (antimetabolite and infliximab) and stable clinical remission (diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors cohort, n=102). From shotgun proteomics experiment (discovery step), biomarker candidates were identified and further targeted by selected reaction monitoring (verification step). The dataset was stratified to search for markers of short-term (<6 months) or mid/long-term relapse (>6 months). The risk of relapse and the predicting capacity associated with biomarker candidates were evaluated using univariate Cox model and log-rank statistic, respectively. To test their complementary predicting capacity, biomarker candidates were systematically combined in pairs. RESULTS: Distinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9

Glycosylation deficiency of lipopolysaccharide-binding protein and corticosteroid-binding globulin associated with activity and response to treatment for rheumatoid arthritis.

BACKGROUND: Serum protein glycosylation is an area of investigation in inflammatory arthritic disorders such as rheumatoid arthritis (RA). Indeed, some studies highlighted abnormalities of protein glycosylation in RA. Considering the numerous types of enzymes, monosaccharides and glycosidic linkages, glycosylation is one of the most complex post translational modifications. By this work, we started with a preliminary screening of glycoproteins in serum from RA patients and controls. METHODS: In order to isolate glycoproteins from serum, lectin wheat germ agglutinin was used and quantitative differences between patients and controls were investigated by LC-MS/MS. Consequently, we focused our attention on two glycoproteins found in this explorative phase: corticosteroid-binding globulin (CBG) and lipopolysaccharide-binding protein (LBP). The subsequent validation with immunoassays was widened to a larger number of early RA (ERA) patients (n = 90) and well-matched healthy controls (n = 90). RESULTS: We observed a significant reduction of CBG and LBP glycosylation in ERA patients compared with healthy controls. Further, after 12 months of treatment, glycosylated CBG and LBP levels increased both to values comparable to those of controls. In addition, these changes were correlated with clinical parameters. CONCLUSIONS: This study enables to observe that glycosylation changes of CBG and LBP are related to RA disease activity and its response to treatment.

Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

Accelerated pre-senile systemic amyloidosis in PACAP knockout mice - a protective role of PACAP in age-related degenerative processes.

Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal, and esophageal involvement. Mass spectrometry-based proteomic analysis, reconfirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposits in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene-deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Precise co-registration of mass spectrometry imaging, histology, and laser microdissection-based omics.

Mass spectrometry imaging (MSI) is an analytical technique for the unlabeled and multiplex imaging of molecules in biological tissue sections. It therefore enables the spatial and molecular annotations of tissues complementary to histology. It has already been shown that MSI can guide subsequent material isolation technologies such as laser microdissection (LMD) to enable a more in-depth molecular characterization of MSI-highlighted tissue regions. However, with MSI now reaching spatial resolutions at the single-cell scale, there is a need for a precise co-registration between MSI and the LMD. As proof-of-principle, MSI of lipids was performed on a breast cancer tissue followed by a segmentation of the data to detect molecularly distinct segments within its tumor areas. After image processing of the segmentation results, the coordinates of the MSI-detected segments were passed to the LMD system by three co-registration steps. The errors of each co-registration step were quantified and the total error was found to be less than 13 µm. With this link established, MSI data can now accurately guide LMD to excise MSI-defined regions of interest for subsequent extract-based analyses. In our example, the excised tissue material was then subjected to ultrasensitive microproteomics in order to determine predominant molecular mechanisms in each of the MSI-highlighted intratumor segments. This work shows how the strengths of MSI, histology, and extract-based omics can be combined to enable a more comprehensive molecular characterization of in situ biological processes.

Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells.

The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.

Proteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma.

Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV-related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region-specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease-free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages.

BACKGROUND: Despite recent advances in colorectal cancer (CRC) diagnosis and population screening programs, the identification of patients with preneoplastic lesions or with early CRC stages remains challenging and is important for reducing CRC incidence and increasing patient's survival. METHODS: We analysed 76 colorectal tissue samples originated from early CRC stages, normal or inflamed mucosa by label-free proteomics. The characterisation of three selected biomarker candidates was performed by immunohistochemistry on an independent set of precancerous and cancerous lesions harbouring increasing CRC stages. RESULTS: Out of 5258 proteins identified, we obtained 561 proteins with a significant differential distribution among groups of patients and controls. KNG1, OLFM4 and Sec24C distributions were validated in tissues and showed different expression levels especially in the two early CRC stages compared to normal and preneoplastic tissues. CONCLUSION: We highlighted three proteins that require further investigations to better characterise their role in early CRC carcinogenesis and their potential as early CRC markers.

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-ß binding protein 1, TGF-ß RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-ß activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing.

Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.

Standardized Workflow for Mass-Spectrometry-Based Single-Cell Proteomics Data Processing and Analysis Using the scp Package.

Mass-spectrometry (MS)-based single-cell proteomics (SCP) explores cellular heterogeneity by focusing on the functional effectors of the cells-proteins. However, extracting meaningful biological information from MS data is far from trivial, especially with single cells. Currently, data analysis workflows are substantially different from one research team to another. Moreover, it is difficult to evaluate pipelines as ground truths are missing. Our team has developed the R/Bioconductor package called scp to provide a standardized framework for SCP data analysis. It relies on the widely used QFeatures and SingleCellExperiment data structures. In addition, we used a design containing cell lines mixed in known proportions to generate controlled variability for data analysis benchmarking. In this chapter, we provide a flexible data analysis protocol for SCP data using the scp package together with comprehensive explanations at each step of the processing. Our main steps are quality control on the feature and cell level, aggregation of the raw data into peptides and proteins, normalization, and batch correction. We validate our workflow using our ground truth data set. We illustrate how to use this modular, standardized framework and highlight some crucial steps.

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.

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.