Deuterium Exchange during Proteoform

Subscriber access provided by UNIV OF TEXAS DALLAS

Letter

Differential hydrogen/deuterium exchange during proteoform separation enables characterization of conformational differences between coexisting protein states Yue Shen, Xiuxiu Zhao, Guanbo Wang, and David D. Y. Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00558 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Differential hydrogen/deuterium exchange during proteoform separation enables characterization of conformational differences between coexisting protein states Yue Shen1,#, Xiuxiu Zhao1,#, Guanbo Wang1,, and David D. Y. Chen2, 1 School

of Chemistry and Materials Science, Nanjing Normal University, Nanjing, Jiangsu Province,

210023, China 2 Department

of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada

# Equal contribution to this work.

address correspondence to: Guanbo Wang School of Chemistry and Materials Science Nanjing Normal University 1 Wenyuan Rd, Qixia District Nanjing, Jiangsu Province 210023, P. R. China Email: [email protected] 

address correspondence to: David D. Y. Chen Department of Chemistry University of British Columbia 2036 Main Mall Vancouver, BC, V6T 2G9, Canada Email: [email protected] 

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 19

ABSTRACT Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents, and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor difference in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb), and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted 2H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with 2H

during separation based on their disulfide-dependent sizes. The resulting differences in deuteration

pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of heme group by comparing the deuteration patterns of holo- and apo-Mb.


Page 3 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conformational heterogeneity is almost always encountered in biological events including protein folding, binding and aggregation. The efficiencies of functions ranging from molecular recognition to enzymatic catalysis differ significantly among coexisting conformational states. Distinguishing these states and characterizing their structural differences are important for determining the pathways of conformational transitions critical for these biological functions, and the efficacy of protein therapeutics. Conventional biophysical techniques are insufficient due to the unsatisfactory resolution (e.g. optical spectrometry and cryo-electron microscopy), loss of dynamic information in solution (e.g. X-ray crystallography) or overlapped signals from ensemble of proteins (e.g. NMR).1 Hydrogen/deuterium exchange (HDX) reaction coupled with mass spectrometry (MS) analysis2-3 performs conformationdependent 2H-labeling under physiologically relevant conditions and interrogates the protein higherorder structures of proteins on a fast time-scale at high spatial resolution4, thereby becoming a powerful tool that highly complements the conventional techniques.5-6 After HDX, different conformers are deuterated to different extents, exhibiting distinguishable distributions of molecular weights (MW). In classical “bottom-up” approach, the ensemble of conformers is proteolyzed prior to MS analysis, making it difficult to unambiguously attribute all detected peptide signals to proper conformers. In contrast, the “top-down” approach allows characterization in a conformer-specific manner.1,

7-9

Since 2H-labeled

species are sent for MS analysis in their intact forms, they can be separated in the gas phase through mass-selection of subpopulations corresponding to individual MW distributions using a mass filter. Structural protection of amide hydrogens at local sites can then be determined through tandem MS, i.e. analysis of deuteration of the gas-phase fragments of the mass-selected species. This strategy has been successfully applied to characterize structural differences between coexisting conformers1, monomeric and oligomeric forms of small amyloidogenic peptides7-8, and proteoforms with different 3 ACS Paragon Plus Environment


Page 4 of 19

posttranslational modifications9. In this strategy, however, the MW-based separation is compromised by the partially overlapped protein populations with modest differences in deuteration level. Incomplete conformer separation introduces interference from other conformers. Moreover, proteoforms that adopt different conformations but exibit similar overall deuteration levels are difficult to distinguish.

In this work, we circumvented this problem by performing differential HDX during separation of proteoforms in solution, followed by conformational characterization of the sequentially eluted 2Hlabeled species in real-time using top-down MS. These species are separated by capillary electrophoreis (CE) according to their differing charge to size ratios10-14, regardless of their overall deuteration level difference. The small volume of the CE capillary allows the use of fully deuterated reagents as the background solvent, thereby enabling HDX during separation. Although there were occasional reports of coupling CE with HDX, except for measurements of peptides15, previous CE separation was performed either after the proteolysis of deuterated proteins in bottom-up approach16, or prior to HDX of small molecules that is achieved by a rapid mixing of outflow with deuterating solution17.

Figure 1A shows the schematic setup of the differential HDX-MS experiment. We modified the inner surface of silica capillary with hydroxylpropyl cellulose (see SI) to mask the silanol groups and minimize protein-capillary interaction.18-19 Since this modification eliminates the electroosmotic flow, protein migration is driven by electrophoretic effect and backpressure of BGE infusion. The positively charged proteins migrate faster than the neutral solvent molecules in background electrolyte solution (BGE). When deuterated BGE is used, the surface-modified capillary can serve as an effective reactor of HDX. Mixing the acidified and denaturing modifier solution with the CE outflow at the flow-through microvial inside the CE-MS interface20-22 quenches the reactions and unfolds the 2H-labeled proteins to facilitate 4 ACS Paragon Plus Environment

Page 5 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the subsequent tandem MS analysis. When the capillary is completely filled with deuterated BGE, the HDX reaction time equals to the migration time of protein, which is dependent on the length of capillary and the flow-rate. With the capillary length kept constant, increase of infusion pressure effectively decreased the HDX time of holo-myoglobin (hMb), resulting in lowered deuteration level of hMb (Figure 1B and 1C; BGE pH: 6.6). We also tested a scheme that can further reduce the effective HDX time (Figure 1D). Prior to sample injection, we injected a desired quantity of non-deuterated BGE to reduce the length of deuterated BGE section retained in the capillary. Thus HDX only takes place at the late stage of protein migration. The small diameter of capillary minimizes the lateral diffusion23 between deuterated and nondeuterated BGE sections at the boundary. Using this approach, we regulated the deuteration levels of hMb over a wide range (Figure 1E), which correspond to effective reaction time scales ranging from seconds to minutes (see SI for determination of the effective reaction time).

Figure 1. (A) Schematic illustration of the CE-HDX-MS setup. (B) Migration time (black) and deuteration level (gray) of hMb as functions of infusion pressure. The error bars show the standard deviation for 5 ACS Paragon Plus Environment


Page 6 of 19

three measurements. (C) Mass spectra of 2H-labeled [hMb]18+ acquired with different HDX times. (D) Schematics of the scheme that further reduces the HDX time. The upper and bottom sectional views of the capillary show the material positions at early and late stages of electrophoresis, respectively. (E) Deuteration level of hMb as a function of volume of deuterated BGE retained in capillary. The partial deuteration of hMb with no deuterated BGE in capillary is due to the use of deuterated modifier solution at the CE-MS interface. Conformational features of the 2H-labeled protein species were characterized by determining the backbone amide deuteration levels at local sites. A lower level indicates a higher structural protection of backbone amide hydrogen, and vice versa. In our top-down MS analysis of local deuteration, we fragmented the 2H-labeled protein ions using electron-transfer dissociation (ETD)24 with minimal collisional heating (see SI) to minimize the hydrogen scrambling that can compromise the reliability of results under improper experimental conditions25-26. Despite the narrow migration window of a protein species (typically < 2 min), the high ionization efficiency provided by the CE-MS interface20-22 and improved scan rate with modern mass spectrometers allow accumulation of sufficient tandem MS scans and detection of abundant fragments (Figure S1). For 2H-labeled hMb, we observed 25 c–type fragment ions and 22 z-type ions upon accumulation of ca. 400 tandem MS scans within 2 min. The deuteration map (backbone-amide deuteration level vs. residue number) of hMb (Figure 2; numeric values are presented in Table S1) shows excellent agreement with the structure of hMb determined with X-ray crystallography27 (PDB ID: 1WLA), as regions displaying low levels of deuteration (i.e. highly protected from HDX) align with the structured helical segments. The appearance of this deuteration pattern also resembles that deduced from a normal top-down HDX-MS measurement28, although the latter was acquired with a shorter HDX time (5 s) and shows a lower deuteration level. These agreements demonstrate the validity of data acquired with this top-down CE-HDX-MS strategy. 6 ACS Paragon Plus Environment

Page 7 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Comparison of deuteration maps (deuteration levels of backbone amide at individual amino acid residues) of hMb determined by top-down CE-HDX-MS (black; BGE pD: 7.0; HDX time: 20 min) and HDX MS (gray28). Experimental data are derived from fragments of mass-selected [hMb]18+ ions. Secondary structural elements of hMb (PDB ID: 1WLA27) are shown schematically on top of the graph. Prior studies of pure hMb and aMb using NMR suggest that in the absence of heme, aMb largely adopts a conformation resembling that of hMb, while its Helix F and the adjacent segments undergo exchange between holo-like conformation and partially unfolded states.28-29 However, in these studies such conformational differences were not revealed in a complex system where both proteoforms coexisted. We prepared a mixture of hMb and aMb (see SI for detail), whose integrities were verified with native MS30-31 measurements (Figure 3A inset). Using the top-down CE-MS setup, we effectively separated aMb and hMb as indicated by the nearly baseline-resolved peaks (Figure 3A). Although the two sequentially eluted species were both detected as heme-free species (Figure 3B, gray traces) due to denaturation by the modifier solution, we identified these species as aMb and hMb respectively through a titration-like measurement (Figure S2). The change of peak area with the varied quantities of hMb or aMb also ruled out the potential interference of artificial peak. In differential HDX, 2H-labeled aMb and hMb were separated in the same manner, except that the elution of both species were slightly delayed (Figure 3A). The delay is caused by increase in viscosity of deuterated BGE32. The earlier eluted aMb shows a higher deuteration level than hMb (Figure 3B), clearly suggesting that the difference in deuterium uptake 7 ACS Paragon Plus Environment


Page 8 of 19

between aMb and hMb is the result of conformational difference, rather than their slightly different HDX time in capillary. We performed online top-down MS analysis for sequentially eluted aMb and hMb, and plotted their deuteration maps (Figure 3C; numeric values are presented in Table S1). While the deuteration pattern of separated hMb resembles that of pure hMb as shown in Figure 2, aMb exhibits significantly higher deuteration level at regions corresponding to the highly structured helices of hMb. Such dramatic difference in deuteration patterns between aMb and hMb agrees with the prior reports that the absence of heme leads to more dynamic conformational fluctuations at helical regions of aMb.2829

Figure 3. (A) Electropherograms (extracted ion current, EIC) of unlabeled (upper colored trace) and 2Hlabeled aMb-hMb mixture (bottom colored trace) respectively. The gray traces show the appearance of 8 ACS Paragon Plus Environment

Page 9 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the corresponding total ion current (TIC) graphs. The insets in red and blue show native MS spectra of aMb and hMb, respectively. (B) Mass spectra of unlabeled (gray) and 2H-labeled [Mb]18+ (red and blue) eluted from Peak I (upper traces) and Peak II (bottom traces) shown in (A). (C) Comparison of deuteration maps of aMb (red) and hMb (blue). Data are derived from fragments of mass-selected [aMb]18+ and [hMb]18+ ions. The error bars show the standard deviation for three measurements. The electrophoretic mobility of a given molecule is proportional to its charge and inversely proportional to its hydrodynamic radius. Throughout the separation stage (at pH 6.8), both hMb and aMb are positively charged. aMb possesses a larger size due to its higher structural flexibility as evidenced by its higher deuteration level. Accordingly, the earlier elution of aMb in CE suggests that aMb accommodates more net positive charges in solution in comparison with hMb, and their charge difference dwarfs their size difference, playing a dominant role in affecting the observed mobility. The capability of revealing charging natures of separated protein states is distinctive for CE compared with size-exclusion chromatography.

For characterization focusing on the relationship between conformational flexibility and covalent structural constraints, proteoforms can be separated predominantly based on their different hydrodynamic sizes by minimizing their charge difference via regulating the BGE condition. In a measurement of a mixture of native lysozyme (Lyz) and its disulfide isomers, we used acidified BGE (pD 3.0) for separation. All isomers underwent acid-induced unfolding, exposing possible charging sites. However, due to the structural constraints set by the disulfide bonds, the extents of unfolding differed among different isomers. Intact Lyz was eluted earlier than its disulfide isomers (Figure 4), and the later eluted species exhibited not only higher level of deuteration but also broader distributions of MW, which indicate higher conformational heterogeneity. This observation is in line with the disulfide mapping 9 ACS Paragon Plus Environment


Page 10 of 19

results that in these isomers, the disulfide bonds connecting distal Cys residues (e.g. Cys6-Cys127 and Cys30-Cys115) are rearranged into those connecting proximal Cys residues (e.g. Cys6-Cys30)33, with the covalent structural constraints significantly reduced.

Figure 4. Mass spectra of unlabeled (gray) and 2H-labeled [Lyz]9+ (red and blue) eluted from 3 peaks of the electropherogram shown in the inset. The shaded widths in the inset indicate the time ranges of MS spectra acquisition. In summary, we developed a differential HDX-MS approach to characterize conformational differences between coexisting protein states. Proteoforms with different conformations exhibited distinguishable charges or hydrodynamic sizes in solution, resulting in distinguishable electrophoretic mobility. The conformational features of these proteoforms were differentially labeled with 2H, and were sequentially characterized by determination of deuteration level at local sites in real time using top-down MS. The electrophoretic stage allows separation of proteoforms regardless of their overall deuteration level difference, thereby enabling integration of proteoform profiling and in-depth proteoform-specific characterization for a complex system. Modern CE instruments allow performance of HDX at low sample consumption in an automated manner, without the need for pre-equilibration in a flow system prior to


Page 11 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


each measurement. This strategy may open up additional possibilities for protein higher-order structure characterization using HDX.

ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation of China (NSFC 21605085 and 21475061) and the Young Elite Scientist Sponsorship Program by China Association for Science and Technology (2017QNRC001). The authors also received support from the Nanjing Qixia Innovation Fund (GC201801 and ZY201813), Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, and Jiangsu Key Laboratory of Biomedical Materials at Nanjing Normal University. The authors are grateful to Prof. Igor A. Kaltashov (University of Massachusetts Amherst) for helpful discussion.



Page 12 of 19

References 1. Wang, G.; Abzalimov, R. R.; Bobst, C. E.; Kaltashov, I. A., Conformer-specific characterization of nonnative protein states using hydrogen exchange and top-down mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (50), 20087-20092. 2. Katta, V.; Chait, B. T.; Carr, S., Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry. Rapid Commun. Mass Spectrom. 1991, 5 (4), 214-217. 3. Englander, S. W., Hydrogen exchange and mass spectrometry: A historical perspective. J. Am. Soc. Mass Spectrom. 2006, 17 (11), 1481-1489. 4. Kaltashov, I. A.; Eyles, S. J., Mass spectrometry in structural biology and biophysics: architecture, dynamics, and interaction of biomolecules. 2nd ed.; John Wiley & Sons: 2012. 5. Engen, J. R.; Wales, T. E., Analytical Aspects of Hydrogen Exchange Mass Spectrometry. Annu. Rev. Anal. Chem. 2015, 8, 127-148. 6. Oganesyan, I.; Lento, C.; Wilson, D. J., Contemporary hydrogen deuterium exchange mass spectrometry. Methods 2018, 144, 27-42. 7. Pan, J.; Han, J.; Borchers, C. H.; Konermann, L., Conformer-specific hydrogen exchange analysis of Abeta(1-42) oligomers by top-down electron capture dissociation mass spectrometry. Anal. Chem. 2011, 83 (13), 5386-5393. 8. Pan, J.; Han, J.; Borchers, C. H.; Konermann, L., Structure and dynamics of small soluble Abeta(140) oligomers studied by top-down hydrogen exchange mass spectrometry. Biochemistry 2012, 51 (17), 3694-3703. 9. Pan, J.; Borchers, C. H., Top-down structural analysis of posttranslationally modified proteins by Fourier transform ion cyclotron resonance-MS with hydrogen/deuterium exchange and electron capture dissociation. Proteomics 2013, 13 (6), 974-981. 10. Jorgenson, J. W.; Lukacs, K. D., Zone electrophoresis in open-tubular glass capillaries. Anal. Chem. 1981, 53 (8), 1298-1302. 11. Dovichi, N. J., DNA sequencing by capillary electrophoresis. Electrophoresis 1997, 18 (12‐13), 2393-2399. 12. Thunecke, F.; Fischer, G., Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis. Electrophoresis 1998, 19 (2), 288-294. 13. Berzas Nevado, J. J.; Contento Salcedo, A. M.; Castaneda Penalvo, G., Simultaneous determination of cis- and trans-resveratrol in wines by capillary zone electrophoresis. Analyst 1999, 124 (1), 61-66. 14. Mironov, G. G.; Clouthier, C. M.; Akbar, A.; Keillor, J. W.; Berezovski, M. V., Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS. Nat. Chem. Biol. 2016, 12 (11), 918-922. 15. Lau, S. S.; Stainforth, N. M.; Watson, D. G.; Skellern, G. G.; Wren, S. A.; Tettey, J. N., CE hydrogen deuterium exchange-MS in peptide analysis. Electrophoresis 2008, 29 (2), 393-400. 16. Black, W. A.; Stocks, B. B.; Mellors, J. S.; Engen, J. R.; Ramsey, J. M., Utilizing Microchip Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry. Anal. Chem. 2015, 87 (12), 6280-6287.


Page 13 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17. Palmer, M. E.; Tetler, L. W.; Wilson, I. D., Hydrogen/deuterium exchange using a coaxial sheathflow interface for capillary electrophoresis/mass spectrometry. Rapid. Commun. Mass Spectrom. 2000, 14 (9), 808-817. 18. Busch, M. H. A.; Kraak, J. C.; Poppe, H., Cellulose acetate-coated fused-silica capillaries for the separation of proteins by capillary zone electrophoresis. J. Chromatogr. A 1995, 695 (2), 287-296. 19. Francois, Y. N.; Biacchi, M.; Said, N.; Renard, C.; Beck, A.; Gahoual, R.; Leize-Wagner, E., Characterization of cetuximab Fc/2 dimers by off-line CZE-MS. Anal. Chim. Acta. 2016, 908, 168-176. 20. Maxwell, E. J.; Zhong, X.; Zhang, H.; van Zeijl, N.; Chen, D. D. Y., Decoupling CE and ESI for a more robust interface with MS. Electrophoresis 2010, 31 (7), 1130-1137. 21. Zhong, X.; Maxwell, E. J.; Ratnayake, C.; Mack, S.; Chen, D. D., Flow-through microvial facilitating interface of capillary isoelectric focusing and electrospray ionization mass spectrometry. Anal. Chem. 2011, 83 (22), 8748-8755. 22. Wang, L.; Bo, T.; Zhang, Z.; Wang, G.; Tong, W.; Da Yong Chen, D., High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface. Anal. Chem. 2018, 90 (15), 9495-9503. 23. Whatley, H., Basic Principles and Modes of Capillary Electrophoresis. In Clinical and Forensic Applications of Capillary Electrophoresis, Petersen, J. R.; Mohammad, A. A., Eds. Humana Press: Totowa, NJ, 2001; pp 21-58. 24. Abzalimov, R. R.; Kaplan, D. A.; Easterling, M. L.; Kaltashov, I. A., Protein conformations can be probed in top-down HDX MS experiments utilizing electron transfer dissociation of protein ions without hydrogen scrambling. J. Am. Soc. Mass Spectrom. 2009, 20 (8), 1514-1517. 25. Hoerner, J. K.; Xiao, H.; Dobo, A.; Kaltashov, I. A., Is there hydrogen scrambling in the gas phase? Energetic and structural determinants of proton mobility within protein ions. J. Am. Chem. Soc. 2004, 126 (24), 7709-7017. 26. Jørgensen, T. J. D.; Gårdsvoll, H.; Ploug, M.; Roepstorff, P., Intramolecular Migration of Amide Hydrogens in Protonated Peptides upon Collisional Activation. J. Am. Chem. Soc. 2005, 127 (8), 27852793. 27. Maurus, R.; Overall, C. M.; Bogumil, R.; Luo, Y.; Mauk, A. G.; Smith, M.; Brayer, G. D., A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket. Biochim. Biophys. Acta 1997, 1341 (1), 1-13. 28. Pan, J.; Han, J.; Borchers, C. H.; Konermann, L., Hydrogen/deuterium exchange mass spectrometry with top-down electron capture dissociation for characterizing structural transitions of a 17 kDa protein. J. Am. Chem. Soc. 2009, 131 (35), 12801-12808. 29. Eliezer, D.; Wright, P. E., Is Apomyoglobin a Molten Globule? Structural Characterization by NMR. J. Mol. Biol. 1996, 263 (4), 531-538. 30. Leney, A. C.; Heck, A. J., Native Mass Spectrometry: What is in the Name? J. Am. Soc. Mass Spectrom. 2017, 28 (1), 5-13. 31. Tong, W.; Wang, G., How can native mass spectrometry contribute to characterization of biomacromolecular higher-order structure and interactions? Methods 2018, 144, 3-13. 32. Jones, G.; Fornwalt, H. J., The Viscosity of Deuterium Oxide and Its Mixtures with Water at 25°C. J. Chem. Phys. 1936, 4 (1), 30-33. 33. Zhao, X.; Shen, Y.; Tong, W.; Wang, G.; Chen, D. D. Y., Deducing disulfide patterns of cysteine-rich proteins using signature fragments produced by top-down mass spectrometry. Analyst 2018, 143 (4), 817-823. 13 ACS Paragon Plus Environment


Table of Contents artwork


Page 14 of 19

Page 15 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. (A) Schematic illustration of the CE-HDX-MS setup. (B) Migration time (black) and deuteration level (gray) of hMb as functions of infusion pressure. The error bars show the standard deviation for three measurements. (C) Mass spectra of 2H-labeled [hMb]18+ acquired with different HDX times. (D) Schematics of the scheme that further reduces the HDX time. The upper and bottom sectional views of the capillary show the material positions at early and late stages of electrophoresis, respectively. (E) Deuteration level of hMb as a function of volume of deuterated BGE retained in capillary. The partial deuteration of hMb with no deuterated BGE in capillary is due to the use of deuterated modifier solution at the CE-MS interface. 205x103mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2. Comparison of deuteration maps (deuteration levels of backbone amide at individual amino acid residues) of hMb determined by top-down CE-HDX-MS (black; BGE pD: 7.0; HDX time: 20 min) and HDX MS (gray28). Experimental data are derived from fragments of mass-selected [hMb]18+ ions. Secondary structural elements of hMb (PDB ID: 1WLA [27]) are shown schematically on top of the graph. 91x37mm (300 x 300 DPI)


Page 16 of 19

Page 17 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (A) Electropherograms (extracted ion current, EIC) of unlabeled (upper colored trace) and 2Hlabeled aMb-hMb mixture (bottom colored trace) respectively. The gray traces show the appearance of the corresponding total ion current (TIC) graphs. The insets in red and blue show native MS spectra of aMb and hMb, respectively. (B) Mass spectra of unlabeled (gray) and 2H-labeled [Mb]18+ (red and blue) eluted from Peak I (upper traces) and Peak II (bottom traces) shown in (A). (C) Comparison of deuteration maps of aMb (red) and hMb (blue). Data are derived from fragments of mass-selected [aMb]18+ and [hMb]18+ ions. The error bars show the standard deviation for three measurements. 91x115mm (300 x 300 DPI)



Figure 4. Mass spectra of unlabeled (gray) and 2H-labeled [Lyz]9+ (red and blue) eluted from 3 peaks of the electropherogram shown in the inset. The shaded widths in the inset indicate the time ranges of MS spectra acquisition. 86x57mm (300 x 300 DPI)


Page 18 of 19

Page 19 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents artwork 82x44mm (300 x 300 DPI)


Deuterium Exchange during Proteoform

Recommend Documents