Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.

• Klíčová slova
• Cryo-CLEM, FIB/SEM tomography, cryo-ET, cryo-FIB, cryo-FIB/SEM, cryo-LM, cryo-electron, cryo-focused ion beam, cryo-lift-out, electron-tomography, focused ion beam, lift-out, tomography workflow,
• MeSH
• automatizace MeSH
• elektronová kryomikroskopie * MeSH
• mikroskopie elektronová rastrovací MeSH
• průběh práce MeSH
• tomografie elektronová * MeSH
• transmisní elektronová mikroskopie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

• Klíčová slova
• M-protein, data independent acquisition, mass spectrometry, multiple myeloma, quantitative biomarker,
• MeSH
• automatizace MeSH
• individualizovaná medicína metody   MeSH
• lidé MeSH
• mnohočetný myelom * diagnóza   krev   MeSH
• průběh práce * MeSH
• reziduální nádor * diagnóza   MeSH
• rychlé screeningové testy metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Poor lifestyle leads potentially to chronic diseases and low-grade physical and mental fitness. However, ahead of time, we can measure and analyze multiple aspects of physical and mental health, such as body parameters, health risk factors, degrees of motivation, and the overall willingness to change the current lifestyle. In conjunction with data representing human brain activity, we can obtain and identify human health problems resulting from a long-term lifestyle more precisely and, where appropriate, improve the quality and length of human life. Currently, brain and physical health-related data are not commonly collected and evaluated together. However, doing that is supposed to be an interesting and viable concept, especially when followed by a more detailed definition and description of their whole processing lifecycle. Moreover, when best practices are used to store, annotate, analyze, and evaluate such data collections, the necessary infrastructure development and more intense cooperation among scientific teams and laboratories are facilitated. This approach also improves the reproducibility of experimental work. As a result, large collections of physical and brain health-related data could provide a robust basis for better interpretation of a person's overall health. This work aims to overview and reflect some best practices used within global communities to ensure the reproducibility of experiments, collected datasets and related workflows. These best practices concern, e.g., data lifecycle models, FAIR principles, and definitions and implementations of terminologies and ontologies. Then, an example of how an automated workflow system could be created to support the collection, annotation, storage, analysis, and publication of findings is shown. The Body in Numbers pilot system, also utilizing software engineering best practices, was developed to implement the concept of such an automated workflow system. It is unique just due to the combination of the processing and evaluation of physical and brain (electrophysiological) data. Its implementation is explored in greater detail, and opportunities to use the gained findings and results throughout various application domains are discussed.

• Klíčová slova
• best practices, brain data, data lifecycle, health information system, health-related data, ontology, physical data, workflow,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

OBJECTIVES: The analysis of organic acids in urine is an important part of the diagnosis of inherited metabolic disorders (IMDs), for which gas chromatography coupled with mass spectrometry is still predominantly used. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for urinary organic acids, acylcarnitines and acylglycines was developed and validated. Sample preparation consists only of dilution and the addition of internal standards. Raw data processing is quick and easy using selective scheduled multiple reaction monitoring mode. A robust standardised value calculation as a data transformation together with advanced automatic visualisation tools are applied for easy evaluation of complex data. RESULTS: The developed method covers 146 biomarkers consisting of organic acids (n=99), acylglycines (n=15) and acylcarnitines (n=32) including all clinically important isomeric compounds present. Linearity with r2>0.98 for 118 analytes, inter-day accuracy between 80 and 120 % and imprecision under 15 % for 120 analytes were achieved. Over 2 years, more than 800 urine samples from children tested for IMDs were analysed. The workflow was evaluated on 93 patient samples and ERNDIM External Quality Assurance samples involving a total of 34 different IMDs. CONCLUSIONS: The established LC-MS/MS workflow offers a comprehensive analysis of a wide range of organic acids, acylcarnitines and acylglycines in urine to perform effective, rapid and sensitive semi-automated diagnosis of more than 80 IMDs.

• Klíčová slova
• inherited metabolic disorders, liquid chromatography, mass spectrometry, organic acidurias,
• MeSH
• chromatografie kapalinová metody   MeSH
• dítě MeSH
• lidé MeSH
• metabolické nemoci * MeSH
• organické látky MeSH
• průběh práce MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acylcarnitine MeSH Prohlížeč
• organické látky MeSH

We report an MS-based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysates that is suitable for high-throughput sample analysis. The workflow is based on an in situ enrichment on matrix-assisted laser desorption/ionization (MALDI) plates that were functionalized by TiO2 using automated ion landing apparatus that can operate unsupervised. The MALDI plate can be functionalized by TiO2 into any array of predefined geometry (here, 96 positions for samples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high-performance liquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn-based separation and achieved comparable or better results than the standard method. The performance of this new workflow was demonstrated on a model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal-to-noise ratio in a single MS spectrum, which resulted in better identification by MS/MS and a subsequent database search. Using the workflow, we also found specific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12-myristate 13-acetate. These phosphorylations concerned the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and its targets and were in agreement with the current knowledge of this signaling cascade. Control sample of non-activated cells was devoid of these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events in minimally processed mammalian cells while using only a short high-performance liquid chromatography gradient.

• Klíčová slova
• MALDI, automation, cell lysates, enrichment, phosphopeptides,
• MeSH
• chromatografie kapalinová metody   MeSH
• design vybavení MeSH
• fosfopeptidy analýza   chemie   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• rychlé screeningové testy metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice přístrojové vybavení   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfopeptidy MeSH

The ability of plants to sense and orient their root growth towards gravity is studied in many laboratories. It is known that manual analysis of image data is subjected to human bias. Several semi-automated tools are available for analysing images from flatbed scanners, but there is no solution to automatically measure root bending angle over time for vertical-stage microscopy images. To address these problems, we developed ACORBA, which is an automated software that can measure root bending angle over time from vertical-stage microscope and flatbed scanner images. ACORBA also has a semi-automated mode for camera or stereomicroscope images. It represents a flexible approach based on both traditional image processing and deep machine learning segmentation to measure root angle progression over time. As the software is automated, it limits human interactions and is reproducible. ACORBA will support the plant biologist community by reducing labour and increasing reproducibility of image analysis of root gravitropism.

• Klíčová slova
• UNET, deep machine learning, image segmentation, python, root gravitropism,
• Publikační typ
• časopisecké články MeSH

Capillary electrophoresis-frontal analysis (CE-FA) together with mobility shift affinity CE is the most frequently used mode of affinity CE for a study of plasma protein-drug interactions, which is a substantial part of the early stage of drug discovery. Whereas in the classic CE-FA setup the sample is prepared by off-line mixing of the interaction partners in the sample vial outside the CE instrument and after a short incubation period loaded into the capillary and analysed, in this work a new methodological approach has been developed that combines CE-FA with the mixing of interacting partners directly inside the capillary. This combination gives rise to a fully automated and versatile methodology for the characterization of these binding interactions besides a substantial reduction in the amounts of sample compounds used. The minimization of possible experimental errors due to the full involving of sophisticated CE instrument in the injection procedure, mixing and separation instead of manual manipulation is another fundamental benefit. The in-capillary mixing is based on the transverse diffusion of laminar flow profile methodology introduced by Krylov et al. using its multi-zone injection modification presented by Řemínek at al.. Actually, after the method optimization, the alternate introduction of six plugs of drug and six plugs of bovine serum protein in BGE, each injected for 3 s at a pressure of -10 mbar (-1 kPa) into the capillary filled by BGE, was found to be the best injection procedure. The method repeatability calculated as RSDs of plateau highs of bovine serum albumin and propranolol as model sample compounds were better than 3.44 %. Its applicability was finally demonstrated on the determination of apparent binding parameters of bovine serum albumin for basic drugs propranolol and lidocaine and acid drug phenylbutazone. The values obtained by a new on-line CE-FA methodology are in agreement with values estimated by classic off-line CE-FA, as well as with literature data obtained using different techniques.

• Klíčová slova
• Binding constant, Bovine serum albumin, Capillary electrophoresis-frontal analysis, On-line method, Transverse diffusion of laminar flow profile,
• MeSH
• difuze MeSH
• elektroforéza kapilární * MeSH
• farmaceutická chemie metody   MeSH
• krevní proteiny chemie   metabolismus   MeSH
• léčivé přípravky metabolismus   MeSH
• propranolol chemie   metabolismus   MeSH
• průběh práce MeSH
• sérový albumin hovězí chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• krevní proteiny MeSH
• léčivé přípravky MeSH
• propranolol MeSH
• sérový albumin hovězí MeSH

BACKGROUND: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. OBJECTIVE: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. METHODS: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. RESULTS: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. CONCLUSION: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

• Klíčová slova
• CD marker, cluster of differentiation (CD), expression profiling, flow cytometry, surfaceome,
• MeSH
• antigeny povrchové * metabolismus   MeSH
• buňky NK metabolismus   MeSH
• CD antigeny metabolismus   MeSH
• leukocyty MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• přirozená imunita * MeSH
• průběh práce MeSH
• průtoková cytometrie metody   MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové * MeSH
• CD antigeny MeSH
• monoklonální protilátky MeSH

The inherent diversity of approaches in proteomics research has led to a wide range of software solutions for data analysis. These software solutions encompass multiple tools, each employing different algorithms for various tasks such as peptide-spectrum matching, protein inference, quantification, statistical analysis, and visualization. To enable an unbiased comparison of commonly used bottom-up label-free proteomics workflows, we introduce WOMBAT-P, a versatile platform designed for automated benchmarking and comparison. WOMBAT-P simplifies the processing of public data by utilizing the sample and data relationship format for proteomics (SDRF-Proteomics) as input. This feature streamlines the analysis of annotated local or public ProteomeXchange data sets, promoting efficient comparisons among diverse outputs. Through an evaluation using experimental ground truth data and a realistic biological data set, we uncover significant disparities and a limited overlap in the quantified proteins. WOMBAT-P not only enables rapid execution and seamless comparison of workflows but also provides valuable insights into the capabilities of different software solutions. These benchmarking metrics are a valuable resource for researchers in selecting the most suitable workflow for their specific data sets. The modular architecture of WOMBAT-P promotes extensibility and customization. The software is available at https://github.com/wombat-p/WOMBAT-Pipelines.

• Klíčová slova
• benchmarking, data analysis, label-free proteomics, quality metrics, workflow,
• MeSH
• analýza dat MeSH
• proteiny MeSH
• proteomika * MeSH
• průběh práce MeSH
• software MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH

SUMMARY: We present the LipidQuant 1.0 tool for automated data processing workflows in lipidomic quantitation based on lipid class separation coupled with high-resolution mass spectrometry. Lipid class separation workflows, such as hydrophilic interaction liquid chromatography or supercritical fluid chromatography, should be preferred in lipidomic quantitation due to the coionization of lipid class internal standards with analytes from the same class. The individual steps in the LipidQuant workflow are explained, including lipid identification, quantitation, isotopic correction and reporting results. We show the application of LipidQuant data processing to a small cohort of human serum samples. AVAILABILITY AND IMPLEMENTATION: The LipidQuant 1.0 is freely available at Zenodo https://doi.org/10.5281/zenodo.5151201 and https://holcapek.upce.cz/lipidquant.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

• MeSH
• chromatografie kapalinová MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• lipidomika * MeSH
• lipidy * analýza   MeSH
• průběh práce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipidy * MeSH