Skip to main content
SpringerLink
Log in

Studying Protein–DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry

  • Protocol
  • First Online:
Book cover Multiprotein Complexes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2247))

Abstract

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein–DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 149.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Katta V, Chait BT, Carr S (1991) Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry. Rapid Commun Mass Spectrom 5:214–217

    Article  CAS  PubMed  Google Scholar 

  2. Zhang Z, Smith DL (1993) Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci 2:522–531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bai YW, Milne JS, Mayne L et al (1993) Primary structure effects on peptide group hydrogen-exchange. Proteins 17:75–86

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Engen JR, Wales TE (2015) Analytical aspects of hydrogen exchange mass spectrometry. Annu Rev Anal Chem 8:127–148

    Article  CAS  Google Scholar 

  5. Oganesyan I, Lento C, Wilson DJ (2018) Contemporary hydrogen deuterium exchange mass spectrometry. Methods 144:27–42

    Article  CAS  PubMed  Google Scholar 

  6. Sorokin VA, Gladchenko GO, Valeev VA (1986) DNA protonation at low ionic strength of solution. Die Makromol Chemie 187:1053–1063

    Article  CAS  Google Scholar 

  7. Ma L, Fitzgerald MC (2003) A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions. Chem Biol 10:1205–1213

    Article  CAS  PubMed  Google Scholar 

  8. Sperry JB, Wilcox JM, Gross ML (2008) Strong anion exchange for studying protein-DNA interactions by H/D exchange mass spectrometry. J Am Soc Mass Spectrom 19:887–890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sperry JB, Shi X, Rempel DL et al (2008) A mass spectrometric approach to the study of DNA-binding proteins: interaction of human TRF2 with telomeric DNA. Biochemistry 47:1797–1807

    Article  CAS  PubMed  Google Scholar 

  10. Poliakov A, Jardine P, Prevelige PE (2008) Hydrogen/deuterium exchange on protein solutions containing nucleic acids: utility of protamine sulfate. Rapid Commun Mass Spectrom 22:2423–2428

    Article  CAS  PubMed  Google Scholar 

  11. Roberts VA, Pique ME, Hsu S et al (2012) Combining HD exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase. Nucleic Acids Res 40:6070–6081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Graham BW, Tao Y, Dodge KL et al (2016) DNA interactions probed by hydrogen-deuterium exchange (HDX) Fourier transform ion cyclotron resonance mass spectrometry confirm external binding sites on the minichromosomal maintenance (MCM) helicase. J Biol Chem 291:12467–12480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Boura E, Silhan J, Herman P et al (2007) Both the N-terminal loop and wing W2 of the forkhead domain of transcription factor Foxo4 are important for DNA binding. J Biol Chem 282:8265–8275

    Article  CAS  PubMed  Google Scholar 

  14. Slavata L, Chmelik J, Kavan D et al (2019) MS-based approaches enable the structural characterization of transcription factor/DNA response element complex. Biomol Ther 9:E535

    Google Scholar 

  15. Anbanandam A, Albarado DC, Nguyen CT et al (2006) Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain. Proc Natl Acad Sci U S A 103:17225–17230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wang L, Pan H, Smith DL (2002) Hydrogen exchange-mass spectrometry. Mol Cell Proteomics 1:132–138

    Article  CAS  PubMed  Google Scholar 

  17. Kadek A, Mrazek H, Halada P et al (2014) Aspartic protease nepenthesin-1 as a tool for digestion in hydrogen/deuterium exchange mass spectrometry. Anal Chem 86:4287–4294

    Article  CAS  PubMed  Google Scholar 

  18. Kochert BA, Iacob RE, Wales TE et al (2018) Hydrogen-deuterium exchange mass spectrometry to study protein complexes. In: Methods in molecular biology (Clifton, N.J.). Humana Press, New York, NY, pp 153–171

    Google Scholar 

  19. Rand KD, Zehl M, Jensen ON et al (2009) Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry. Anal Chem 81:5577–5584

    Article  CAS  PubMed  Google Scholar 

  20. Mistarz UH, Bellina B, Jensen PF et al (2018) UV Photodissociation mass spectrometry accurately localize sites of backbone Deuteration in peptides. Anal Chem 90:1077–1080

    Article  CAS  PubMed  Google Scholar 

  21. Mayne L, Kan ZY, Sevugan Chetty P et al (2011) Many overlapping peptides for protein hydrogen exchange experiments by the fragment separation-mass spectrometry method. J Am Soc Mass Spectrom 22:1898–1905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kan Z-Y, Walters BT, Mayne L et al (2013) Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis. Proc Natl Acad Sci U S A 110:16438–16443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cravello L, Lascoux D, Forest E (2003) Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteins. Rapid Commun Mass Spectrom 17:2387–2393

    Article  CAS  PubMed  Google Scholar 

  24. Rey M, Man P, Brandolin G et al (2009) Recombinant immobilized rhizopuspepsin as a new tool for protein digestion in hydrogen/deuterium exchange mass spectrometry. Rapid Commun Mass Spectrom 23:3431–3438

    Article  CAS  PubMed  Google Scholar 

  25. Kadek A, Tretyachenko V, Mrazek H et al (2014) Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis. Protein Expr Purif 95:121–128

    Article  CAS  PubMed  Google Scholar 

  26. Yang M, Hoeppner M, Rey M et al (2015) Recombinant Nepenthesin II for hydrogen/deuterium exchange mass spectrometry. Anal Chem 87:6681–6687

    Article  CAS  PubMed  Google Scholar 

  27. Kadek A, Kavan D, Marcoux J et al (2017) Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics. Biochim Biophys Acta Gen Subj 1861:157–167

    Article  CAS  PubMed  Google Scholar 

  28. Moroco JA, Engen JR (2015) Replication in bioanalytical studies with HDX MS: aim as high as possible. Bioanalysis 7:1065–1067

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Houde D, Berkowitz SA, Engen JR (2011) The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies. J Pharm Sci 100:2071–2086

    Article  CAS  PubMed  Google Scholar 

  30. Wales TE, Poe JA, Emert-Sedlak L et al (2016) Hydrogen exchange mass spectrometry of related proteins with divergent sequences: a comparative study of HIV-1 Nef allelic variants. J Am Soc Mass Spectrom 27:1048–1061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pascal BD, Willis S, Lauer JL et al (2012) HDXWorkbench: software for the analysis of H/D exchange MS data. J Am Soc Mass Spectrom 23:1512–1521

    Article  CAS  PubMed  Google Scholar 

  32. Guttman M, Weis DD, Engen JR et al (2013) Analysis of overlapped and noisy hydrogen/deuterium exchange mass spectra. J Am Soc Mass Spectrom 24:1906–1912

    Article  CAS  PubMed  Google Scholar 

  33. Lindner R, Lou X, Reinstein J et al (2014) Hexicon 2: automated processing of hydrogen-deuterium exchange mass spectrometry data with improved deuteration distribution estimation. J Am Soc Mass Spectrom 25:1018–1028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Rey M, Sarpe V, Burns KM et al (2014) Mass spec studio for integrative structural biology. Structure 22:1538–1548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kan ZY, Ye X, Skinner JJ et al (2019) ExMS2: an integrated solution for hydrogen-deuterium exchange mass spectrometry data analysis. Anal Chem 91:7474–7481

    Article  CAS  PubMed  Google Scholar 

  36. Claesen J, Burzykowski T (2017) Computational methods and challenges in hydrogen/deuterium exchange mass spectrometry. Mass Spectrom Rev 36:649–667

    Article  CAS  PubMed  Google Scholar 

  37. Eggertson MJ, Fadgen K, Engen JR et al (2020) Considerations in the analysis of hydrogen exchange mass spectrometry data. Methods Mol Biol 2051:407–435

    Article  CAS  PubMed  Google Scholar 

  38. Kavan D, Man P (2011) MSTools - web based application for visualization and presentation of HXMS data. Int J Mass Spectrom 302:53–58

    Article  CAS  Google Scholar 

  39. Strohalm M, Kavan D, Novak P et al (2010) mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal Chem 82:4648–4651

    Article  CAS  PubMed  Google Scholar 

  40. Majumdar R, Manikwar P, Hickey JM et al (2012) Minimizing carry-over in an online pepsin digestion system used for the H/D exchange mass spectrometric analysis of an IgG1 monoclonal antibody. J Am Soc Mass Spectrom 23:2140–2148

    Article  CAS  PubMed  Google Scholar 

  41. Rey M, Mrazek H, Pompach P et al (2010) Effective removal of nonionic detergents in protein mass spectrometry, hydrogen/deuterium exchange, and proteomics. Anal Chem 82:5107–5116

    Article  CAS  PubMed  Google Scholar 

  42. Glasoe PK, Long FA (1960) Use of glass electrodes to measure acidities in deuterium oxide 1,2. J Phys Chem 64:188–190

    Article  CAS  Google Scholar 

  43. Guttman M, Wales TE, Whittington D et al (2016) Tuning a high transmission ion guide to prevent gas-phase proton exchange during H/D exchange MS analysis. J Am Soc Mass Spectrom 27:662–668

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Czech Science Foundation projects 16-24309S and 16-20860S are gratefully acknowledged. Additional support was obtained from EU/MEYS projects BioCeV (CZ.1.05/1.1.00/02.0109) and NPU II (LQ1604). R.F. also thanks Charles University Grant Agency (project 1618218) and SVV260427/2019.

Author information

Authors and Affiliations

  1. Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

    Ruzena Filandrova, Daniel Kavan, Alan Kadek, Petr Novak & Petr Man

  2. Faculty of Sciences, Charles University, Prague, Czech Republic

    Ruzena Filandrova

  3. Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany

    Alan Kadek

Authors
  1. Ruzena Filandrova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Kavan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alan Kadek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Petr Novak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Petr Man
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Petr Man .

Editor information

Editors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Center for Integrated Biology, Integrated Structural Biology Department, Equipe labellisée Ligue Contre le Cancer, CNRS UMR 7104 - Inserm U 1258, University of Strasbourg, Illkirch, France

    Arnaud Poterszman

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Filandrova, R., Kavan, D., Kadek, A., Novak, P., Man, P. (2021). Studying Protein–DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry. In: Poterszman, A. (eds) Multiprotein Complexes. Methods in Molecular Biology, vol 2247. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1126-5_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1126-5_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1125-8

  • Online ISBN: 978-1-0716-1126-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation