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.
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References
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
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
Bai YW, Milne JS, Mayne L et al (1993) Primary structure effects on peptide group hydrogen-exchange. Proteins 17:75–86
Engen JR, Wales TE (2015) Analytical aspects of hydrogen exchange mass spectrometry. Annu Rev Anal Chem 8:127–148
Oganesyan I, Lento C, Wilson DJ (2018) Contemporary hydrogen deuterium exchange mass spectrometry. Methods 144:27–42
Sorokin VA, Gladchenko GO, Valeev VA (1986) DNA protonation at low ionic strength of solution. Die Makromol Chemie 187:1053–1063
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
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
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
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
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
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
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
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
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
Wang L, Pan H, Smith DL (2002) Hydrogen exchange-mass spectrometry. Mol Cell Proteomics 1:132–138
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
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
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
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
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
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
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
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
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
Yang M, Hoeppner M, Rey M et al (2015) Recombinant Nepenthesin II for hydrogen/deuterium exchange mass spectrometry. Anal Chem 87:6681–6687
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
Moroco JA, Engen JR (2015) Replication in bioanalytical studies with HDX MS: aim as high as possible. Bioanalysis 7:1065–1067
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
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
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
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
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
Rey M, Sarpe V, Burns KM et al (2014) Mass spec studio for integrative structural biology. Structure 22:1538–1548
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
Claesen J, Burzykowski T (2017) Computational methods and challenges in hydrogen/deuterium exchange mass spectrometry. Mass Spectrom Rev 36:649–667
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
Kavan D, Man P (2011) MSTools - web based application for visualization and presentation of HXMS data. Int J Mass Spectrom 302:53–58
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
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
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
Glasoe PK, Long FA (1960) Use of glass electrodes to measure acidities in deuterium oxide 1,2. J Phys Chem 64:188–190
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
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.
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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
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DOI: https://doi.org/10.1007/978-1-0716-1126-5_11
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