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Review
. 2015 Jan 6;87(1):99-118.
doi: 10.1021/ac5040242. Epub 2014 Nov 14.

Applications of hydrogen/deuterium exchange MS from 2012 to 2014

Gregory F Pirrone  1 , Roxana E Iacob, John R Engen
Affiliations
Review

Applications of hydrogen/deuterium exchange MS from 2012 to 2014

Gregory F Pirrone et al. Anal Chem. .
No abstract available

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Figures

Figure 1
Figure 1
Synopsis of the published applications of HDX MS from January 2012 to June 2014. (A) A total of two-hundred thirty-four (234) articles were published and classified according to the scheme shown, based on author affiliations. The number of articles in each category is shown in parentheses; review articles were not included. (B) The global distribution of the articles in panel A, based on the home institution of the communicating author of each article. (C) Similar to panel A, but for the largest source of articles, the United States of America. (D) Breakdown of the non-USA publications according to country and to sector. The data are divided roughly by continent going from left to right. (E) The six topical classifications chosen for the Review are shown as pie graphs, with the size of each pie equivalent to the number of articles (shown in parentheses) in each topic. Further subdivision within topics follows the order of the main text of the article.
Figure 2
Figure 2
Application to the study of protein folding and unfolding. (A) Example of determining the folding order of a protein with HDX MS, here the order of α1-antitrypsin. Modified with permission from ref (1). Copyright 2012 National Academy of Sciences of the United States of America. (B) Example of monitoring protein unfolding via conformational dynamics. Totally deuterated β2-microglobin (β2m) was incubated in H2O buffer and intact mass spectra acquired at the times shown. Exchange through EX1+EX2 kinetics (blue peaks), representing a very unfolded unprotected form, was observed from which a rate of EX1 unfolding could be extracted (right graph). (C) The EX1 rate of β2m unfolding was measured for a host of other conditions, including mutation and solution additives. Panels B and C modified and used with permission from ref (12). Copyright 2012 John Wiley and Sons.
Figure 3
Figure 3
HDX MS in the study of chaperone-assisted protein folding. The TIM-barrel protein DapA was unfolded with denaturant and, upon dilution of the denaturant, allowed to fold and assemble into its native tetramer spontaneously (A) or in the presence of GroEL/ES (B). After various periods of refolding, pulse labeling, pepsin digestion, and mass analysis were performed. The protection half-times (colored by the categories shown: red, yellow, blue) for segments of the protein (left) were greatly accelerated by the chaperones compared to the spontaneous folding. The chaperones also changed the order of folding. The locations of each protection category are shown at the right on the crystal structure of the assembled tetramer. Reprinted with permission from ref (31). Copyright 2014 Elsevier.
Figure 4
Figure 4
Observing structural changes during function. It was proposed that the LOV (light–oxygen–voltage) domain protein VIVID (VVD) underwent a structural change upon illumination and HDX MS was used to show that this was the case. (A) Continuous labeling of the dark state (VVDD) and light state (VVDL) revealed unfolding with EX1 kinetics and very different time scales of unfolding for the two states. The location of the unfolding was determined with analysis of pepsin fragments (example spectra shown in panel A inset). (B) Comparison of deuterium levels in peptides and (C) interpretation of the data on the structure of the protein and its dimer indicated that all structural elements of VVDD incorporate more deuterium in nearly all regions except for the N-cap. See ref (32) for full details. Modified with permission from ref (32). Copyright 2013 Elsevier.
Figure 5
Figure 5
Application of HDX MS to the study of viruses and virus-related proteins. (A) Analysis of the binding to peripentonal hexons of the 33-residue N-terminal fragment of the precursor VI protein (pVIn) from human adenovirus. Exchange into purified hexons was compared with and without pVIn and the affected regions were localized. Modified with permission from ref (72). Copyright 2014 Elsevier. (B) Binding-induced changes in HIV Env trimers were determined when complexed with two different small molecule HIV entry inhibitors (NBD-556 or BMS-806) that block the CD4 binding site. Changes in exchange were compared to those seen when Env trimmers were bound to CD4 alone (not shown here). Modified with permission from ref (81). Copyright 2014 Elsevier.
Figure 6
Figure 6
HDX MS for epitope mapping of a monoclonal antibody (mAb) against factor H binding protein (fHbp), a virulence factor, and vaccine antigen of the causative agent of bacterial meningitis, Neisseria meningitidis. Comparisons were made against (A) the known interface between factor H (fH) and fHbp, interface residues colored yellow in the structure at the left, and (B–E) epitope mapping data, colored red on the structure and linear representation, for mAb 12C1 and fHbp by various methods. HDX MS mapping (panel D) identified all regions but not with the resolution of the cocrystal structure. Reprinted with permission from ref (86). Copyright 2013 National Academy of Sciences of the United States of America.
Figure 7
Figure 7
Example of mapping protein/protein interactions by HDX MS. (A) The effects of binding the nucleosome assembly protein 1 (Nap1) to the histone H2A–H2B heterodimer were shown by comparing exchange into the H2B portion of H2A–H2B alone (left) to exchange into H2B when bound to Nap1 (middle). The regions most affected by binding were mapped to the crystal structure of H2B (colored blue, right panel). (B) Analysis by HDX MS suggested regions where mutations might be made and tested in other assays; mutants 3 and 4 (residues changed indicated in yellow and red, respectively) later showed reduced binding to Nap1 in FRET assays. Modified with permission from ref (97). Copyright 2013 Elsevier.
Figure 8
Figure 8
Small molecule interactions with a protein. The AMP-activated protein kinase (AMPK) was incubated with various small molecules (A769662, beta cyclodextrin, and staurosporine), and HDX MS was performed. Changes in deuteration relative to unbound AMPK were both (A) mapped to the crystal structure of AMPK and (B) displayed schematically. The percentages of deuterium differences were mapped according to the key shown. Reprinted with permission from ref (124). Copyright 2013 Elsevier.
Figure 9
Figure 9
Characterization of Fc fusion proteins and how their parts relate to naturally occurring versions. HDX MS of recombinant factor IX (rFIX) was compared to a fusion of rFIX with an Fc of an antibody (termed rFIX-Fc). The pattern of differences in deuterium levels of rFIX as a result of calcium binding (A) was the same as that observed when the fusion form, rFIX-Fc, bound to calcium (B) meaning that the FIX portion was not impaired in its calcium binding activity by being attached to the Fc. There were essentially no differences (C, D) between exchange into rFIX and rFIX-Fc with calcium or without calcium. Reprinted with permission from ref (158). Copyright 2012 Wiley.
Figure 10
Figure 10
Membrane protein investigations with HDX MS. (A) Exchange into phosphoinositide 3-kinase γ (also termed p110γ) was compared to exchange during interactions with its regulatory/adaptor subunit p101 (left). The complex of p110γ/p101 was then labeled with and without empty liposomes (middle) or liposomes containing G-proteins (Gβγ) (right) to ascertain the role of the membrane and effects of binding. See ref (229) for full details. Reprinted with permission from ref (229). Copyright 2013 National Academy of Sciences of the United States of America. (B) HDX MS of BmrA, a bacterial multidrug ABC transporter, was performed while the protein was in n-dodecyl-β-d-maltoside (DDM) detergent. HDX results for analysis of an apo form were superimposed on the 3D model of the open conformation (left), and analysis of a closed form ATP-Mg-bound mutant were superimposed on a 3D model of the closed conformation (right). Coloring is based on the percentage of deuterium after 1 h of labeling, according to the color scale at the right. Reprinted with permission from ref (181). Copyright 2012 National Academy of Sciences of the United States of America.
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