This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Fonds de Recherche du Quebec Nature et Technologie, the Canadian Foundation for Innovation, and McGill University start-up funds.

1. Preparation of deuterated reagents and enzyme stock solutions
<ol>
	<li>Prepare reagents needed for the HDX reactions (including any buffers, salts, substrates, ligands, etc.) as 100&#8722;200x concentrated stock solutions in D2O (99.9% atom fraction D). Prepare at least 50 mL of buffer stock solution. 
	NOTE: For characterization of HalM2, the following solutions were prepared: 500 mM MgCl2, 100 mM tris(2-carboxyethyl)phosphine (TCEP), 750 mM ATP (in HEPES buffer), 800 mM HEPES pD 7.1, 50...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+dynamism+and+evolvability.">Protein dynamism and evolvability.</a> Science. 324 (5924), 203-207 (2009).</li><li>Wales, T. E., Engen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+mass+spectrometry+for+the+analysis+of+protein+dynamics.">Hydrogen exchange mass spectrometry for the analysis of protein dynamics.</a> Mass Spectrometry Reviews. 25 (1), 158-170 (2006).</li><li>Konermann, L., Pan, J., Liu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+mass+spectrometry+for+studying+protein+structure+and+dynamics.">Hydrogen exchange mass spectrometry for studying protein structure and dynamics.</a> Chemical Society Reviews. 40 (3), 1224 (2011).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 5 (4), 214 (1991).</li><li>Zhang, Z., Smith, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+effects+on+peptide+group+hydrogen+exchange.">Primary structure effects on peptide group hydrogen exchange.</a> Proteins. 17 (1), 75-86 (1993).</li><li>Xiao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+architecture+of+protein+complexes+by+an+orthogonal+approach+combining+HDXMS,+CXMS,+and+disulfide+trapping.">Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping.</a> Nature Protocols. 13 (6), 1403-1428 (2018).</li><li>Smith, D. L., Deng, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+non-covalent+structure+of+proteins+by+amide+hydrogen+exchange+and+mass+spectrometry.">Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry.</a> Journal of Mass Spectrometry. 32 (2), 135-146 (1997).</li><li>Masson, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+performing,+interpreting+and+reporting+hydrogen+deuterium+exchange+mass+spectrometry+(HDX-MS)+experiments.">Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.</a> Nature Methods. 16 (7), 595-602 (2019).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Efficient+Site-Specific+Method+for+Irreversible+Covalent+Labeling+of+Proteins+with+a+Fluorophore.">An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.</a> Scientific Reports. 5, 16883 (2015).</li><li>Marion, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+overlap+problem+in+the+assignment+of+proton+NMR+spectra+of+larger+proteins+by+use+of+three-dimensional+heteronuclear+proton-nitrogen-15+Hartmann-Hahn-multiple+quantum+coherence+and+nuclear+Overhauser-multiple+quantum+coherence+spectroscopy:+application+to+interleukin+1.beta.">Overcoming the overlap problem in the assignment of proton NMR spectra of larger proteins by use of three-dimensional heteronuclear proton-nitrogen-15 Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1.beta.</a> Biochemistry. 28 (15), 6150-6156 (1989).</li><li>Balasubramaniam, D., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen-exchange+mass+spectrometry+for+the+study+of+intrinsic+disorder+in+proteins.">Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins.</a> Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1834 (6), 1202-1209 (2013).</li><li>Goswami, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+window+expansion+for+HDX+analysis+of+an+intrinsically+disordered+protein.">Time window expansion for HDX analysis of an intrinsically disordered protein.</a> Journal of the American Society for Mass Spectrometry. 24 (10), 1584-1592 (2013).</li><li>Keppel, T. R., Howard, B. A., Weis, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+Unstructured+Regions+and+Synergistic+Folding+in+Intrinsically+Disordered+Proteins+with+Amide+H/D+Exchange+Mass+Spectrometry.">Mapping Unstructured Regions and Synergistic Folding in Intrinsically Disordered Proteins with Amide H/D Exchange Mass Spectrometry.</a> Biochemistry. 50 (40), 8722-8732 (2011).</li><li>Mitchell, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Characterization+and+Conformational+Analysis+of+the+Herpesvirus+saimiri+Tip-C484+Protein.">Functional Characterization and Conformational Analysis of the Herpesvirus saimiri Tip-C484 Protein.</a> Journal of Molecular Biology. 366 (4), 1282-1293 (2007).</li><li>Pan, J., Han, J., Borchers, C. H., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen/deuterium+exchange+mass+spectrometry+with+top-down+electron+capture+dissociation+for+characterizing+structural+transitions+of+a+17+kDa+protein.">Hydrogen/deuterium exchange mass spectrometry with top-down electron capture dissociation for characterizing structural transitions of a 17 kDa protein.</a> Journal of the American Chemical Society. 131 (35), 12801-12808 (2009).</li><li>Habibi, Y., Uggowitzer, K. A., Issak, H., Thibodeaux, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+Dynamic+Structural+Properties+of+a+Lanthipeptide+Synthetase+using+Hydrogen-Deuterium+Exchange+Mass+Spectrometry.">Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen-Deuterium Exchange Mass Spectrometry.</a> Journal of the American Chemical Society. 141 (37), 14661-14672 (2019).</li><li>Hebling, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+analysis+of+membrane+proteins+in+phospholipid+bilayer+nanodiscs+by+hydrogen+exchange+mass+spectrometry.">Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.</a> Analytical Chemistry. 82 (13), 5415-5419 (2010).</li><li>Duc, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+application+of+bicelles+for+conformational+analysis+of+G+protein-coupled+receptors+by+hydrogen/deuterium+exchange+mass+spectrometry.">Effective application of bicelles for conformational analysis of G protein-coupled receptors by hydrogen/deuterium exchange mass spectrometry.</a> Journal of the American Society for Mass Spectrometry. 26 (5), 808-817 (2015).</li><li>Canul-Tec, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+allosteric+inhibition+of+excitatory+amino+acid+transporter+1.">Structure and allosteric inhibition of excitatory amino acid transporter 1.</a> Nature. 544 (7651), 446-451 (2017).</li><li>Reading, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+Membrane+Protein+Conformational+Dynamics+within+Native+Lipid+Compositions.">Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions.</a> Angewandte Chemie International Edition. 56 (49), 15654-15657 (2017).</li><li>Hentze, N., Mayer, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+dynamics+using+hydrogen+exchange+mass+spectrometry.">Analyzing protein dynamics using hydrogen exchange mass spectrometry.</a> Journal of Visualized Experiments. (81), e50839 (2013).</li><li>Lento, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+ElectroSpray+Ionization+Hydrogen-deuterium+Exchange+Mass+Spectrometry+for+Studying+Protein+Structure+and+Dynamics.">Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics.</a> Journal of Visualized Experiments. (122), e55464 (2017).</li><li>Schowen, K. B., Schowen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+isotope+effects+on+enzyme+systems.">Solvent isotope effects on enzyme systems.</a> Methods in Enzymology. 87, 551-606 (1982).</li><li>Lau, A. M. C., Ahdash, Z., Martens, C., Politis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deuteros:+software+for+rapid+analysis+and+visualization+of+data+from+differential+hydrogen+deuterium+exchange-mass+spectrometry.">Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry.</a> Bioinformatics. 35 (7), 3171-3173 (2019).</li><li>Houde, D., Berkowitz, S. A., Engen, J. 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We have nothing to disclose.

The HDX-MS workflow presented in this protocol provides a remarkably robust platform for mapping the spatial distribution of structurally dynamic elements in proteins and for investigating how these dynamics change in response to perturbation (ligand binding, enzyme mutagenesis, etc.). HDX-MS holds several distinct advantages over other structural biology approaches that are commonly used to investigate conformational dynamics. Most notably, only small quantities of protein are needed. Using the workflow described herein...

Proteins are structurally dynamic molecules that sample different conformations on time scales ranging from femtosecond-scale bond vibration to rearrangements of entire protein domains which can occur over many seconds1. These conformational fluctuations are often critical aspects of enzyme/protein function. For example, conformational changes induced by ligand binding are often critically important for modulating enzyme function, either by organizing active site residues needed for catalysis, defining substrate binding sites in sequential kinetic mechanisms, shielding reactive intermediates from the environment, or by modulating enzyme function via allosteric networks. Recent studies have also shown that conformational dynamics can be conserved throughout evolution and that perturbations to conserved molecular motions can be correlated with changes in substrate specificity and the emergence of new enzyme functions2,3.
In recent years, hydrogen-deuterium exchange mass spectrometry (HDX-MS) has rapidly emerged as a powerful technique to probe how protein conformational landscapes respond to perturbations such as ligand binding or mutagenesis4,5,6,7. In a typical HDX-MS experiment (Figure 1), a protein of interest is placed into buffer prepared in D2O, which triggers replacement of solvent-exchangeable protons with deuteria. The rate of exchange of the amide moiety of the peptide bonds depends strongly on the pH, the local amino acid sequence, and on the local structural environment of the amide8. Amides that are engaged in hydrogen bonding interactions (such as those present in &#945;-helices and &#946;-sheets) exchange more slowly than amides in unstructured regions of the protein that are exposed to bulk solvent. Thus, the extent of deuterium uptake is a reflection of the structure of the enzyme. Enzymes that are conformationally dynamic, or that undergo structural transitions upon ligand binding, would be expected to yield a measurable HDX response.
The mechanistic basis for the slow exchange rate of a structured amide is shown in Figure 25,8,9. In order to undergo HDX, the structured region must first transiently sample an unfolded conformation, such that the solvent molecules that catalyze HDX exchange via a specific acid/base chemical mechanism, have access to the exchangeable amide. Ultimately, the relative magnitudes of the chemical exchange rate (kchem) and the folding and refolding rates (kopen and kclose) determine the HDX rate measured in the experiment5,8. From this simple kinetic model, it is clear that extent of deuterium uptake will reflect the underlying conformational dynamics (as defined by kopen and kclose). Most HDX-MS experiments are performed in a bottom-up workflow where, following the exchange reaction, the protein of interest is digested into peptides and the deuterium uptake by each peptide is measured as an increase in mass7. In this way, HDX-MS allows perturbations to enzyme conformational dynamics to be mapped on the local spatial scale of peptides, allowing the researcher to assess how the perturbation alters dynamics in different regions of the enzyme of interest.
The advantages of the HDX-MS approach for elucidating protein structural dynamics are numerous. First, the method can be performed with small quantities of native protein or on protein complexes in systems with quaternary structure10. It is not even necessary for the enzyme preparation used in the assay to be highly purified11,12, as long as the bottom-up HDX-MS workflow provides a sufficient number of confidently identified peptides that cover the protein sequence of interest. Moreover, HDX-MS can provide information on conformational dynamics under near native conditions without the need for site-specific protein labeling as would be used in single molecule fluorescence studies13, and there is no size limit to the protein or protein complex that can be investigated (which makes approaches such as nuclear magnetic resonance [NMR] spectroscopy challenging)7,14. Finally, time-resolved HDX-MS methods can be employed to study intrinsically disordered proteins, which are difficult to study with X-ray crystallography15,16,17,18. The main limitation of HDX-MS is that the data is of low structural resolution. HDX-MS data are useful for pointing to where conformational dynamics are changing and for revealing coupled conformational changes, but they do not often provide much insight into the precise molecular mechanism driving the observed change. Recent advances in the combination of electron capture dissociation methods with protein HDX-MS data have shown promise for mapping exchange sites to single amino acid residues19, but follow up biochemical and structural studies are still often needed to provide clarity to structural models forwarded by HDX-MS data.
Below, a detailed protocol for the development of an HDX-MS assay is presented20. The sample preparation protocols presented below should be generally applicable to any protein that exhibits good solubility in aqueous buffers. More specialized sample preparation methods and HDX-MS workflows are available for proteins than need to be assayed in the presence of detergent or phospholipids21,22,23,24. Instrumental settings for HDX-MS data collection are described for a high-resolution quadrupole time-of-flight mass spectrometer coupled to liquid chromatography system. Data of similar complexity and resolution could be collected on any one of a number of commercially available liquid chromatography-mass spectrometry (LC-MS) systems. Key aspects of the data processing using a commercially available software package are also provided. We also present guidelines for data collection and analysis that are consistent with recommendations made by the broader HDX-MS community12. The described protocol is used to study the dynamic structural properties of HalM2, a lanthipeptide synthetase that catalyzes the multistep maturation of an antimicrobial peptide natural product20. We illustrate how HDX-MS can be used to reveal substrate binding sites and allosteric properties that have eluded previous characterization. Several other protocols on protein HDX-MS have been published in recent years25,26. Together with the present work, these earlier contributions should provide the reader some flexibility in experimental design.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[glu-1]-fibrinopeptide B (Glu-Fib)</td>
			<td>BioBasic</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Amicon Ultracel 10k centrifugal filtration device (Millipore)</td>
			<td>Milipore Sigma</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Fisher</td>
			<td>A955-1</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP-PNP</td>
			<td>SIGMA</td>
			<td>A2647-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>SIGMA</td>
			<td>a2383-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O</td>
			<td>ALDRICH</td>
			<td>435767-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Thermo Fisher</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidine-HCl</td>
			<td>VWR</td>
			<td>97063-764</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>SiGMA-Aldrich</td>
			<td>63068-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>BioBasic</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>BioBasic</td>
			<td>PB0445</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP Hydrochloride</td>
			<td>TRC Canada</td>
			<td>T012500</td>
			<td>peptide was synthesized upon request</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deuteros</td>
			<td>Andy M C Lau, et al</td>
			<td>version 1.08</td>
			<td></td>
		</tr>
		<tr>
			<td>DynamX</td>
			<td>Waters</td>
			<td>version 3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MassLynx</td>
			<td>Waters</td>
			<td>version 4.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Lynx 
			Global Server (PLGS)</td>
			<td>Waters</td>
			<td>version 3.0.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>Schr&#246;dinger</td>
			<td>version 2.2.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Instrument and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C18 analytical Column</td>
			<td>Waters</td>
			<td>186002346</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C8 VanGuard Pre-column</td>
			<td>Waters</td>
			<td>186003978</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC M-Class HDX System</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDX Manager</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microtip pH electrode</td>
			<td>Thermo Fisher</td>
			<td>13-620-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Enzymate BEH column or Pepsin solumn</td>
			<td>Waters</td>
			<td>186007233</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Synapt G2-Si</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogen-deuterium exchange mass spectrometry (hdx-ms) platform for investigating peptide biosynthetic enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

It is necessary to assess the quality of the proteolytic digestion and the reproducibility of the workflow for each set of sample injections. Thus, prior to performing HDX-MS assays, it is essential to establish effective conditions for the proteolysis of the protein of interest, for the separation of peptides using reverse phase liquid chromatography and gas phase ion mobility, and for the detection of peptides using MS. For this purpose, the reference samples for the protein of interest (collected in the absence of deu...

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A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

This protocol can provide specially resolved information about protein conformational dynamics. Which can help to identify functionally important regions in protein of interest. This technique probes protein conformational dynamic under near-native conditions.Without the need for protein labeling using nanomolar amount of material for each reaction. Begin by preparing undeuterated reference samples for the protein of interest in triplicate, in 500 microliter tubes. Quench the samples with the appropriate volume of quench buffer per tube to achieve a pH meter reading of 2.5.Flash freeze the samples in liquid nitrogen for storage at minus 80 degrees Celsius until their analysis. Then analyze the protiated enzyme reference samples using a bottom-up liquid chromatography mass spectrometry workflow according to standard protocols. To analyze the raw mass spectrometry data in the appropriate Proteomic Software Program.Navigate to Libraries and Protein Sequence Databanks to import the amino acid sequence of the protein of interest. Enter a name for the protein sequence of interest and import the protein sequence in FASTA format. To define the processing parameters under the Library menu, select Electrospray MSE as the data acquisition type.In the Lock Mass for Charge 2 field, enter 785.8426 for the master charge ratio for the two plus ion of glucan one fiber no peptide B and click Finish. To define the workflow parameters, select Electrospray MSE for the search type. Under the Workflow and Database search query heading, select the database protein created in the Databank field and change the primary digest reagent to nonspecific.To clear the fixed modifier reagent field, hold the control button while clicking Carbamidomethyl C.To specify the output directory, select Options, Automation Setup, and Identify E.Then check the apex 3D and peptide 3D output and ion accounting output boxes. And specify the desired directory. To process the referenced sample data, right-click on the microtiter plate to create a new plate.Left-click in one well and drag to three other wells to highlight one well for each referenced sample. Next, right-click and select Add Raw Data. In the window, navigate to the directory containing the three referenced files and select all three files at the same time.Click Next and select the defined processing parameters. Click Next again and select the defined workflow parameters. Then click Finish.Once the raw data, processing parameters, and workflow parameters have been assigned to each well on the plate, the wells will turn blue. Select the wells, right-click and select Process Latest Raw Data. To track the processing of the data, click the right bottom corner of the window.When no job to run appears, the processing is complete and the wells in the microtiter plate will turn green. Right-click on the wells and select View Workflow Results. A separate window will open for each referenced data file.Inspect the data to ensure that the majority of the mass spectrometry signals in the referenced sample data were successfully mapped to the peptides predicted from the in silico digestion of the protein of interest. Matched peptides will appear blue in the output spectrum. Double-click the okay filter and check that the percent coverage is greater than 99%Import the Proteomic Software output into the Hydrogen Deuterium Exchange Processing Software for additional thresholding.And click Data and import PLGS results. Click the Add icon and navigate to the appropriate directory to select the processed data files. Click Next and set the minimum consecutive ions to greater than or equal to two.The mass error to five parts per million. And the file threshold to three, then click Finish. To conduct hydrogen deuterium exchange reactions, aliquot quench buffer in 500 microliter tubes.And briefly centrifuge to transfer all of the quench buffer to the bottom of each tube. Place the tubes containing quenched solution on ice. Next, premix all of the deuterated reaction components in a separate 1.5 milliliter tube.Then transfer the reaction mixture to a 25 degrees Celsius temperature controlled waterbath for a 10-minute incubation. Add enzyme to the deuterated reaction components to initiate the exchange reaction. Mix the reaction contents thoroughly by pipetting up and down.At the appropriate experimental exchange time points remove 50 microliter aliquots from each hydrogen deuterium exchange reaction. And mix the reactions quickly and evenly with one aliquot of ice-cold quench buffer in individual 500 microliter tubes. Then immediately cap the tubes for flash-freezing in liquid nitrogen.Store the samples at minus 80 degrees Celsius until ready for analysis. After thawing the frozen samples, analyze the deuterated enzyme reference samples using the same bottom-up liquid chromatography mass spec workflow developed for the undeuterated reference samples. After collecting the LCMS data for the deuterated samples, import the data into the previously created hydrogen deuterium exchange project for data processing.And click New State and New Exposure to define the biochemical states and deuterium exposure times respectively pertinent to the analysis. Click New Raw to select the hydrogen deuterium exchange data files to be analyzed. And assign the appropriate exchange times and biochemical state to each raw data file that is imported.For analysis and visualization of the data, select the first peptide in the Peptide list and open the stacked spectral plot from the Views menu. Click the mouse to assign and unassign sticks as necessary to ensure that the proper isotope distribution has been located in the data. And that each isotope peak has been assigned.To check the stick assignments for each charge state toggle the charge state at the top of the stacked spectral plot window. To check the standard deviation of the peptide deuterium uptake values in the Views menu, select the coverage map. Which displays each peptide in the Peptide list mapped along the amino acid sequence of the protein of interest.Color the peptides according to relative standard deviation and visually search the map for outlier peptides with the high relative standard deviation. Click the outlier peptides in the coverage map to populate the stacked spectral plots and the data viewer window with the target peptide. Display the difference of interest in the coverage map and right-click on the map to export the difference data to a CSV file.Import the difference, and state data, and the PDB file of the protein of interest into Deuteros. And select the 99%confidence interval and Enable Sum and process the data. Then under pi mol select Export Uptake and Export to generate a PyMOL script to map the regions of significant exchange difference onto the PDB structure of the protein of interest in the PyMOL software.These representative total ion chromatograms for three reference samples of Hal-M2 lambda peptide synthetase show the time-dependent changes in the sum of all of the ion counts at all of the mass-to-charge ratio values included in the mass spectral scan. The shape and intensity of the total ion chromatogram profile should be similar. Indicating that the proteolytic digestion of Hal-M2 is reproducible.And is of similar efficiency in all three reference samples. The mass spectra over a given time interval should also be similar. Providing confidence that similar peptides are present within each sample and are alluding at similar times from the C18 column.Most of the peptides in the deuterium exchanged samples should exhibit obvious shifts in there isotopic distributions toward higher mass-to-charge ratio values. For example, these data indicate that a substantial fraction of the deuterium label was being maintained throughout the course of the acid quench, pepsin digestion, and liquid chromatography mass spectrometry data collection. After characterization of the biochemical properties of the enzyme as demonstrated, representative hydrogen deuterium exchange mass spectrometry results such as these can be generated.To illustrate the binding of the Hal-A2 precursor peptide to the Hal-M2 AMP, PMP complex. The hydrogen deuterium exchange reactions must be prepared and quenched in a consistent manner. And you must be methodical when determining the deuterium exchange values for numerous peptide detected.Hydrogen deuterium exchange mass spectrometry provide peptide-level information and dynamic protein conformational changes. The functional relevance of any hydrogen deuterium exchange should be validated for Meter Genesis. And orthogonal activity assays.

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7.7K vistas.  McGill University. This protocol can provide specially resolved information about protein conformational dynamics. Which can help to identify functionally important regions in protein of interest. This technique probes protein conformational dynamic under near-native conditions.Without the need for protein labeling using nanomolar amount of material for each reaction. Begin by preparing undeuterated reference samples for the protein of interest in triplicate, in 500 microliter tubes. Quench the samples w...