Research
Focus
Structural mass spectrometry
method development
- novel covalent labeling and cross-linking chemistry of proteins /nucleic acids
- new acid proteases as a tool for protein digestion
- automation of HDX-MS and CX-MS workflow including software design
Functionalized surfaces for mass spectrometry
- ambient ion landing protein immobilization
- surface Immuno-affinity substrates for clinical diagnostics
- biochemically active plates for desorption mass spectrometry
Current projects
- HIPPOSTRUCT (MSCA-IF): Structural insights into binding signatures of transcription factors regulated by HIPPO signalling
- EPIC-XS: European Proteomics Infrastructure Consortium providing access
- EU FT-ICR MS: European Network of Fourier-Transform Ion-Cyclotron-Resonance Mass Spectrometry Centers
- Mapping the protein surface accessible area utilizing Top Down mass spectrometry and reactive radical footprinting.
- ReCognitiON - Recognition and Validation of Druggable Targets from the Response to Cognitive Behaviour Therapy in Myotonic Dystrophy type 1 patients from Integrated -Omics Networks.
- Functional Assays for Rapid Microbiological Diagnostics of Selected Health-Care Associated Infections .
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Structural insights into binding signatures of transcription factors regulated by HIPPO signalling
Transcription factors (TFs) are proteins which recognize specific DNA-sequence to orchestrate various gene expression programs according to cellular requirements. Currently, approximately 1,600 TFs along with their binding motifs have been annotated. Further, recent studies enabled classification of many of them according to their binding modes across nucleosome assembly. Despite such progress, interplay of different TFs in the context of adjacent DNA sequence or spatial organisation of chromatin is still largely unknown. This project aims to dissect crosstalk between different TFs that are targeted by HIPPO signalling pathway, a tumour suppressor pathway representing potential target for anti-tumour therapies. Preliminary data from host lab combined with previous studies from other groups lead us to postulate hypothesis that TFs from TEAD and FOX groups cooperate on regulation of transcription programs controlled by HIPPO pathway. Specifically, FOX TFs act as chromatin re-modellers enabling TEADs binding to the naked part of DNA which then triggers desired gene expression programs. To test this hypothesis, we will combine bioinformatic searches across human genome with experimental work assessing intermolecular binding between TFs and DNA, followed by structural characterization of selected macromolecular complexes. We will identify genomic loci which contains binding motifs for both TEAD and FOX TFs. These DNA fragments will be tested experimentally in terms of their ability to physically interact with both TFs, which will provide mechanistic insight into their cooperation. Finally, 3-D structure of the reconstituted complexes containing TF pairs bound to DNA will be determined using advanced mass spectrometry combined with high-resolution structural techniques such as X-ray crystallography and cryo-electron microscopy. This project will provide insight how TFs cooperate in the relevant genomic context with physiological chromatin architecture.
Funded by EU MSCA-IF (widening), project number 101003406 (HIPPOSTRUCT)
European Network of Fourier-Transform Ion-Cyclotron-Resonance Mass Spectrometry Centers
The European Proteomics Infrastructure Consortium providing access (EPIC-XS) consists of a unified network of experienced access providers and research groups who share a common goal; to facilitate the development and sustainability of proteomics exploration to all life science researchers within the European Union. The initiative is coordinated by Albert Heck, professor of Biomolecular Mass Spectrometry and Proteomics at Utrecht University. EPIC-XS will provide scientists transnational access to high end proteomics technologies and resources. The project will strengthen and expand the European proteomics community through its expertise in proteomics. It’s excellent training courses and programs, available to both novices and experts, will attract new research communities who will be schooled in advanced proteomics technologies. The initiative brings together some of the most world renowned proteomics laboratories and researchers. The community has expertise in accommodating and guiding researchers within the life sciences, as many of the members also participated in the pan- European PRIME-XS initiative, which ended in 2015.
Funded by EU H2020 (EPIC-XS), project number 82383
European Network of Fourier-Transform Ion-Cyclotron-Resonance Mass Spectrometry Centers
EU_FT-ICR_MS proposal aims to establish a European network of FT-ICR (Fourier Transform Ion Cyclotron Resonance) mass spectrometry (MS) centers in association with a manufacturer and a SME software company. Mass spectrometry (MS) has become the most ubiquitous analytical techniques in use today, providing more information on the composition and the structure of a substance from a smaller amount of sample than any other techniques. Unlike other analytical techniques, such as NMR, which mainly rely on a unique technology, MS is characterized by the existence of a large range of mass analyzers. FT-ICR MS is the most powerful MS technique. It offers up to 100 fold higher mass resolving power and mass accuracy than any other MS technique. On the contrary to NMR community with which the FT-ICR MS shares several features, FT-ICR MS has never been involved in a European INFRA network and so will be a legitimate candidate to the Integrating Activities for Starting Communities call. The EU_FT-ICR_MS network includes 10 FT-ICR MS centers from 8 different European countries (Belgium, Czech Republic, Finland, France, Germany, Italy, Portugal, and United Kingdom) and 1 third country (Russia), a European FT-ICR MS manufacturer and 2 SMEs. It includes centers equipped with up-to-date FT-ICR MS and expertise which will cover most of the field in which FT-ICR mass spectrometry is involved: BioOrganic & BioInorganic, Cultural heritage, Glycomics, Environment, Imaging, InfraRed Spectroscopy of Ions in the Gas Phase, Lipidomics, Medicine, Petroleum & Coal Oil, Nanoparticles, Organic chemistry, Physical chemistry, Proteomics, Structural biology. The EU_FT-ICR_MS proposal contains six work-packages which cover all the aspects of the INFRAIA-02-2017 (RIA) Integrating Activities for Starting Communities (WP1 Transnational access; WP2 Training and Education; WP3 Open Data and e-Infrastructure; WP 4 Joint Research Action; WP 5 Dissemination; WP6 Consortium management).
Funded by EU H2020 (EU_FT-ICR_MS), project number 731077
Mapping the protein surface accessible area utilizing Top Down mass spectrometry and reactive radical footprinting.
Structural proteomics has become an emerging technique of current structural biology. It covers very broad range of analytical methods from hydrogen/deuterium exchange to chemical crosslinking. Although, the limitations and potentials of both techniques are well studied, foundations of covalent labelling are still poorly described. While H/D exchange offers information about backbone accessibility, covalent labelling targets amino acid side chains and thus provides complementary structural information. In this proposal we will develop chemical reagents with novel reactivity that will allow targeting of more amino acid side chains and expand the spatial resolution of the method. We will also pursue hydroxyradical footprinting where we aim mainly at the development of top-down mass spectrometric approaches allowing to assign not only the site of the modification but also to assess the reactivity scale of individual modified residues. Such advancements will greatly facilitate protein structure characterization and will provide valuable information for protein structure modelling.
Funded by Czech Science Foundation (19-16084S)
Recognition and Validation of Druggable Targets from the Response to Cognitive Behaviour Therapy in Myotonic Dystrophy type 1 patients from Integrated -Omics Networks.
Myotonic dystrophy type 1 (DM1), the most common adult form of muscular dystrophy, affects virtually all tissues; the noncurable condition carries significant morbidity and mortality impacting patient and family quality of life and socio-economic status. The OPTIMISTIC clinical trial has shown that Cognitive Behaviour Therapy (CBT), a patient-tailored intervention to increase activity and enable patients to deal with their disease, imparts significant beneficial impact on activity and participation. We now propose a multi-omic approach to identify the molecular signatures of the response to this clinical intervention, taking advantage of the thorough clinical characterization of the enrolled patients and the comprehensive set of serum samples at baseline and two follow-up time points. Our central hypothesis is that pathways associated with the positive response to CBT can be consolidated or reinforced by conventional drug therapies targeting the same pathways. A network-based bioinformatics approach shall be used to identify drug targets in the molecular signatures. We shall repurpose clinically approved drugs measuring impact on molecular profiles of patients cells and the behaviour of DM1 mouse models. Repurposed drugs with effects similar to CBT can be evaluated in isolation or combination with other treatments in future clinical trials for DM1 and other neurological conditions. The drug repurposing strategy based on the reverse engineering of a positive response to a behavioural intervention may set the scene for future drug development trajectories for rare diseases.
Funded by EU - E-Rare (ReCognitiON)
Functional Assays for Rapid Microbiological Diagnostics of Selected Health-Care Associated Infections.
Health-care associated infections (HCAIs) significantly influence current medicine and may limit the progress in many medical procedures. Treatment of such infections has been increasingly complicated by the spread of antimicrobial resistance (AMR). To reduce risks of inappropriate initial therapy, the availability of rapid diagnostic microbiological assays is of utmost importance. Especially, functional diagnostics (i.e., detection of enzyme activity) is important in era of horizontal gene transfer. Functional assays for detection of selected bacterial toxins (ToxB of C. difficile and YopT of Y. enterocolitica) using affinity MALDI will be developed and validated. Simultaneously, functional assay for detection of carbapenemase activity developed in the laboratory of co-investigator will be further optimized and validated. Further, a new assay for functional detection of lipid A modification detecting colistin resistance will be also developed. All of those assays and tests represent a challenge for symptom-based microbiology and functional detection of important antibiotic resistance. Introduction of such assays to routine diagnostics will help to detect potentially unknown pathogens emerged because of horizontal gene transfer.
Funded by AZV (NV19-05-00541)
Past projects
- Czech Science Foundation (16-24309S) Mass Spectrometric 3D Structure Analysis of DNA Response Elements / Transcription Factor Association and Modeling (2016-2018)
- Czech Science Foundation (16-20860S) Structural and functional analysis of Hsp70/Hsp90 chaperone complexes (2016-2018)
- Ministry of Education, Youth, and Sports (COST CZ LH15010) A new tool for structure biology: Combination of high resolution mass spectrometry, chemical cross-linking and H/D exchange (2015-2017)
- Ministry of Education, Youth, and Sports (Kontakt II LH15010) Structural mass spectrometry analysis for complexes of transcription factors with DNA response elements (2015-2017)
- Czech Science Foundation and FWF (Austria) (16-34818L / I 2385-N28) Electron Transfer in Cellulose Degrading Enzymes (2016-2018)