Method Validation and Standards in Hydrogen Exchange Mass Spectrometry

Hudgens, Jeffrey W.; Huang, Richard Y.‐C.; D’Ambro, Emma L.

doi:10.1002/9781118703748.ch4

Cited by 4 publications

(8 citation statements)

References 43 publications

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“…What were once considered pipedreams of structural biology are now within reach. Furthermore, as HDX‐MS becomes used by a larger population of researchers, significant efforts have been made across the field to standardize experimental procedures and data processing and reporting practices …”

Section: Discussionmentioning

confidence: 99%

“…It can also be indicative of conformational heterogeneity resulting from sample impurity or degradation, mixed populations from unsaturated ligand binding, and even back exchange from chromatographic carryover and improper sample handling. 14,26,27 Similarly where EX2 kinetics are observed, it does not necessarily prove that the local protein motion across those residues is uncorrelated, only that it is occurring faster than the labeling rate. 17,28 Critical analysis of HDX-MS data using the proper and most robust analytical tools is essential for making meaningful conclusions about protein structural dynamics and motion.…”

Section: The Biophysics Of Hdx-msmentioning

confidence: 99%

“…The presence of bimodal spectra in HDX‐MS data does not always correspond to EX1 kinetics arising from large scale conformational changes, reversible interconversion between states, or local coordinated motion. It can also be indicative of conformational heterogeneity resulting from sample impurity or degradation, mixed populations from unsaturated ligand binding, and even back exchange from chromatographic carryover and improper sample handling . Similarly where EX2 kinetics are observed, it does not necessarily prove that the local protein motion across those residues is uncorrelated, only that it is occurring faster than the labeling rate .…”

Section: The Biophysics Of Hdx‐msmentioning

confidence: 99%

“…Furthermore, as HDX-MS becomes used by a larger population of researchers, significant efforts have been made across the field to standardize experimental procedures and data processing and reporting practices. 3,26 ORCID Mark A. Benhaim https://orcid.org/0000-0003-2906-5599…”

Section: Conformational Switching Through Correlated Motionsmentioning

confidence: 99%

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Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

2019

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Much of our understanding of protein structure and mechanistic function has been derived from static high‐resolution structures. As structural biology has continued to evolve it has become clear that high‐resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX‐MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX‐MS to monitor these dynamic changes in proteins. We examine how HDX‐MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. Statement for a Broad Audience The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

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Section: Discussionmentioning

confidence: 99%

Section: The Biophysics Of Hdx-msmentioning

confidence: 99%

Section: The Biophysics Of Hdx‐msmentioning

confidence: 99%

Section: Conformational Switching Through Correlated Motionsmentioning

confidence: 99%

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Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

2019

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“…HDX-MS studies have reported daily repeatability ranging between 0.3% and 2.9% of maximum deuterium uptake and IMP’s ranging between 1% and 9% over periods of 37 d to eight months. ,− Cummins et al compared HDX-MS measurements for two laboratories harmonized by employing identical procedures and equipment. For ligand-vitamin D nuclear receptor complexes immersed for 30 s in D 2 O their results exhibited D-uptake repeatability and reproducibility of 0.54% for a set of 35 peptide sequences …”

Section: Introductionmentioning

confidence: 99%

Interlaboratory Comparison of Hydrogen–Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb

et al. 2019

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Descriptions of materials, metrological methods, computational methods, and supplementary results. Figures of HDX-MS publications and citations versus publication year, histogram of peptide sequence lengths, sequence coverage maps, performance of instrumentsoftware configurations, repeatability plots, %E corrected peptide t HDX versus log 10 (t HDX ) for eight peptides. Tables of instrumentation,software, peptide search methodology, and operating conditions of proteolytic, chromatographic components, and effects of peptide charge on deuterium uptake (PDF)

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Imidazolium Compounds as Internal Exchange Reporters for Hydrogen/Deuterium Exchange by Mass Spectrometry

et al. 2020

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Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis that is well suited for biotherapeutic development and characterization. Because HDX is strongly dependent on solution conditions, even small variations in temperature or pH can have a pronounced effect on the observed kinetics that can manifest in significant run-to-run variability and compromise reproducibility. Recent attention has been given to the development of internal exchange reporters (IERs), which directly monitor changes to exchange reaction conditions. However, the currently available small peptide IERs are only capable of sampling a very narrow temporal window and are understood to exhibit complex solution dependent exchange behavior. Here we demonstrate the use of imidazolium carbon acids as superior IERs for HDX-MS. These compounds exhibit predictable exchange behavior under a wide variety of reaction conditions, are highly stable, and can be readily modified to exchange over a broad temporal window. The use of these compounds as IERs for solution based HDX-MS could considerably extend the utility of the technique by allowing for more robust empirical exchange correction, thereby improving reproducibility.

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Method Validation and Standards in Hydrogen Exchange Mass Spectrometry

Cited by 4 publications

References 43 publications

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

Interlaboratory Comparison of Hydrogen–Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb

Imidazolium Compounds as Internal Exchange Reporters for Hydrogen/Deuterium Exchange by Mass Spectrometry

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