Corynebacterium (C.) durum je součástí rezidentní flóry dutiny ústní. Jeho podíl na etiologii infekčních onemocnění je nejednoznačný. S vyšším počtem imunoalterovaných pacientů je nutné s ním počítat jako s potenciálním oportunním patogenem. Nejčastěji je izolováno ze sputa, bronchoalveolární lavážní tekutiny, ale také z krve, zejména u imunosuprimovaných pacientů s pneumonií. V tom případě je nutné bakterii přesně identifikovat a nález správně interpretovat. Dříve velmi využívaný komerční test pro určení korynebakterií (API Coryne, BioMerieux) nelze použít pro všechna korynebakteria včetně C. durum. Tento druh není obsažen v databázi biotypových čísel. Lze provést porovnání biotypového čísla s údaji v literatuře. K přesnému odlišení od jiných korynebakterií je nutná chemotaxonomická a proteomická analýzy (MALDI-TOF MS), nebo sekvenace genu 16S rRNA. Klíčový je polyfázový přístup využívající poznatky z jednotlivých laboratorních vyšetření.

Corynebaterium (C.) durum is a part of the resident human oral microbiota. Its role in the aetiology of infectious diseases is ambiguous. With the increasing number of immunocompromised patients, it must be considered a potential opportunistic pathogen. It is isolated from the sputum, bronchoalveolar-lavage fluid, as well as blood, especially from immunocompromised patients with pneumonia. In that case, the critical steps involve a correct identification of Corynebacterium to the species level and right interpretation of the findings. The previously widely used commercial test for the identification of Corynebacteria (API Coryne, BioMerieux) is not suitable for all species, including C. durum, as its biotype number is not included in the database. But the obtained result can be compared with the available literature data. Chemotaxonomic and proteomic analysis (matrix-assisted laser desorption/ ionization – time of flight, MALDI-TOF MS) or 16S rRNA sequencing allow for accurate differentiation from the other Corynebacteria species. Nevertheless, these methods are not routinely used in clinical laboratories. A polyphasic approach to the taxonomy based on the data from combined laboratory tests is crucial.

• MeSH
• Bronchoalveolar Lavage Fluid microbiology   MeSH
• Corynebacterium * isolation & purification   MeSH
• Immunocompromised Host MeSH
• Corynebacterium Infections diagnosis   MeSH
• Blood microbiology   MeSH
• Humans MeSH
• Microbiological Techniques methods   MeSH
• Opportunistic Infections microbiology   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Sputum microbiology   MeSH
• Check Tag
• Humans MeSH

Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel mass spectrometry (MS)-cleavable reagents. At present, only water-insoluble MS-cleavable cross-linkers are commercially available. However, to comprehensively analyse the various chemical and structural motifs making up proteins, it is necessary to target different protein sites with varying degrees of hydrophilicity. Here we introduce the new MS-cleavable cross-linker disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house for this purpose. DSSBU contains an N-hydroxysulfosuccinimide (sulfo-NHS) reactive group, so it can serve as a water-soluble counterpart to the widely used cross-linker disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers: bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU and DSSBU with bovine serum albumin. In addition, we compared the efficacy of DSBU and DSSBU with human haemoglobin. Our results demonstrate that the sulfo-NHS group ensures the superior water solubility of DSSBU and thus negates the need for organic solvents such as dimethyl sulfoxide while preserving the effectivity of urea-based MS-cleavable crosslinkers such as DSBU. Additionally, it makes it possible to target polar regions in proteins. The data gathered are available via ProteomeXchange under identifier PXD055284. SIGNIFICANCE: We have synthesized the novel protein cross-linker DSSBU, which combines sulfo-NHS ester chemistry with a mass spectrometry-cleavable urea group. This makes DSSBU a water-soluble, MS-cleavable cross-linker that reacts with amino groups. To our knowledge, it is the first cross-linker which combines all three of these characteristics. We have tested the performance of our novel cross-linker on bovine serum albumin, a model widely used by the cross-linking mass spectrometry community, and on human haemoglobin. We have comprehensively assessed the performance of DSSBU and compared its efficacy with that of three other cross-linkers in current use (BS3, DSS and DSBU). We conclude that our novel cross-linker surpasses its MS-non-cleavable analogue BS3 in performance and that its water solubility eliminates the need for organic solvents while its hydrophilicity allows for the targetting of polar regions in proteins. Therefore, it will likely become a significant addition to the portfolio of N-hydroxysuccinimide ester cross-linkers.

• MeSH
• Mass Spectrometry methods   MeSH
• Humans MeSH
• Urea chemistry   MeSH
• Proteomics methods   MeSH
• Cross-Linking Reagents * chemistry   MeSH
• Serum Albumin, Bovine chemistry   MeSH
• Cattle MeSH
• Succinimides * chemistry   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Cylindrospermopsin, a potent hepatotoxin produced by harmful cyanobacterial blooms, poses environmental and human health concerns. We used a 3D human liver in vitro model based on spheroids of HepG2 cells, in combination with molecular and biochemical assays, automated imaging, targeted LC-MS-based proteomics, and lipidomics, to explore cylindrospermopsin effects on lipid metabolism and the processes implicated in hepatic steatosis. Cylindrospermopsin (1 μM, 48 h) did not significantly affect cell viability but partially reduced albumin secretion. However, it increased neutral lipid accumulation in HepG2 spheroids while decreasing phospholipid levels. Simultaneously, cylindrospermopsin upregulated genes for lipogenesis regulation (SREBF1) and triacylglycerol synthesis (DGAT1/2) and downregulated genes for fatty acid synthesis (ACLY, ACCA, FASN, SCD1). Fatty acid uptake, oxidation, and lipid efflux genes were not significantly affected. Targeted proteomics revealed increased levels of perilipin 2 (adipophilin), a major hepatocyte lipid droplet-associated protein. Lipid profiling quantified 246 lipid species in the spheroids, with 28 significantly enriched and 15 downregulated by cylindrospermopsin. Upregulated species included neutral lipids, sphingolipids (e.g., ceramides and dihexosylceramides), and some glycerophospholipids (phosphatidylethanolamines, phosphatidylserines), while phosphatidylcholines and phosphatidylinositols were mostly reduced. It suggests that cylindrospermopsin exposures might contribute to developing and progressing towards hepatic steatosis or metabolic dysfunction-associated steatotic liver disease (MASLD).

• MeSH
• Alkaloids * pharmacology   MeSH
• Bacterial Toxins * metabolism   MeSH
• Spheroids, Cellular drug effects   metabolism   MeSH
• Hep G2 Cells MeSH
• Homeostasis drug effects   MeSH
• Liver * metabolism   drug effects   MeSH
• Humans MeSH
• Lipidomics MeSH
• Lipogenesis drug effects   MeSH
• Lipid Metabolism * drug effects   MeSH
• Proteomics MeSH
• Cyanobacteria Toxins * MeSH
• Uracil * analogs & derivatives   metabolism   MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Feochormocytomy a pargangliomy (PHEO/PGL) jsou vzácné neuro-endokrinní nádory původem z chromafinních buněk dřeně nadledvin (PHEO) nebo extra-drenálních sympatických a parasympatických ganglií (PGL). Cílem projektu je budoucí zpřesnění diagnózy a zefektivnění terapie PHEO/PGL cestou podrobné charakterizace molekulárních procesů, drah a molekul spojených se vznikem a progresí PHEO/PGL. Detailní proteomická analýza (LC-MS/MS) pacientských vzorků PHEO/PGL kombinovaná s profilováním kompletního transkriptomu metodou sekvenace RNA (RNA-Seq) poskytne informaci o specifických procesech zodpovědných nebo asociovaných s tímto onemocněním. Tyto specifické molekuly a procesy jsou potenciálními zásahovými místy „druggable targets“ budoucích terapií a rovněž možnými diagnostickými či prognostickými markery.; Pheochromocytoma and paraganglioma (PHEO/PGL) are rare neuroendocrine tumors arising from chromaffin cells of adrenal medulla and from extra-adrenal sympathetic and parasympathetic ganglia, respectively. The aim of the project is to characterize the molecular pathways (both, causal and associated) involved in PHEO/PGL development and progression in order to provide the basis for future sensitive diagnosis and knowledge-based targeted therapy of this rare disease. Top-notch proteomic analyses of patient tumor samples combined with transcriptome profiling using RNA sequencing technology will provide detailed and comprehensive quantitative proteome and transcriptome description of individual PHEO/PGL subtypes and will identify the underlying molecular processes and molecules exploitable as drug targets. Additionally, the proposed proteo-genomic “tour de force” will identify molecules that provide specific information about disease presence, type or progression – diagnostic and/or prognostic biomarkers of PHEO/PGL.

• Keywords
• feochromocytom, paragangliom, pheochromocytoma, paraganglioma, proteom, transkriptom, transcriptomics, proteomics, Hmotnostní spektrometrie, Mass Spectrometry,

• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease with a complex etiology that lacks biomarkers predicting disease progression. The objective of this study was to use longitudinal cerebrospinal fluid (CSF) samples to identify biomarkers that distinguish fast progression (FP) from slow progression (SP) and assess their temporal response. METHODS: We utilized mass spectrometry (MS)-based proteomics to identify candidate biomarkers using longitudinal CSF from a discovery cohort of SP and FP ALS patients. Immunoassays were used to quantify and validate levels of the top biomarkers. A state-transition mathematical model was created using the longitudinal MS data that also predicted FP versus SP. RESULTS: We identified a total of 1148 proteins in the CSF of all ALS patients. Pathway analysis determined enrichment of pathways related to complement and coagulation cascades in FPs and synaptogenesis and glucose metabolism in SPs. Longitudinal analysis revealed a panel of 59 candidate markers that could segregate FP and SP ALS. Based on multivariate analysis, we identified three biomarkers (F12, RBP4, and SERPINA4) as top candidates that segregate ALS based on rate of disease progression. These proteins were validated in the discovery and a separate validation cohort. Our state-transition model determined that the overall variance of the proteome over time was predictive of the disease progression rate. INTERPRETATION: We identified pathways and protein biomarkers that distinguish rate of ALS disease progression. A mathematical model of the CSF proteome determined that the change in entropy of the proteome over time was predictive of FP versus SP.

• MeSH
• Amyotrophic Lateral Sclerosis * MeSH
• Biomarkers cerebrospinal fluid   MeSH
• Humans MeSH
• Retinol-Binding Proteins, Plasma MeSH
• Disease Progression MeSH
• Proteome metabolism   MeSH
• Proteomics methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

• Publication type
• Journal Article MeSH

Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

• MeSH
• Apoptosis * MeSH
• Chondrocytes * metabolism   MeSH
• Caspase 9 metabolism   pharmacology   MeSH
• Caspases metabolism   pharmacology   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).

• MeSH
• Chromatography, Liquid methods   MeSH
• Cysteine metabolism   MeSH
• Isotope Labeling methods   MeSH
• Oxidation-Reduction MeSH
• Proteome * metabolism   MeSH
• Proteomics * methods   MeSH
• Tandem Mass Spectrometry methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture-based LC-MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2-PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein-protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells.

• MeSH
• Chromatography, Liquid MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Mucoproteins * metabolism   MeSH
• Neoplasms * MeSH
• Oncogene Proteins * metabolism   MeSH
• Protein Disulfide-Isomerases * MeSH
• Molecular Docking Simulation MeSH
• Tandem Mass Spectrometry MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.

• MeSH
• Cell Differentiation MeSH
• Mass Spectrometry methods   MeSH
• Humans MeSH
• Neural Stem Cells * chemistry   metabolism   MeSH
• Proteome analysis   MeSH
• Proteomics * methods   MeSH
• Quality Control MeSH
• Software * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH