This work was supported by grants from the National Natural Science Foundation of China (NSFC 21974069). The authors also received support from the Institute for Cell Analysis, Shenzhen Bay Laboratory, China; Jiangsu Collaborative Innovation Center of Biomedical Functional Materials; and Jiangsu Key Laboratory of Biomedical Materials at Nanjing Normal University, China.

<ol><li>Kaltashov, I. A., Bobst, C. E., Pawlowski, J., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry-based+methods+in+characterization+of+the+higher+order+structure+of+protein+therapeutics%5BTitle%5D)+AND+%22Journal+of+Pharmaceutical+and+Biomedical+Analysis%22%5BJournal%5D)">Mass spectrometry-based methods in characterization of the higher order structure of protein therapeutics.</a> Journal of Pharmaceutical and Biomedical Analysis. 184, 113169(2020).</li>
<li>Engen, J. R., Botzanowski, T., Peterle, D., Georgescauld, F., Wales, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developments+in+hydrogen%2Fdeuterium+exchange+mass+spectrometry%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Developments in hydrogen/deuterium exchange mass spectrometry.</a> Analytical Chemistry. 93 (1), 567-582 (2021).</li>
<li>Pan, J., Han, J., Borchers, C. H., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conformer-specific+hydrogen+exchange+analysis+of+Abeta%281-42%29+oligomers+by+top-down+electron+capture+dissociation+mass+spectrometry%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Conformer-specific hydrogen exchange analysis of Abeta(1-42) oligomers by top-down electron capture dissociation mass spectrometry.</a> Analytical Chemistry. 83 (13), 5386-5393 (2011).</li>
<li>Pan, J., Han, J., Borchers, C. H., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+dynamics+of+small+soluble+Abeta%281-40%29+oligomers+studied+by+top-down+hydrogen+exchange+mass+spectrometry%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Structure and dynamics of small soluble Abeta(1-40) oligomers studied by top-down hydrogen exchange mass spectrometry.</a> Biochemistry. 51 (17), 3694-3703 (2012).</li>
<li>Pan, J., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Top-down+structural+analysis+of+posttranslationally+modified+proteins+by+Fourier+transform+ion+cyclotron+resonance-MS+with+hydrogen%2Fdeuterium+exchange+and+electron+capture+dissociation%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Top-down structural analysis of posttranslationally modified proteins by Fourier transform ion cyclotron resonance-MS with hydrogen/deuterium exchange and electron capture dissociation.</a> Proteomics. 13 (6), 974-981 (2013).</li>
<li>Wang, G., Abzalimov, R. R., Bobst, C. E., Kaltashov, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conformer-specific+characterization+of+nonnative+protein+states+using+hydrogen+exchange+and+top-down+mass+spectrometry%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United.+States+of+America%22%5BJournal%5D)">Conformer-specific characterization of nonnative protein states using hydrogen exchange and top-down mass spectrometry.</a> Proceedings of the National Academy of Sciences of the United. States of America. 110 (50), 20087-20092 (2013).</li>
<li>Mironov, G. G., Clouthier, C. M., Akbar, A., Keillor, J. W., Berezovski, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+analysis+of+enzyme+structure+and+activity+by+kinetic+capillary+electrophoresis-MS%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS.</a> Nature Chemical Biology. 12 (11), 918-922 (2016).</li>
<li>Shen, Y., Zhao, X., Wang, G., Chen, D. D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+hydrogen%2Fdeuterium+exchange+during+proteoform+separation+enables+characterization+of+conformational+differences+between+coexisting+protein+states%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Differential hydrogen/deuterium exchange during proteoform separation enables characterization of conformational differences between coexisting protein states.</a> Analytical Chemistry. 91 (6), 3805-3809 (2019).</li>
<li>Kontopidis, G., Holt, C., Sawyer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invited+review%3A+%CE%B2-lactoglobulin%3A+binding+properties%2C+structure%2C+and+function%5BTitle%5D)+AND+%22Journal+of+Dairy+Science%22%5BJournal%5D)">Invited review: β-lactoglobulin: binding properties, structure, and function.</a> Journal of Dairy Science. 87 (4), 785-796 (2004).</li>
<li>Qin, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+basis+of+the+Tanford+transition+of+bovine+%CE%B2-lactoglobulin%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Structural basis of the Tanford transition of bovine β-lactoglobulin.</a> Biochemistry. 37 (40), 14014-14023 (1998).</li>
<li>Qin, B. Y., Bewley, M. C., Creamer, L. K., Baker, E. N., Jameson, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+implications+of+structural+differences+between+variants+A+and+B+of+bovine+beta-lactoglobulin%5BTitle%5D)+AND+%22Protein+Science%22%5BJournal%5D)">Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin.</a> Protein Science. 8 (1), 75-83 (1999).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+resolution+capillary+isoelectric+focusing+mass+spectrometry+analysis+of+peptides%2C+proteins%2C+and+monoclonal+antibodies+with+a+flow-through+microvial+interface%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">High resolution capillary isoelectric focusing mass spectrometry analysis of peptides, proteins, and monoclonal antibodies with a flow-through microvial interface.</a> Analytical Chemistry. 90 (15), 9495-9503 (2018).</li>
<li>Busch, M. H. A., Kraak, J. C., Poppe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellulose+acetate-coated+fused-silica+capillaries+for+the+separation+of+proteins+by+capillary+zone+electrophoresis%5BTitle%5D)+AND+%22Journal+of+Chromatography+A%22%5BJournal%5D)">Cellulose acetate-coated fused-silica capillaries for the separation of proteins by capillary zone electrophoresis.</a> Journal of Chromatography A. 1695 (2), 287-296 (1995).</li>
<li>Zhao, X., Shen, Y., Tong, W., Wang, G., Chen, D. D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deducing+disulfide+patterns+of+cysteine-rich+proteins+using+signature+fragments+produced+by+top-down+mass+spectrometry%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">Deducing disulfide patterns of cysteine-rich proteins using signature fragments produced by top-down mass spectrometry.</a> Analyst. 143 (4), 817-823 (2018).</li>
<li>Sutera, S. P., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+history+of+Poiseuille%27s+law%5BTitle%5D)+AND+%22Annual+Review+of+Fluid+Mechanics%22%5BJournal%5D)">The history of Poiseuille's law.</a> Annual Review of Fluid Mechanics. 25 (1), 1-20 (1993).</li>
<li>Wang, G., Kaltashov, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Approach+to+characterization+of+the+higher+order+structure+of+disulfide-containing+proteins+using+hydrogen%2Fdeuterium+exchange+and+top-down+mass+spectrometry%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry.</a> Analytical Chemistry. 86 (15), 7293-7298 (2014).</li>
<li>Wang, G., Johnson, A. J., Kaltashov, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+electrospray+ionization+mass+spectrometry+as+a+tool+for+characterization+of+small+soluble+protein+aggregates%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Evaluation of electrospray ionization mass spectrometry as a tool for characterization of small soluble protein aggregates.</a> Analytical Chemistry. 84 (3), 1718-1724 (2012).</li>
<li>Fellers, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ProSight+Lite%3A+graphical+software+to+analyze+top-down+mass+spectrometry+data%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">ProSight Lite: graphical software to analyze top-down mass spectrometry data.</a> Proteomics. 15 (7), 1235-1238 (2015).</li>
<li>Cai, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MASH+Suite+Pro%3A+A+comprehensive+software+tool+for+top-down+proteomics%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Proteomics%22%5BJournal%5D)">MASH Suite Pro: A comprehensive software tool for top-down proteomics.</a> Molecular & Cellular Proteomics. 15 (2), 703-714 (2016).</li>
<li>Paterson, G. R., Hill, J. P., Otter, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+%CE%B2-lactoglobulin+A%2C+B+and+C+variants+of+bovine+whey+using+capillary+zone+electrophoresis%5BTitle%5D)+AND+%22Journal+of+Chromatography+A%22%5BJournal%5D)">Separation of β-lactoglobulin A, B and C variants of bovine whey using capillary zone electrophoresis.</a> Journal of Chromatography A. 700 (1), 105-110 (1995).</li>
<li>Wang, G., Kaltashov, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Approach+to+characterization+of+the+higher+order+structure+of+disulfide-containing+proteins+using+hydrogen%2Fdeuterium+exchange+and+top-down+mass+spectrometry%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry.</a> Analytical Chemistry. 86 (15), 7293-7298 (2014).</li>
<li>Nicolardi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On-line+electrochemical+reduction+of+disulfide+bonds%3A+improved+FTICR-CID+and+-ETD+coverage+of+oxytocin+and+hepcidin%5BTitle%5D)+AND+%22Journal+of+the+American+Society+for+Mass+Spectrometry%22%5BJournal%5D)">On-line electrochemical reduction of disulfide bonds: improved FTICR-CID and -ETD coverage of oxytocin and hepcidin.</a> Journal of the American Society for Mass Spectrometry. 24 (12), 1980-1987 (2013).</li>
<li>Adhikari, S., Xia, Y., McLuckey, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Top-down+analysis+of+disulfide-linked+proteins+using+photoinduced+radical+reactions+and+ET-DDC%5BTitle%5D)+AND+%22International+Journal+of+Mass+Spectrometry%22%5BJournal%5D)">Top-down analysis of disulfide-linked proteins using photoinduced radical reactions and ET-DDC.</a> International Journal of Mass Spectrometry. 444, 116173(2019).</li>
<li>Rush, M. J. P., Riley, N. M., Westphall, M. S., Coon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Top-down+characterization+of+proteins+with+intact+disulfide+bonds+using+activated-ion+electron+transfer+dissociation%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Top-down characterization of proteins with intact disulfide bonds using activated-ion electron transfer dissociation.</a> Analytical Chemistry. 90 (15), 8946-8953 (2018).</li>
<li>Zhong, X., Maxwell, E. J., Chen, D. D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+transport+in+a+micro+flow-through+vial+of+a+junction-at-the-tip+capillary+electrophoresis-mass+spectrometry+interface%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Mass transport in a micro flow-through vial of a junction-at-the-tip capillary electrophoresis-mass spectrometry interface.</a> Analytical Chemistry. 83 (12), 4916-4923 (2011).</li>
</ol>

D. D. Y. Chen is one of the founders of the Knowledge for Health Institute for Biomolecules, which is commercializing the flow-through microvial CE-MS interface. Other authors have nothing to disclose.

The objectives of coating the inner wall of the CE capillary include the minimization of the electroosmotic flow and protein absorption during the CE process13. Although electroosmotic flow is beneficial for conventional CE analysis of small molecules owing to its capability of driving neutral or oppositely charged species to the detector, it compromises the separation efficiency of protein species with similar sizes and net charges in solution. Coating the capillary with HPC minimizes the electro...

Distinguishing protein species in different conformational, binding, or modification states and characterizing their structural differences are important for monitoring the pathways of transitions between these species involved in biological events, ranging from molecular recognition to enzymatic catalysis, and understanding the mechanisms underlying these events. Conventional biophysical techniques do not provide a complete solution due to the limitations such as insufficient resolution and loss of dynamic information in solution. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) is a technique that labels the structural and conformational features of proteins with deuterium (2H) via the exchange between labile hydrogen atoms of proteins and 2H from the deliberately introduced 2H2O solution. Protons involved in hydrogen bonding or that are sequestered from the solvent in the protein interior do not exchange readily1. Thus, as the exchange rate at an exchangeable site is highly dependent on its involvement in higher-order structures, the protein structures can be revealed at high spatial resolution by MS that probes the extent and rate of 2H-uptake based on the differing atomic masses between 1H and 2H. Over the recent decades, HDX MS has become an outstandingly successful technique for studying protein conformations and dynamics2.
In the classical bottom-up approach of HDX MS, the ensemble of protein species in different conformational, binding, or modification states is proteolyzed without separation at the intact protein level, making it infeasible to characterize individual species by analyzing the resulting proteolytic fragments with convoluted deuterium contents. In contrast, in the top-down approach, different protein states or proteoforms that have incorporated different deuterium contents give rise to multiple distributions of intact protein masses in an MS scan. This allows individual species to be separated by mass-selection of ions corresponding to each mass distribution using a proper mass filter (such as a quadrupole) and the characterization of their conformational differences in the subsequent tandem MS analysis3,4,5,6. However, the efficiency of separating protein states or proteoforms in this strategy is limited by the extent of difference in their corresponding mass distributions.
Capillary electrophoresis (CE) provides a means to separate protein species based on their differing charges and hydrodynamic sizes in the solution phase with high efficiency7. Combining CE with HDX offers additional separation of protein states or proteoforms in the solution phase. In addition, the small volume of the CE capillary allows the utilization of a fully deuterated solution as the background electrolyte solution (BGE), i.e., the running buffer, rendering the capillary as an HDX reactor for protein samples. Due to the difference in electrophoretic mobility between proteins and protic reagents in the electrophoresis process, conducting HDX during CE results in a nearly complete deuterating environment for the protein analytes with minimal residual non-deuterated contents, thereby enhancing the sensitivity of the structural analysis using HDX data. As such, we developed a CE-based differential HDX approach coupled with top-down MS to characterize protein higher-order structures in a state- or proteoform-specific manner8.
This paper describes protocols for this approach by detailing the steps of material preparation, experimental procedure, and data analysis. Factors that may affect the method performance or data quality are listed in short notes. The representative results presented here include differential HDX data of mixtures of different proteins and natural variants of bovine &#946;-lactoglobulin (&#946;-lg), the major whey protein present in milk9. We demonstrate separation efficiency, reproducibility, and 2H-labeling performance of the two abundant variants of &#946;-lg, i.e., A and B10,11 during the CE-based HDX and variant-specific characterization of their conformations.

<table><tbody><tr><td>ammonium acetate</td><td>Fisher Chemical</td><td>A/3446/50</td><td>&amp;ge;99%</td></tr><tr><td>CESI 8000 plus capillary electrophoresis system</td><td>Sciex, USA</td><td></td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5406000097</td><td></td></tr><tr><td>centrifugal filter</td><td>Merck</td><td>UFC201024</td><td>10 kDa cutoff</td></tr><tr><td>deuterium oxide</td><td>Energy Chemical</td><td>E090001</td><td>99.9 % D</td></tr><tr><td>formic acid</td><td>Acros Organics</td><td>&amp;nbsp;270480250</td><td></td></tr><tr><td>fused silica glass capillary</td><td>Polymicro Technologies</td><td>1068150017</td><td>ID 50&amp;mu;m, OD 360&amp;mu;m</td></tr><tr><td>gas chromatography</td><td>Agilent</td><td>GC6890N</td><td></td></tr><tr><td>hydrochloric acid</td><td>Sigma Aldrich</td><td>258148</td><td></td></tr><tr><td>hydroxypropyl cellulose</td><td>Aladdin</td><td>H113415</td><td>MW 100000</td></tr><tr><td>magnetic stirrers</td><td>DLAB</td><td>8030101212</td><td></td></tr><tr><td>methanol</td><td>Fisher Chemical</td><td>A456-4</td><td>MS grade</td></tr><tr><td>microvolume UV-Vis spectrophotometer</td><td>DeNovix</td><td>84677JK7731</td><td></td></tr><tr><td>myoglobin</td><td>Sigma Aldrich</td><td>M1882</td><td></td></tr><tr><td>Orbitrap Fusion Lumos mass spectrometer</td><td>Thermo Fisher Scientific, USA</td><td></td><td></td></tr><tr><td>PA 800 Plus Pharmaceutical Analysis CE System</td><td>Beckman Coulter, USA</td><td></td><td></td></tr><tr><td>Q Exactive UHMR mass Spectrometer</td><td>Thermo Fisher Scientific, Germany</td><td></td><td></td></tr><tr><td>sodium&amp;nbsp;hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td></td></tr><tr><td>ubiquitin</td><td>Sigma Aldrich</td><td>U6253</td><td></td></tr><tr><td>ultrasonicator</td><td>SCIENTZ</td><td>SB-5200</td><td></td></tr><tr><td>&amp;beta;-lactoglobulin</td><td>Sigma Aldrich</td><td>L0130</td><td></td></tr></tbody></table>

capillary electrophoresis-based hydrogen/deuterium exchange for conformational characterization of proteins with top-down mass spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Changing the infusion pressure of BGE allows the adjustment of both separation efficiency and migration time, which is equivalent to the HDX reaction time of the proteins to be separated (Figure 3). A lower infusion pressure results in better separation of CE peaks at the cost of the duration of the experiment (Figure 3A). A longer migration/HDX reaction time results in a higher level of deuteration of the protein analytes (Figure 3B</strong...

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Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

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3.2K Views.  Nanjing Normal University. Deze methode maakt het mogelijk om de scheiding van naast elkaar bestaande eiwitsoorten, inclusief verschillende conformers of varianten en online karakterisering van hun hogere orde structuurverschillen, in een enkele CEMS-meting te voltooien. Het maakt gebruik van het elektroforetische effect om een bijna 100% langdurige omgeving te bieden voor de HDX-reacties en orthogonale scheiding van de naast elkaar bestaande eiwitsoorten tijdens HDX voor top-down MS-analyse. Begin met het conditionere...

Video: Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca2+ binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (&#964; &#60; 20 &#956;sec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.

Here we report an application of the photothermal beam deflection technique in combination with a caged calcium compound, DM-nitrophen, to monitor microsecond and millisecond dynamics and energetics of structural changes associated with the calcium association to a neuronal calcium sensor, Downstream Regulatory Element Antagonist Modulator.

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biochemistry

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

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.

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.

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

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Biology

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

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