Contemporary hydrogen deuterium exchange mass spectrometry

Oganesyan, Irina; Lento, Cristina; Wilson, Derek J.

doi:10.1016/j.ymeth.2018.04.023

Cited by 105 publications

(106 citation statements)

References 154 publications

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“…Broad applicability and label-free sample preparation have made HDX-MS an increasingly attractive biophysical technique to study global biomolecular structure and dynamics under native conditions, as demonstrated by the variety of reported applications on both globular and membrane proteins and frequently-updated reviews (3,4,62). The major challenge, however, has been how to objectively translate the HDX data into structural information, so as to be able to derive quantitative mechanistic insights.…”

Section: Discussionmentioning

confidence: 99%

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Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

Bradshaw

Marinelli

Faraldo‐Gómez

et al. 2019

Preprint

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Statement of significanceHDX-MS experiments are a powerful approach for probing the conformational dynamics and mechanisms of proteins. However, the mechanistic implications of HDX-MS observations are frequently difficult to interpret, due to the limited spatial resolution of the technique as well as the lack of quantitative tools to translate measured data into structural information. To overcome these problems, we have developed a computational approach to construct structural ensembles that are maximally diverse while reproducing target experimental HDX-MS data within a given level of uncertainty. Using artificial test data, we demonstrate that the approach can correctly discern distinct structural ensembles reflected in the target data, and thereby facilitate statistically robust evaluations of competing mechanistic interpretations of HDX-MS experiments.

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Section: Discussionmentioning

confidence: 99%

“…Consequently, measurements of Hydrogen-Deuterium exchange (HDX) rates are increasingly used as a direct probe of protein dynamics. Moreover, by combining HDX with mass spectrometry (HDX-MS), this approach has become feasible also for large complexes and membrane proteins, even at low concentrations (3).…”

Section: Introductionmentioning

confidence: 99%

Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

Bradshaw

Marinelli

Faraldo‐Gómez

et al. 2019

Preprint

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“…Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) has emerged as a useful tool to monitor the conformation and dynamics of therapeutic proteins (10)(11)(12)(13). Conventional biophysical techniques such as circular dichroism and fluorescence (14)(15)(16) provide only general information, and it is difficult to detect subtle conformational changes or changes related only to specific regions of proteins with these techniques.…”

Section: Introductionmentioning

confidence: 99%

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss

Wang

Mel

et al. 2019

Preprint

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Asparagine deamidation is a common posttranslational modification in which asparagine is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics.We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kilodalton (kDa) truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which suggests a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogendeuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide a possible explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest an approach that can be used for process control to ensure product quality and process consistency.3 Statement of SignificanceAsparagine deamidation can have a potentially significant impact on protein therapeutics.Previous studies have revealed that deamidation at a single site significantly reduces the biological activity of moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin developed for the treatment of B-cell malignancies. Surprisingly, despite the fact that deamidation introduced a negative charge, the deamidated variant eluted earlier than its unmodified form on anion exchange chromatography. In order to understand these observations, we isolated the deamidated variant using an anion exchange column and characterized it by differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry. The results revealed the conformational change caused by the deamidation, which explains the diminished biological activity of the variant and its early elution time on anion exchange chromatography.

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“…Tandem MS/MS peptide sequencing is used to locate the position of chemical crosslinks to help define protein interactions (19). It can also be used to quantify hydrogen/deuterium exchange to estimate solvent accessibility and flexibility of protein regions (22). Similarly, chemical probing (covalent labeling) exploits differential reactivity to reagents that non-specifically modify protein side chains, such as hydroxy radicals, or that target specific amino acids depending on a residues exposure to solvent vs. participation in interactions within the macromolecule (23,24).…”

Section: Introductionmentioning

confidence: 99%

A two-step probing method to compare lysine accessibility across macromolecular complex conformations

MacRae

Coltri²,

Hrabeta-Robinson³

et al. 2018

Preprint

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Structural models of multi-megadalton molecular complexes are appearing in increasing numbers, in large part because of technical advances in cryo-electron microscopy realized over the last decade. However, the inherent complexity of large biological assemblies comprising dozens of components often limits the resolution of structural models. Furthermore, multiple functional configurations of a complex can leave a puzzle as to how one intermediate moves to the next stage. Orthogonal biochemical information is crucial to understanding the molecular interactions that drive those rearrangements. We present a two-step method for chemical probing detected by tandem mass-spectrometry to globally assess the reactivity of lysine residues within purified macromolecular complexes. Because lysine side chains often balance the negative charge of RNA in ribonucleoprotein complexes, the method is especially powerful for detecting changes in protein-RNA interactions. Probing the E. coli 30S ribosome subunit showed that the reactivity pattern of lysine residues quantitatively reflects structure models from X-ray crystallography. We assessed differences in two conformations of purified human spliceosomes. Our results demonstrate that this method supplies powerful biochemical information that aids in functional interpretation of atomic models of macromolecular complexes at the intermediate resolution often provided by cryo-electron microscopy.

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Contemporary hydrogen deuterium exchange mass spectrometry

Cited by 105 publications

References 154 publications

Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss

A two-step probing method to compare lysine accessibility across macromolecular complex conformations

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