Examination of the Complex Molecular Landscape in Obesity and Type 2 Diabetes.

The escalating prevalence of metabolic disorders, notably type 2 diabetes (T2D) and obesity, presents a critical global health challenge, necessitating deeper insights into their molecular underpinnings. Our study integrates proteomics and metabolomics analyses to delineate the complex molecular landscapes associated with T2D and obesity. Leveraging data from 130 subjects, including individuals with T2D and obesity as well as healthy controls, we elucidate distinct molecular signatures and identify novel biomarkers indicative of disease progression. Our comprehensive characterization of cardiometabolic proteins and serum metabolites unveils intricate networks of biomolecular interactions and highlights differential protein expression patterns between T2D and obesity cohorts. Pathway enrichment analyses reveal unique mechanisms underlying disease development and progression, while correlation analyses elucidate the interplay between proteomics, metabolomics, and clinical parameters. Furthermore, network analyses underscore the interconnectedness of cardiometabolic proteins and provide insights into their roles in disease pathogenesis. Our findings may help to refine diagnostic strategies and inform the development of personalized interventions, heralding a new era in precision medicine and healthcare innovation. Through the integration of multi-omics approaches and advanced analytics, our study offers a crucial framework for deciphering the intricate molecular underpinnings of metabolic disorders and paving the way for transformative therapeutic strategies.

Hypercitrullination and anti-citrullinated protein antibodies in chronic apical periodontitis, a laboratory investigation. Does autoimmunity contribute to the pathogenesis?

AIM: The presence of Gram-negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P. gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti-citrullinated autoantibodies (ACPA-s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. METHODOLOGY: Twenty-five periapical granulomas (n = 25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n = 6). The peptidyl-citrulline level was determined with the dot blot method. ACPA levels were analysed using anti-citrullinated cyclic peptide (anti-CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann-Whitney U tests. p Values <.05 were considered as statistically significant. RESULTS: Protein concentrations, peptidyl-citrulline levels and anti-CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p = .042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p = .01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. CONCLUSIONS: This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.

Salivary Chemical Barrier Proteins in Oral Squamous Cell Carcinoma-Alterations in the Defense Mechanism of the Oral Cavity.

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of head and neck cancer. Despite the genetic and environmental risk factors, OSCC is also associated with microbial infections and/or dysbiosis. The secreted saliva serves as the chemical barrier of the oral cavity and, since OSCC can alter the protein composition of saliva, our aim was to analyze the effect of OSCC on the salivary chemical barrier proteins. Publicly available datasets regarding the analysis of salivary proteins from patients with OSCC and controls were collected and examined in order to identify differentially expressed chemical barrier proteins. Network analysis and gene ontology (GO) classification of the differentially expressed chemical barrier proteins were performed as well. One hundred and twenty-seven proteins showing different expression pattern between the OSCC and control groups were found. Protein-protein interaction networks of up- and down-regulated proteins were constructed and analyzed. The main hub proteins (IL-6, IL-1B, IL-8, TNF, APOA1, APOA2, APOB, APOC3, APOE, and HP) were identified and the enriched GO terms were examined. Our study highlighted the importance of the chemical barrier of saliva in the development of OSCC.

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.

Cross-Linking Mass Spectrometry on P-Glycoprotein.

The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp's conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp's solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins.

Comparative Analysis of Differential Cellular Transcriptome and Proteome Regulation by HIV-1 and HIV-2 Pseudovirions in the Early Phase of Infection.

In spite of the similar structural and genomic organization of human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2), striking differences exist between them in terms of replication dynamics and clinical manifestation of infection. Although the pathomechanism of HIV-1 infection is well characterized, relatively few data are available regarding HIV-2 viral replication and its interaction with host-cell proteins during the early phase of infection. We utilized proteo-transcriptomic analyses to determine differential genome expression and proteomic changes induced by transduction with HIV-1/2 pseudovirions during 8, 12 and 26 h time-points in HEK-293T cells. We show that alteration in the cellular milieu was indeed different between the two pseudovirions. The significantly higher number of genes altered by HIV-2 in the first two time-points suggests a more diverse yet subtle effect on the host cell, preparing the infected cell for integration and latency. On the other hand, GO analysis showed that, while HIV-1 induced cellular oxidative stress and had a greater effect on cellular metabolism, HIV-2 mostly affected genes involved in cell adhesion, extracellular matrix organization or cellular differentiation. Proteomics analysis revealed that HIV-2 significantly downregulated the expression of proteins involved in mRNA processing and translation. Meanwhile, HIV-1 influenced the cellular level of translation initiation factors and chaperones. Our study provides insight into the understudied replication cycle of HIV-2 and enriches our knowledge about the use of HIV-based lentiviral vectors in general.

Tissue Transglutaminase Knock-Out Preadipocytes and Beige Cells of Epididymal Fat Origin Possess Decreased Mitochondrial Functions Required for Thermogenesis.

Beige adipocytes with thermogenic function are activated during cold exposure in white adipose tissue through the process of browning. These cells, similar to brown adipocytes, dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). Recently, we have shown that tissue transglutaminase (TG2) knock-out mice have decreased cold tolerance in parallel with lower utilization of their epididymal adipose tissue and reduced browning. To learn more about the thermogenic function of this fat depot, we isolated preadipocytes from the epididymal adipose tissue of wild-type and TG2 knock-out mice and differentiated them in the beige direction. Although differentiation of TG2 knock-out preadipocytes is phenotypically similar to the wild-type cells, the mitochondria of the knock-out beige cells have multiple impairments including an altered electron transport system generating lower electrochemical potential difference, reduced oxygen consumption, lower UCP1 protein content, and a higher portion of fragmented mitochondria. Most of these differences are present in preadipocytes as well, and the differentiation process cannot overcome the functional disadvantages completely. TG2 knock-out beige adipocytes produce more iodothyronine deiodinase 3 (DIO3) which may inactivate thyroid hormones required for the establishment of optimal mitochondrial function. The TG2 knock-out preadipocytes and beige cells are both hypometabolic as compared with the wild-type controls which may also be explained by the lower expression of solute carrier proteins SLC25A45, SLC25A47, and SLC25A42 which transport acylcarnitine, Co-A, and amino acids into the mitochondrial matrix. As a consequence, the mitochondria in TG2 knock-out beige adipocytes probably cannot reach the energy-producing threshold required for normal thermogenic functions, which may contribute to the decreased cold tolerance of TG2 knock-out mice.

Metabolomic Analysis of Serum and Tear Samples from Patients with Obesity and Type 2 Diabetes Mellitus.

Metabolomics strategies are widely used to examine obesity and type 2 diabetes (T2D). Patients with obesity (n = 31) or T2D (n = 26) and sex- and age-matched controls (n = 28) were recruited, and serum and tear samples were collected. The concentration of 23 amino acids and 10 biogenic amines in serum and tear samples was analyzed. Statistical analysis and Pearson correlation analysis along with network analysis were carried out. Compared to controls, changes in the level of 6 analytes in the obese group and of 10 analytes in the T2D group were statistically significant. For obesity, the energy generation, while for T2D, the involvement of NO synthesis and its relation to insulin signaling and inflammation, were characteristic. We found that BCAA and glutamine metabolism, urea cycle, and beta-oxidation make up crucial parts of the metabolic changes in T2D. According to our data, the retromer-mediated retrograde transport, the ethanolamine metabolism, and, consequently, the endocannabinoid signaling and phospholipid metabolism were characteristic of both conditions and can be relevant pathways to understanding and treating insulin resistance. By providing potential therapeutic targets and new starting points for mechanistic studies, our results emphasize the importance of complex data analysis procedures to better understand the pathomechanism of obesity and diabetes.

Tailoring to Search Engines: Bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence.

Bottom-up proteomics relies on identification of peptides from tandem mass spectra, usually via matching against sequence databases. Confidence in a peptide-spectrum match can be characterized by a score value given by the database search engines, and it depends on the information content and the quality of the spectrum. The latter are influenced by experimental parameters, of which the collision energy is the most important one in the case of collision-induced dissociation. We examined how the identification score of the Byonic and Andromeda (MaxQuant) engines varies with collision energy for more than a thousand individual peptides from a HeLa tryptic digest on a QTof instrument. We thereby extended our earlier study on Mascot scores and corroborated its findings on the potential bimodal nature of this energy dependence. Optimal energies as a function of m/z show comparable linear trends for the three engines. On the basis of peptide-level results, we designed methods with one or two liquid chromatography-tandem mass spectrometry (LC-MS/MS) runs and various collision energy settings and assessed their practical performance in peptide and protein identification from the HeLa standard sample. A 10-40% gain in various measures, such as the number of identified proteins or sequence coverage, was obtained over the factory default settings. Best performing methods differ for the three engines, suggesting that the experimental parameters should be fine-tuned to the choice of the engine. We also recommend a simple approach and provide reference data to ease the transfer of the optimized methods to other mass spectrometers relevant for proteomics. We demonstrate the utility of this approach on an Orbitrap instrument. Data sets can be accessed via the MassIVE repository (MSV000086379).

Detection of Antimicrobial Peptides in Stratum Corneum by Mass Spectrometry.

Antimicrobial and immunomodulatory peptides (AMPs) are considered as the key players in the maintenance of skin barrier functions. Here, we developed a novel approach for the examination of AMPs in the outermost layer of the epidermis, namely stratum corneum (SC). The SC sample collection by tape stripping was coupled with detection by highly specific and sensitive parallel reaction monitoring (PRM)-based mass spectrometry. We found that hexane-free processing of SC samples produced higher protein yield compared to hexane-based extraction. Of the 18 investigated peptides, 9 could be detected either in healthy or in inflamed skin specimens. Regarding the amount of S100A8, LCN2, LACRT and LYZ significant topographical differences were described among gland poor (GP), sebaceous gland rich (SGR) and apocrine gland rich (AGR) healthy skin regions. We applied a minimally invasive, reproducible approach for sampling, which can be assessed for research and diagnostic purposes and for monitoring the effectiveness of therapies in skin diseases.

Structural Characterization of Daunomycin-Peptide Conjugates by Various Tandem Mass Spectrometric Techniques.

The use of peptide-drug conjugates has generated wide interest as targeted antitumor therapeutics. The anthracycline antibiotic, daunomycin, is a widely used anticancer agent and it is often conjugated to different tumor homing peptides. However, comprehensive analytical characterization of these conjugates via tandem mass spectrometry (MS/MS) is challenging due to the lability of the O-glycosidic bond and the appearance of MS/MS fragment ions with little structural information. Therefore, we aimed to investigate the optimal fragmentation conditions that suppress the prevalent dissociation of the anthracycline drug and provide good sequence coverage. In this study, we comprehensively compared the performance of common fragmentation techniques, such as higher energy collisional dissociation (HCD), electron transfer dissociation (ETD), electron-transfer higher energy collisional dissociation (EThcD) and matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) activation methods for the structural identification of synthetic daunomycin-peptide conjugates by high-resolution tandem mass spectrometry. Our results showed that peptide backbone fragmentation was inhibited by applying electron-based dissociation methods to conjugates, most possibly due to the "electron predator" effect of the daunomycin. We found that efficient HCD fragmentation was largely influenced by several factors, such as amino acid sequences, charge states and HCD energy. High energy HCD and MALDI-TOF/TOF combined with collision induced dissociation (CID) mode are the methods of choice to unambiguously assign the sequence, localize different conjugation sites and differentiate conjugate isomers.

Hemoglobin oxidation generates globin-derived peptides in atherosclerotic lesions and intraventricular hemorrhage of the brain, provoking endothelial dysfunction.

The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC-MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1ß and nuclear translocation of the NF-κß transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.

Compounds with Antiviral, Anti-Inflammatory and Anticancer Activity Identified in Wine from Hungary's Tokaj Region via High Resolution Mass Spectrometry and Bioinformatics Analyses.

(1) Background: Wine contains a variety of molecules with potential beneficial effects on human health. Our aim was to examine the wine components with high-resolution mass spectrometry including high-resolution tandem mass spectrometry in two wine types made from grapes with or without the fungus Botrytis cinerea, or "noble rot". (2) For LC-MS/MS analysis, 12 wine samples (7 without and 5 with noble rotting) from 4 different wineries were used and wine components were identified and quantified. (3) Results: 288 molecules were identified in the wines and the amount of 169 molecules was statistically significantly different between the two wine types. A database search was carried out to find the molecules, which were examined in functional studies so far, with high emphasis on molecules with antiviral, anti-inflammatory and anticancer activities. (4) Conclusions: A comprehensive functional dataset related to identified wine components is also provided highlighting the importance of components with potential health benefits.

Heme-Induced Oxidation of Cysteine Groups of Myofilament Proteins Leads to Contractile Dysfunction of Permeabilized Human Skeletal Muscle Fibres.

Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20-300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.

Analysis of networks of host proteins in the early time points following HIV transduction.

BACKGROUND: Utilization of quantitative proteomics data on the network level is still a challenge in proteomics data analysis. Currently existing models use sophisticated, sometimes hard to implement analysis techniques. Our aim was to generate a relatively simple strategy for quantitative proteomics data analysis in order to utilize as much of the data generated in a proteomics experiment as possible. RESULTS: In this study, we applied label-free proteomics, and generated a network model utilizing both qualitative, and quantitative data, in order to examine the early host response to Human Immunodeficiency Virus type 1 (HIV-1). A weighted network model was generated based on the amount of proteins measured by mass spectrometry, and analysis of weighted networks and functional sub-networks revealed upregulation of proteins involved in translation, transcription, and DNA condensation in the early phase of the viral life-cycle. CONCLUSION: A relatively simple strategy for network analysis was created and applied to examine the effect of HIV-1 on host cellular proteome. We believe that our model may prove beneficial in creating algorithms, allowing for both quantitative and qualitative studies of proteome change in various biological and pathological processes by quantitative mass spectrometry.

The Expressional Pattern of Invasion-Related Extracellular Matrix Molecules in CNS Tumors.

Aim of the study: Astrocytomas are primary CNS malignancies which infiltrate the peritumoral tissue, even when they are low-grade. Schwannomas are also primary CNS tumors, however, they do not show peritumoral infiltration similarly to brain metastases which almost never invade the neighboring parts of brain. Extracellular matrix is altered in composition in various cancer types and is proposed to play an important role in the development of invasiveness of astrocytic tumors. This study aims to identify differences in the ECM composition of CNS tumors with different invasiveness.Materials and methods: The mRNA and protein levels of ECM components were measured by QRT-PCR and mass-spectrometry, respectively, in grade II astrocytoma, NSCLC brain metastasis, schwannomas, and non-tumor brain control samples. Expressional data was analyzed statistically with ANOVA and nearest neighbor search.Results: There is a significant difference in the expressional pattern of invasion-related ECM components among various CNS tumors, especially among those of different embryonic origin. Non-invasive tumors show only slight differences in the expressional pattern of ECM molecules. Tumor samples can be separated based on their expressional pattern using statistical classifiers, therefore the ECM composition seems to be typical of various cancer types.Conclusions: Differences in the expressional pattern of the ECM could be responsible for the different invasiveness of various CNS tumors.

Wound-Healing Markers Revealed by Proximity Extension Assay in Tears of Patients following Glaucoma Surgery.

Tears are a constantly available and highly valuable body fluid collectable by non-invasive techniques. Although it can give information on ocular status and be used for follow-ups, tear analysis is challenging due to the low amount of sample that is available. Proximity extension assay (PEA) allows for a sensitive and scalable analysis of multiple proteins in a single run from a one-µL sample, so we applied this technique and examined the amount of 184 proteins in tears collected at different time points after trabeculectomy. The success rate of this surgical intervention highly depends on proper wound healing; therefore, information on the process is indispensable. We observed significantly higher levels of IL-6 and MMP1 at the early time points (day one, two, and four) following trabeculectomy, and the protein amounts went back to the level observed before the surgery three months after the intervention. Patients with or without complications were tested, and proteins that have roles in the immune response and wound healing could be observed with altered frequency and amounts in the cases of patients with complications. Our results highlight the importance of inflammation in wound-healing complications, and at the same time, indicate the utility of PEA in tear analysis.

Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device.

Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15-70 kDa) were digested efficiently with ∼50 s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2 h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.

Relative quantification of human ß-defensins by a proteomics approach based on selected reaction monitoring.

RATIONALE: A targeted proteomics method based on selected reaction monitoring (SRM) is a relevant approach for the analysis of multiple analytes in biological samples. Defensins are phylogenetically conserved small antimicrobial peptides contributing to innate host defense and exhibiting low immunogenicity, resistance to proteolysis and a broad range of antimicrobial activities. The goal of the present study was to develop and optimize SRM-based targeted proteomics methods for the detection of human ß-defensins 1-4 in various biological fluids. METHODS: An SRM-based targeted proteomics method was developed and validated for the detection of human ß-defensins 1-4. The supernatants of resting and IL-1ß-stimulated Caco2, HT-29 and SW-1116 colonic epithelial cells (CEC), cell lysates of CECs and tear samples of human healthy individuals were analyzed and the feasibility of the developed method was validated by ELISA and dot-blot analysis complemented by RT-qPCR. RESULTS: Our results demonstrate that the developed SRM method offers an alternative approach for the cost-effective and rapid analysis of human ß-defensins in samples with biological relevance. CONCLUSIONS: A semi-quantitative targeted mass spectrometry method was developed and validated for the relative quantification of ß-defensins 1-4 in cell culture supernatants and body fluid analyses.

A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects.

The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1-2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.