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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+methods+in+characterization+of+the+higher+order+structure+of+protein+therapeutics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+hydrogen/deuterium+exchange+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformer-specific+hydrogen+exchange+analysis+of+Abeta(1-42)+oligomers+by+top-down+electron+capture+dissociation+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+dynamics+of+small+soluble+Abeta(1-40)+oligomers+studied+by+top-down+hydrogen+exchange+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+structural+analysis+of+posttranslationally+modified+proteins+by+Fourier+transform+ion+cyclotron+resonance-MS+with+hydrogen/deuterium+exchange+and+electron+capture+dissociation.">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. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+hydrogen/deuterium+exchange+during+proteoform+separation+enables+characterization+of+conformational+differences+between+coexisting+protein+states.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invited+review:+&#946;-lactoglobulin:+binding+properties,+structure,+and+function.">Invited review: &#946;-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+the+Tanford+transition+of+bovine+&#946;-lactoglobulin.">Structural basis of the Tanford transition of bovine &#946;-lactoglobulin.</a> Biochemistry. 37 (40), 14014-14023 (1998).</li><li>Qin, B. Y., Bewley, M. C., Creamer, L. K., Baker, E. N., Jameson, G. 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P., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+Poiseuille's+law.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+characterization+of+the+higher+order+structure+of+disulfide-containing+proteins+using+hydrogen/deuterium+exchange+and+top-down+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electrospray+ionization+mass+spectrometry+as+a+tool+for+characterization+of+small+soluble+protein+aggregates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProSight+Lite:+graphical+software+to+analyze+top-down+mass+spectrometry+data.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MASH+Suite+Pro:+A+comprehensive+software+tool+for+top-down+proteomics.">MASH Suite Pro: A comprehensive software tool for top-down proteomics.</a> Molecular &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+&#946;-lactoglobulin+A,+B+and+C+variants+of+bovine+whey+using+capillary+zone+electrophoresis.">Separation of &#946;-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+characterization+of+the+higher+order+structure+of+disulfide-containing+proteins+using+hydrogen/deuterium+exchange+and+top-down+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-line+electrochemical+reduction+of+disulfide+bonds:+improved+FTICR-CID+and+-ETD+coverage+of+oxytocin+and+hepcidin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+analysis+of+disulfide-linked+proteins+using+photoinduced+radical+reactions+and+ET-DDC.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+characterization+of+proteins+with+intact+disulfide+bonds+using+activated-ion+electron+transfer+dissociation.">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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ammonium acetate</td>
			<td>Fisher Chemical</td>
			<td>A/3446/50</td>
			<td>&#8805;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>&#160;270480250</td>
			<td></td>
		</tr>
		<tr>
			<td>fused silica glass capillary</td>
			<td>Polymicro Technologies</td>
			<td>1068150017</td>
			<td>ID 50&#956;m, OD 360&#956;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 colspan="2">Thermo Fisher Scientific, USA</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&#160;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>&#946;-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...

Watch this Scientific Journal Video about Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry at JoVE.com

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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3.1K Views.  Nanjing Normal University. 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.

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 ...

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 ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 &#181;g/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 &#181;g/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was &#60;3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

This iron redox speciation method is based on capillary electrophoresis-inductively coupled plasma mass spectrometry with sample stacking combined with short analysis in one run. The method quickly analyzes and provides low limits of quantification for iron redox species across a diverse range of tissues and biofluid samples.

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...