Laboratories

Michał Dadlez, PhD, DSc, Prof.

Laboratory of Mass Spectrometry

Research Scope

Our laboratory is interested in applying mass spectrometry techniques to solve biomedically oriented problems, with a focus on structural studies of protein assemblies, structural consequences of posttranslational modifications, and targeted proteomic analyses of protein panels with potential use in medicine. Our long-term goal is to popularize new mass spectrometry-based methods to solve problems that are difficult or impossible to tackle using other approaches.

Research

Main Scientific Achievements

  • We uncovered a new scheme for tetramer-tetramer contact regions in vimentin for their lateral association into unit-length filaments, leading to a new model of an intermediate filament assembly pathway that is possibly canonical for the entire class of this fundamental cellular scaffold and protein signaling assemblies.
  • We discovered new interactions within participating proteins of the histone pre-mRNA processing complex, eEF1B translation elongation complex, RAGE, and centriolar and kinetochore complexes, providing a basis for a new assembly scheme.

Research Description

The protein structurome is only partially tractable using classic methods of structure analysis (X-ray, nuclear magnetic resonance [NMR], and cryo-electron microscopy) because of the high content of high dynamics (intrinsic disorder [ID]) and structural heterogeneity. An estimated 70% of signaling proteins contain large ID regions. This is one reason for the increasing gap between the number of protein sequences and their structures. Mass spectrometry (MS) methods help close this gap because they (1) provide access to dynamic properties of proteins and their assemblies by monitoring the kinetics of hydrogen-deuterium exchange (HDX), (2) can characterize coexisting structural variants (ion mobility), and (3) provide structural constraints for modeling even the largest molecular complexes (native MS, X-linking, and HDX). In recent years, we provided unique insights into structural properties of several protein systems, including centrioles, kinetochores, the histone pre-mRNA processing complex, the eEF1B translation elongation complex, intermediate filament proteins (IFs), receptor for advanced glycation endproducts (RAGE), and Alzheimer’s disease amyloid b (Ab) peptide oligomers. We are continuing several research lines and engage in new projects that lead to new experiment-based structural models of challenging protein targets.

One of the fundamental cellular regulation/signaling mechanisms is the tuning of proteins through oxidation-reduction (redox)-based post translational modifications (PTM). We examine structural and functional aspects of PTMs of proteins, particularly two redox modifications of protein cysteines (S-nitrosylation and S-glutathionylation). We seek to provide insights into the molecular aspects of cellular control mechanisms that are performed by multiple protein proteoforms in an interplay between selective modifications of cysteines and protein oligomerization, metal cations, ligands, and target protein binding. Our research subjects are vital human proteins that are related to innate immune response processes and cancer, including the S100 family and IFs. Our long-term goals are to create foundations for the development of new diagnostic methods using various protein proteoforms as biomarkers of inflammation and cancer processes. Our group’s research became the basis of protein/protein interaction-based electrochemical biosensors to detect RAGE ligands, which are biomarkers of many human diseases, including diabetes, rheumatoid arthritis, Alzheimer’s disease, and cancer.

Targeted Proteomics and Medical Applications

The field of MS-based targeted proteomics is on the cusp of entering clinical use and is undeniably the future of clinical protein-biomarker diagnostics1,2. Mass spectrometry is already routinely used clinically for small-molecule quantification. Analyses of peptides/proteins are recently replacing and complementing aging antibody-based tests3. Clinicians are aware of the current diagnostic limitations and are keen to develop new technologies with proteomics researchers to improve patient care. We seek to advance the technology and produce applications to help expedite the process by which proteomics enter the clinic. This has been increasingly happening in our laboratory since 2012 with the arrival of Dr. Dominik Domanski, a specialist in targeted proteomics and its application in translational research. This has occurred through direct financing from institutional grants and national and international collaborations that support the development of targeted proteomics tools and technologies to advance the measurement of biomarkers in medicine with potential clinical application toward cancer diagnostics and personalized medicine. Dr. Domanski has contributed to the field in the standardization of liquid chromatography-MS quantification and sample procurement, improvements in analytical robustness and sensitivity, the development of bioinformatics tools, and novel methods for quantifying the phosphorylation of cancer signaling proteins. Highlights of this work in our laboratory include the development of a panel of cytokeratin cancer markers to improve pleural effusion (PE) diagnosis in patients4,5. The plan for the next 2 years is to move the panel for PE classification toward a validated diagnostic test and develop other technologies for personalized medicine for breast cancer that involves CDK4/6 inhibitors in collaboration with Dr. Basik from McGill.

  • Bibliography
  1. Annesley et al. Clin. Chem. 2015.
  2. Duarte et al. Proteomes
  3. Annesley et al. Chem. 2016.
  4. Domanski et al. Neoplasia
  5. Perzanowska et al. Proteomics – Clin. Appl.
  6. García-Bailo et al. J. Clin. Nutr. 2012.
  7. Parker et al. Curr. Chem. 2014.
  8. Domanski et al. Lab. Med. 2011.
  9. Gillette et al. Methods 2013.

Methodology

For the structural MS of various proteoforms, such as those that are caused by PTMs, we use MS-based methods (HDX, X-linking, native MS, ion mobility), accompanied by a set of more classic approaches: size-exclusion chromatography coupled with multiangle light scattering mass analysis, analytical ultracentrifugation, electron microscopy, atomic force microscopy, calorimetry, dynamic light scattering, circular dichroism, nuclear magnetic resonance, etc. Using these methods, we study the consequences of PTMs on the conformation, oligomerization properties, metal ions, ligands, and protein binding to the studied proteoforms. HDX maps protein chain dynamics in different protein regions and its changes that accompany structural transformations that are induced by ligand binding. X-linking maps the contact region between protein domains in protein complexes. Native MS characterizes complex stoichiometries. Ion mobility characterizes structural heterogeneity of the protein assemblies. We use classic proteomic methods to identify targets of redox-based modification of protein cysteine thiols. We utilize semi-synthetic strategies to obtain highly homogeneous full-length recombinant proteins, also those that are observed in vivo, that bear site-specific modifications of cysteine thiols.

Mass spectrometry-based targeted proteomics approaches (multiple/parallel reaction monitoring [MRM/PRM]) can overcome the many issues (e.g., cross-reactivity, patient auto-antibodies, low multiplexing, expensive development, etc.) of immunological assays that have been the gold-standard for protein quantification. Our developed MRM/PRM panels, combined with isotope-labeled peptide standards, provide greater detail and specific biological information. They are also less expensive and quicker to develop. Moreover, they are characterized by higher analytical specificity and precision, a wider dynamic range, and the possibility of measuring numerous proteins within a single rapid analysis6-9. Our laboratory houses unique MS systems that are required for these methods.

Selected Publications

    • Structural Dynamics of the Vimentin Coiled-coil Contact Regions Involved in Filament Assembly as Revealed by Hydrogen-Deuterium Exchange. Premchandar A, Mücke N, Poznański J, Wedig T, Kaus-Drobek M, Herrmann H, Dadlez M. J Biol Chem. 2016 Nov 25; 291(48): 24931-24950. doi: 10.1074/jbc.M116.748145.
    • Protein Composition of Catalytically Active U7-Dependent Processing Complexes Assembled on Histone Pre-MRNA Containing Biotin and a Photo-Cleavable Linker. Skrajna, A.; Yang, X.; Dadlez, M.; Marzluff, W. F.; Dominski, Z. Nucleic Acids Research 2018, 46 (9), 4752–4770. https://doi.org/10.1093/nar/gky133.
    • Global analysis of S-nitrosylation sites in the wild type (APP) transgenic mouse brain-clues for synaptic pathology. Zaręba-Kozioł M, Szwajda A, Dadlez M, Wysłouch-Cieszyńska A, Lalowski M. Mol Cell Proteomics. 2014 Sep;13(9):2288-305. doi: 10.1074/mcp.M113.036079.
    • Molecular basis for inner kinetochore configuration through RWD-domain interactions. Florian Schmitzberger, Magdalena M. Richter, Yuliya Gordiyenko, Carol V. Robinson, Michał Dadlez, and Stefan Westermann (2017) EMBO J. 2017 Dec 1; 36(23): 3458-3482. doi: 10.15252/embj.201796636.

Collaborations

    • Harald Herrmann, German Cancer Research Center (DKFZ), Universitätsklinikum Erlangen, Institute of Neuropathology. https://www.researchgate.net/profile/Harald_Herrmann2
    • David Glover, California Institute of Technology, https://gloverlab.caltech.edu/.
    • Zbigniew Domiński, William Marzluff, North Carolina University. https://www.med.unc.edu/biochem/directory/dominski/
    • Michał Hetman, Louisville University. http://louisville.edu/kscirc/basic-research/faculty-1/michal-hetman-m-d-ph-d.
    • Marcin Przewłoka, Southhampton University. https://www.southampton.ac.uk/biosci/about/staff/mrp1m15.page
    • Frank Sobott, Leeds University. https://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=FS
    • Aleksander Edelman, Necker Institute. https://www.researchgate.net/lab/GEIC2O-Aleksander-Edelman
    • Boris Negrutskii, Institute of Molecular Biology and Genetics, NAS of Ukraine. https://www.imbg.org.ua/en/dept/translation/lab_pbs/
    • Paolo d'Avino, University of Cambridge. https://www.path.cam.ac.uk/directory/paolo-davino.
    • Zoltan Lipinszki, Lendület Laboratory of Cell Cycle Regulation, Institute of Biochemistry, Biological Research Centre, Szeged, web: http://www.brc.hu/biochem_cell_cycle_and_transcription_regulation
    • Sasa Kazazic, Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb. https://www.irb.hr/eng/Divisions/Division-of-Physical-Chemistry/Laboratory-for-mass-spectrometry-and-functional-proteomics/Employees/Sasa-Kazazic
    • Andrzej Ożyhar, Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology. https://www.researchgate.net/lab/Andrzej-Ozyhar-Lab
    • Marc Baumann, Meilahti Clinical Proteomics Core Facility, Institute of Biomedicine, Biochemistry and Developmental Biology, University of Helsinki. https://researchportal.helsinki.fi/en/persons/marc-baumann
    • Matthias Bochtler, International Institute of Molecular and Cell Biology, Warsaw. https://www.iimcb.gov.pl/en/research/laboratories/3-laboratory-of-structural-biology-bochtler-laboratory
    • Sylwia Rodziewicz-Motowidło, University of Gdańsk
    • Kasper Rand, Department of Pharmacy, University of Copenhagen. https://pharmacy.ku.dk/employees/?pure=en/persons/130972
    • Artur Krężel, Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław. http://www.chembiolab.uni.wroc.pl/
    • Florian Schmitzberger, Research Institute of Molecular Pathology (IMP), Vienna, Ichnos Sciences.
    • Chris Borchers, Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal. http://www.ladydavis.ca/en/christophborchers
    • Ruth Chicquet-Ehrisman, University of Basel.
    • Anna Niedźwiecka, Institute of Physics, Polish Academy of Sciences. http://www.ifpan.edu.pl/SL-4/index.php?group=niedzwiecka.
    • Katarzyna Koziak, Centre for Preclinical Research and Technology, Department of Immunology, Biochemistry and Nutrition, Medical University of Warsaw
    • prof Hanna i Jerzy Radeccy, Institute of Animal Reproduction and Food Research Polish Academy of Science, Tuwima 10, 10-748 Olsztyn, Poland.
    • Tomasz Bednarczuk, Department of Internal Medicine and Endocrinology, Medical University of Warsaw, Poland.
    • Dr Maciej Lalowski, Biomedicum Helsinki, Institute of Biomedicine, Biochemistry/Developmental Biology, Meilahti Clinical Proteomics Core Unit, University of Helsinki, Finland
    • Magdalena Kowalewska - Maria Sklodowska-Curie Institute Oncology Center,
    • Grzegorz Wojtas (MD) - Mazovian Center of Pulmonary Disease and Tuberculosis Treatment,
    • Katarzyna Wertheim-Tysarowska (MD) - Institute of Mother and Child,
    • Mark Basik (MD) - Jewish General Hospital, McGill, Canada. https://www.mcgill.ca/generalsurgery/dr-mark-basik
    • Katarzyna Pawlak http://www.ch.pw.edu.pl/Pracownicy/Sklad-osobowy/Katedra-Chemii-Analitycznej/Katarzyna-Pawlak

Prizes and Awards

    • Michał Dadlez - awards for a publication as a co-author: Parnas award 2001, Ministry of Health award 2007, 2008, Polish Internal Medicine Society award 2014, 2015, Warsaw University Rector, 2017

Publications (IBB PAS affiliated)

LOBOCKA M.B., HEJNOWICZ M.S., GĄGAŁA U., WEBER-DABROWSKA B., WEGRZYN G., DADLEZ M., The first step to bacteriophage therapy - how to choose the correct phage. Chapter 2 in: Phage Therapy. Current Research and Applications. Ed. Borysowski J., Międzybrodzki R., Górski A., Caister Academic Press; ISBN 978-1-908230-40-9 [378p.] 2014, p. 23-67
SITKIEWICZ E., KLONIECKI M., POZNANSKI J., BAL W., DADLEZ M., Factors influencing compact-extended structure equilibrium in oligomers of Aβ1-40 peptide-an ion mobility mass spectrometry study. Journal of Molecular Biology (2014) 426(15): 2871-2885 IF 3.959
SITKIEWICZ E., OLEDZKI J., POZNANSKI J., DADLEZ M., Di-tyrosine cross-link decreases the collisional cross-section of Aβ peptide dimers and trimers in the gas phase: an ion mobility study. PLOS ONE (2014) 9(6): e100200 (14p.) doi:10.1371/journal.pone.0100200 IF 3.534
NESTERUK M., HENNIG E., MIKUŁA M., KARCZMARSKI J., DZWONEK A., GORYCA K., RUBEL T., PAZIEWSKA A., WOSZCZYŃSKI M., LEDWON J., DĄBROWSKA M., DADLEZ M., OSTROWSKI J., Mitochondrial-related proteomic changes during obesity and fasting inmice are greater in the liver than skeletal muscles. Functional & Integrative Genomics (2014) 14(1): 245–259 IF 2.691
PETKAUSKAITE R., LUKOSIUS D., DEBSKI J., JASILIONIS A., DADLEZ M., KIERAITE L., TIMONINA A., KUISIENE N., Identification of proteins involved in starch and polygalacturonic acid degradation using LC/MS. Central European Journal of Biology (2014) 9(7): 708-716 IF 0.633
TARNOWSKI K.J., FITUCH K., SZCZEPANOWSKI R.H., DADLEZ M., KAUS-DROBEK M., Patterns of structural dynamics in RACK1 protein retained throughout evolution: A hydrogen-deuterium exchange study of three orthologs. Protein Science (2014) 23(5): 639-651 IF 2.861
BAKUN M., SENATORSKI G., RUBEL T., LUKASIK A., ZIELENKIEWICZ P., DADLEZ M., PĄCZEK L., Urine proteomes of healthy aging humans reveal extracellular matrix (ECM) alterations and immune system dysfunction. AGE (2014) 36(1): 299–311 IF 3.445
BARTOSZEWICZ Z., KONDRACKA A., JAŹWIEC R., POPOW M., DADLEZ M., BEDNARCZUK T., Can we accurately measure the concentration of clinically relevant vitamin D metabolites in the circulation? The problems and their consequences. Endokrynologia Polska (2013) 64(3): 238-45 IF 1.070
SABATH I., SKRAJNA A., YANG X-C., DADLEZ M., MARZLUFF W.F., DOMIŃSKI Z., 3`-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors. RNA-A Publication of the RNA Society (2013) 19: 1726-1744 IF 5.088
KARCZMARSKI J., RUBEL T., MIKUŁA M., WOLSKI J., RUTKOWSKI A., ZAGOROWICZ E., DADLEZ M., OSTROWSKI J., Pre-analytical-related variability influencing serum peptide profiles demonstrated in a mass spectrometry-based search for colorectal and prostate cancer biomarkers. Acta Biochimica Polonica (2013) 60(3): 417-425 IF 1.185
SITKIEWICZ E., TARNOWSKI K.J., POZNANSKI J., KULMA M., DADLEZ M., Oligomerization interface of RAGE receptor revealed by MS-monitored hydrogen deuterium exchange. PLOS ONE (2013) 8(10): e 76353 (16p.) DOI: 10.1371/journal.pone.0076353 IF 3.730
ODOLCZYK N., FRITSCH J., NOREZ C., SERVEL N., FARIA da CUNHA M., BITAM S., KUPNIEWSKA-KOZAK A., WISZNIEWSKI L., COLAS J., TARNOWSKI K.J., TONDELIER D., ROLDAN A., SAUSSEREAU E.L., MELIN-HESCHEL P., WIECZOREK G., LUKACS G., DADLEZ M., FAURE G., HERRMANN H., OLLERO M., BECQ F., ZIELENKIEWICZ P., EDELMAN A., Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain. EMBO Molecular Medicine (2013) 5(10): 1484-1501 IF 7.795
HOSER R., ŻURCZAK M., LICHOCKA M., ZUZGA S., DADLEZ M., SAMUEL M.A., ELLIS B.E., STUTTMANN J., PARKER J.E., HENNIG J., KRZYMOWSKA M., Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1. New Phytologist (2013) 200: 158-171 IF 6.736
TSZYRSZNIC W., BOROWIEC A., PAWŁOWSKA E., JAŹWIEC R., ŻOCHOWSKA D., BARTŁOMIEJCZYK I., ŻEGARSKA J., PĄCZEK L., DADLEZ M., Two rapid ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods with common sample pretreatment for therapeutic drug monitoring of immunosuppressants compared to immunoassay. Journal of Chromatography B (2013) 928: 9–15 IF 2.487
SCIFO E., SZWAJDA A., DEBSKI J., UUSI-RAUVA K., KESTI T., DADLEZ M., GINGRAS A.C., TYYNELÄ J., BAUMAN M.H., JALANKO A., LALOWSKI M., Drafting the CLN3 protein interactome in SH-SY5Y human neuroblastoma cells: a label-free quantitative proteomics approach. Journal of Proteome Research (2013) 12(5): 2101-2115 IF 5.056
GISKA F., LICHOCKA M., PIECHOCKI M., DADLEZ M., SCHMELZER E., HENNIG J., KRZYMOWSKA M., Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins. Plant Physiology (2013) 161(4): 2049-2061 IF 6.555
YANG X-C., SABATH I., DEBSKI J., KAUS-DROBEK M., DADLEZ M., MARZLUFF W.F., DOMIŃSKI Z., A complex containing the CPSF73 endonuclease and other polyadenylation factors associates with U7 snRNP and is recruited to histone pre-mRNA for 3’ end processing. Molecular and Cellular Biology (2013) 33(1): 28-37 IF 5.372
BAKUN M., NIEMCZYK M., DOMAŃSKI D., JAŹWIEC R., PERZANOWSKA A., NIEMCZYK S., KISTOWSKI M., FABIJAŃSKA A., BOROWIEC A., PĄCZEK L., DADLEZ M., Urine proteome of autosomal dominant polycystic kidney disease patients. Clinical Proteomics (2012) 9:13
RICHARDSON K., DENNY R., HUGHES CH., SKILLING J., SIKORA J., DADLEZ M., MANTECA A., JUNG H.R., JENSEN O.N., REDEKER V., MELKI R., LANGRIDGE J., ,VISSERS J.P.C., A probabilistic framework for peptide and protein qualification from data-dependent and data-independent LC-MS proteomics experiments. OMICS A Journal of Integrative Biology (2012) 16(9): 468-482 IF 2.441
MALINOWSKA A., KISTOWSKI M., BAKUN M., RUBEL T., TKACZYK M., MIERZEJEWSKA J., DADLEZ M., Diffprot-software for non-parametric statistical analysis of differential proteomics data. Journal of Proteomics (2012) 75(13):4062-73 IF 4.878

Patents

    • An MRM-Based Cytokeratin Marker Assay as a Tool for Cancer Studies: Application to Lung Cancer Pleural Effusions. Perzanowska A, Fatalska A, Wojtas G, Lewandowicz A, Michalak A, Krasowski G, Borchers CH, Dadlez M, Domanski D. Proteomics Clin Appl. 2018 Mar;12(2). doi: 10.1002/prca.201700084. Multiplexed cytokeratin panel, method for preparing multiplexed cytokeratin panel and method for classification of lung cancer patient (Multipleksowy panel cytokeratynowy, sposób wytwarzania multipleksowego panelu cytokeratynowego i sposób klasyfikowania pacjentów z nowotworem płuc). Perzanowska A, Domanski D, Kistowski M, Michalak A, Wojtas G, Dadlez M. 2015. Patent nr P.414159 (UPRP).