Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

Faculty of Science Charles University Prague Prague 128 00 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Prague 142 20 Czech Republic

Zobrazit více v PubMed

Beck A.; Debaene F.; Diemer H.; Wagner-Rousset E.; Colas O.; Van Dorsselaer A.; Cianférani S. Cutting-Edge Mass Spectrometry Characterization of Originator, Biosimilar and Biobetter Antibodies. J. Mass Spectrom. 2015, 50 (2), 285–297. 10.1002/jms.3554. PubMed DOI

Pan J.; Zhang S.; Borchers C. H. Comparative Higher-Order Structure Analysis of Antibody Biosimilars Using Combined Bottom-up and Top-down Hydrogen-Deuterium Exchange Mass Spectrometry. Biochim. Biophys. Acta, Proteins Proteomics 2016, 1864 (12), 1801–1808. 10.1016/j.bbapap.2016.08.013. PubMed DOI

Háda V.; Bagdi A.; Bihari Z.; Timári S. B.; Fizil Á.; Szántay C. Recent Advancements, Challenges, and Practical Considerations in the Mass Spectrometry-Based Analytics of Protein Biotherapeutics: A Viewpoint from the Biosimilar Industry. J. Pharm. Biomed. Anal. 2018, 161, 214–238. 10.1016/j.jpba.2018.08.024. PubMed DOI

Aggarwal S. R. What’s Fueling the Biotech Engine—2012 to 2013. Nat. Biotechnol. 2014, 32 (1), 32–39. 10.1038/nbt.2794. PubMed DOI

Gahoual R.; Biacchi M.; Chicher J.; Kuhn L.; Hammann P.; Beck A.; Leize-Wagner E.; François Y. N. Monoclonal Antibodies Biosimilarity Assessment Using Transient Isotachophoresis Capillary Zone Electrophoresis-Tandem Mass Spectrometry. mAbs 2014, 6 (6), 1464–1473. 10.4161/mabs.36305. PubMed DOI PMC

Zhang J.; Liu H.; Katta V. Structural Characterization of Intact Antibodies by High-Resolution LTQ Orbitrap Mass Spectrometry. J. Mass Spectrom. 2010, 45 (1), 112–120. 10.1002/jms.1700. PubMed DOI

Allocati E.; Bertele V.; Gerardi C.; Garattini S.; Banzi R. Clinical Evidence Supporting the Marketing Authorization of Biosimilars in Europe. Eur. J. Clin. Pharmacol. 2020, 76 (4), 557–566. 10.1007/s00228-019-02805-y. PubMed DOI

Bardelli M.; Livoti E.; Simonelli L.; Pedotti M.; Moraes A.; Valente A. P.; Varani L. Epitope Mapping by Solution NMR Spectroscopy. J. Mol. Recognit. 2015, 28 (6), 393–400. 10.1002/jmr.2454. PubMed DOI

Di Muzio M.; Wildner S.; Huber S.; Hauser M.; Vejvar E.; Auzinger W.; Regl C.; Laimer J.; Zennaro D.; Wopfer N.; et al. Hydrogen/Deuterium Exchange Memory NMR Reveals Structural Epitopes Involved in IgE Cross-Reactivity of Allergenic Lipid Transfer Proteins. J. Biol. Chem. 2020, 295 (51), 17398–17410. 10.1074/jbc.RA120.014243. PubMed DOI PMC

Poljak R. J.; Amzel L. M.; Avey H. P.; Chen B. L.; Phizackerley R. P.; Saul F. Three Dimensional Structure of the Fab’ Fragment of a Human Immunoglobulin at 2.8 Å Resolution. Proc. Natl. Acad. Sci. U.S.A. 1973, 70 (12), 3305–3310. 10.1073/pnas.70.12.3305. PubMed DOI PMC

King M. T.; Brooks C. L.. Epitope Mapping of Antibody-Antigen Interactions with X-Ray Crystallography. In Epitope Mapping Protocols; Springer, 2018; Vol. 1785, pp 13–27. PubMed PMC

Renaud J. P.; Chari A.; Ciferri C.; Liu W. T.; Rémigy H. W.; Stark H.; Wiesmann C. Cryo-EM in Drug Discovery: Achievements, Limitations and Prospects. Nat. Rev. Drug Discovery 2018, 17 (7), 471–492. 10.1038/nrd.2018.77. PubMed DOI

Wigge C.; Stefanovic A.; Radjainia M. The Rapidly Evolving Role of Cryo-EM in Drug Design. Drug Discovery Today: Technol. 2020, 38, 91–102. 10.1016/j.ddtec.2020.12.003. PubMed DOI

Jethva P. N.; Gross M. L. Hydrogen Deuterium Exchange and Other Mass Spectrometry- Based Approaches for Epitope Mapping. Front. Anal. Sci. 2023, 3, 111874910.3389/frans.2023.1118749. PubMed DOI PMC

Suckau D.; Köhl J.; Karwath G.; Schneider K.; Casaretto M.; Bitter-Suermann D.; Przybylski M. Molecular Epitope Identification by Limited Proteolysis of an Immobilized Antigen-Antibody Complex and Mass Spectrometric Peptide Mapping. Proc. Natl. Acad. Sci. U.S.A. 1990, 87 (24), 9848–9852. 10.1073/pnas.87.24.9848. PubMed DOI PMC

Zhao Y.; Chait B. T. Protein Epitope Mapping by Mass Spectrometry. Anal. Chem. 1994, 66 (21), 3723–3726. 10.1021/ac00093a029. PubMed DOI

Fiedler W.; Borchers C.; Macht M.; Deininger S. O.; Przybylski M. Molecular Characterization of a Conformational Epitope of Hen Egg White Lysozyme by Differential Chemical Modification of Immune Complexes and Mass Spectrometric Peptide Mapping. Bioconjugate Chem. 1998, 9 (2), 236–241. 10.1021/bc970148g. PubMed DOI

Yamada N.; Suzuki E. I.; Hirayama K. Identification of the Interface of a Large Protein-Protein Complex Using H/D Exchange and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Rapid Commun. Mass Spectrom. 2002, 16 (4), 293–299. 10.1002/rcm.579. PubMed DOI

Baerga-Ortiz A.; Hughes C. A.; Mandell J. G.; Komives E. A. Epitope Mapping of a Monoclonal Antibody against Human Thrombin by H/D-Exchange Mass Spectrometry Reveals Selection of a Diverse Sequence in a Highly Conserved Protein. Protein Sci. 2002, 11 (6), 1300–1308. 10.1110/ps.4670102. PubMed DOI PMC

Pimenova T.; Meier L.; Roschitzki B.; Paraschiv G.; Przybylski M.; Zenobi R. Polystyrene Beads as an Alternative Support Material for Epitope Identification of a Prion-Antibody Interaction Using Proteolytic Excision-Mass Spectrometry. Anal. Bioanal. Chem. 2009, 395 (5), 1395–1401. 10.1007/s00216-009-3119-8. PubMed DOI

Jones L. M.; Sperry J. B.; Carroll J. A.; Gross M. L. Fast Photochemical Oxidation of Proteins for Epitope Mapping. Anal. Chem. 2011, 83 (20), 7657–7661. 10.1021/ac2007366. PubMed DOI PMC

Cheng M.; Zhang B.; Cui W.; Gross M. L. Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting. Angew. Chem., Int. Ed. 2017, 56 (45), 14007–14010. 10.1002/anie.201706697. PubMed DOI PMC

Cheng M.; Asuru A.; Kiselar J.; Mathai G.; Chance M. R.; Gross M. L. Fast Protein Footprinting by X-Ray Mediated Radical Trifluoromethylation. J. Am. Soc. Mass Spectrom. 2020, 31 (5), 1019–1024. 10.1021/jasms.0c00085. PubMed DOI PMC

Rahimidashaghoul K.; Klimánková I.; Hubálek M.; Korecký M.; Chvojka M.; Pokorný D.; Matoušek V.; Fojtík L.; Kavan D.; Kukačka Z.; et al. Reductant-Induced Free Radical Fluoroalkylation of Nitrogen Heterocycles and Innate Aromatic Amino Acid Residues in Peptides and Proteins. Chem. - Eur. J. 2019, 25 (69), 15779–15785. 10.1002/chem.201902944. PubMed DOI

Fojtík L.; Fiala J.; Pompach P.; Chmelík J.; Matoušek V.; Beier P.; Kukačka Z.; Novák P. Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules. J. Am. Chem. Soc. 2021, 143 (49), 20670–20679. 10.1021/jacs.1c07771. PubMed DOI

Eisenberger P.; Gischig S.; Togni A. Novel 10-I-3 Hypervalent Iodine-Based Compounds for Electrophilic Trifluoromethylation. Chem. - Eur. J. 2006, 12 (9), 2579–2586. 10.1002/chem.200501052. PubMed DOI

Matoušek V.; Václavík J.; Hájek P.; Charpentier J.; Blastik Z. E.; Pietrasiak E.; Budinská A.; Togni A.; Beier P. Expanding the Scope of Hypervalent Iodine Reagents for Perfluoroalkylation: From Trifluoromethyl to Functionalized Perfluoroethyl. Chem. - Eur. J. 2016, 22 (1), 417–424. 10.1002/chem.201503531. PubMed DOI

Vermeire P. J.; Lilina A. V.; Hashim H. M.; Dlabolová L.; Fiala J.; Beelen S.; Kukačka Z.; Harvey J. N.; Novák P.; Strelkov S. V. Molecular Structure of Soluble Vimentin Tetramers. Sci. Rep. 2023, 13 (1), 884110.1038/s41598-023-34814-4. PubMed DOI PMC

Perkins D. N.; Pappin D. J.; Creasy D. M.; Cottrell J. S. Probability-Based Protein Identification by Searching Sequence Databases Using Mass Spectrometry Data. Electrophoresis 1999, 20 (18), 3551–3567. 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2. PubMed DOI

Trcka F.; Durech M.; Vankova P.; Chmelik J.; Martinkova V.; Hausner J.; Kadek A.; Marcoux J.; Klumpler T.; Vojtesek B.; et al. Human Stress-Inducible Hsp70 Has a High Propensity to Form ATP-Dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding. Mol. Cell. Proteomics 2019, 18 (2), 320–337. 10.1074/mcp.RA118.001044. PubMed DOI PMC

Kavan D.; Man P. MSTools - Web Based Application for Visualization and Presentation of HXMS Data. Int. J. Mass Spectrom. 2011, 302 (1–3), 53–58. 10.1016/j.ijms.2010.07.030. DOI

Perez-Riverol Y.; Bai J.; Bandla C.; García-Seisdedos D.; Hewapathirana S.; Kamatchinathan S.; Kundu D. J.; Prakash A.; Frericks-Zipper A.; Eisenacher M.; et al. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50 (D1), D543–D552. 10.1093/nar/gkab1038. PubMed DOI PMC

Hubbard S. J.; Thornton J. M.. NACCESS. Dep. Biochem. Mol. Biol. Univ. Coll. 1993. http://www.bioinf.manchester.ac.uk/naccess

Diwanji D.; Trenker R.; Thaker T. M.; Wang F.; Agard D. A.; Verba K. A.; Jura N. Structures of the HER2–HER3–NRG1β Complex Reveal a Dynamic Dimer Interface. Nature 2021, 600 (7888), 339–343. 10.1038/s41586-021-04084-z. PubMed DOI PMC

Lambert T.; Gramlich M.; Stutzke L.; Smith L.; Deng D.; Kaiser P. D.; Rothbauer U.; Benesch J. L. P.; Wagner C.; Koenig M.; et al. Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS. J. Am. Soc. Mass Spectrom. 2023, 34 (11), 2556–2566. 10.1021/jasms.3c00268. PubMed DOI PMC

Kalaninová Z.; Fojtík L.; Chmelík J.; Novák P.; Volný M.; Man P.. Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry. In Methods in Molecular Biology; Gevaert K., Ed.; Springer, 2023; pp 303–334. PubMed

Trcka F.; Durech M.; Man P.; Hernychova L.; Muller P.; Vojtesek B. The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex. J. Biol. Chem. 2014, 289 (14), 9887–9901. 10.1074/jbc.M113.526046. PubMed DOI PMC