News:

  • 2022/11/18:

    And yet another one piece (last one in 2022) with Angel Pey and Milagros Medina. Our collaborative effort on NQO1 active site mutations is now in FEBS J. And again thanks EU FT-ICR MS (H2020) for support.

  • 2022/10/13:

    This year it looks like a solid harvest of work conducted with Angel L. Pey from Uni of Grenada. Another joint paper, this time on structural effects induced by cavity making mutation on a two domain protein - PGK1. Paper now available in Sci Rep. Thanks EU FT-ICR MS (H2020) for support.

  • 2022/10/10:

    Congratulations to Lukas on BIOCEV's PhD Student Publication Award - our JACS paper on FFAP attracts attention 😉. Great collaboration with the Beier group and CF Plus Chemicals. Thanks CSF and H2020 for financial support and HPST for sponsoring this award.

  • 2022/09/13:

    Insight into the role of site-specific phosphorylation of human antioxidant and disease-associated flavoprotein NQO1 provided through great collaboration with teams of Angel L. Pey and Milagros Medina. Paper now available in Arch Biochem Biophys. Thanks EU FT-ICR MS (H2020) for support.

  • 2022/09/09:

    Congratulations to Bára on succesfull (and very nice) master thesis defense!

  • 2022/08/30:

    Nice work with Moshe and Yoni, this time on tweety proteins is finally out. Freely available in Commun. Biol.! Emelia and guys, thanks for great collaboration and thanks EU FT-ICR MS (H2020) for support.

Horizon 2020
EPIC-XS
EU FT-ICR MS MSCA

Important:

  • 2022/07/22:

    Synopsis of Ales' Hippostruct MSCA project is available here.

  • 2022/04/22:

    Are you interested in structural mass spectrometry, method development ad/or membrane proteins? We are looking for Bc, MSc, PhD students and post-doc! Inquiries should be addressed to Petr Novak.

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
and application to selected biological problems (medicinally or biotechnologically important proteins), membrane proteins, dynamic and heavily modified proteins and their complexes

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


  • Expanding the analytical toolbox for structural mass spectrometry

    In this proposal, we plan to develop bi-functional chemical probes with novel reactivity and functionality that will expand the palette of currently targetable amino acid residues and thus increase the spatial resolution of chemical cross-linking. The probes will be isotopically coded to enable quantitative cross-linking experiments for monitoring of the protein dynamics. The novel reactivity will target aromatic amino acid side chains and will be based on bi-functional Togni reagents. In order to increase the functionality of currently available cross-linking probes we will design, synthetize and test isotopically labeled, MS cleavable disuccinimidyl dipropionic, diimidazolyl dipropionic and dihydrazide dipropionic urea enabling unambiguous quantitative cross-linking through hydroxy and carboxyl bearing amino acids. To further streamline these cross-linking experiments, we also plan to develop specialized algorithms dedicated to data processing of isotopically labeled samples. We will also evaluate complete analytical procedure including enrichment, separation and fragmentation of the cross-linked peptides. The effect of the reagents on the protein structure and their application to selected biologically relevant systems will form the final goal of the project. All these advancements will increase the ability of structural mass spectrometry to provide valuable information addressing structural aspects and dynamics of proteins and protein complexes.

    Funded by Czech Science Foundation (22-27695S)

  • 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.

    Synopsis canbe accessed here.

    Funded by EU MSCA-IF (widening), project number 101003406 (HIPPOSTRUCT)

  • European Proteomics Infrastructure Consortium providing access

    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

  • 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 (19-16084S) Mapping the protein surface accessible area utilizing Top Down mass spectrometry and reactive radical footprinting (2019-2021)
    • 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 LD15089) 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)