scholarly journals Combining small-molecule bioconjugation and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to expose allostery: the case of human cytochrome P450 3A4

2020 ◽  
Author(s):  
Julie Ducharme ◽  
Christopher J. Thibodeaux ◽  
Karine Auclair
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Cytochrome P450 3A4 ◽  
Hydrogen Deuterium Exchange ◽  
Allosteric Site ◽  
Exchange Mass Spectrometry ◽  
Human Cytochrome ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractWe report herein a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4). CYP3A4 is arguably the most important drug-metabolizing enzyme, and as such, is involved in numerous drug interactions. Diverse allosteric ligand effects have been reported for this enzyme, yet the structural mechanism of these phenomena remain poorly understood. We have described different CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors on CYP3A4, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic), or do not show activation while still blocking the allosteric site (antagonistic). These bioonjugates were studied here by HDX-MS, which enabled us to better define the position of the allosteric site, and to identify important regions involved in CYP3A4 activation.

scholarly journals Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 286
Author(s):  
Oliver Ozohanics ◽  
Attila Ambrus
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Ligand Interaction ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

scholarly journals Construction of a Dual Protease Column, Subzero (-30 oC) Chromatography System and Multi-channel Precision Temperature Controller for Hydrogen-Deuterium Exchange Mass Spectrometry

2020 ◽  
Vol 125 ◽  
Author(s):  
Jeffrey W. Hudgens
Keyword(s):  
Mass Spectrometry ◽  
Three Dimensional ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Precision Temperature ◽  
Hydrogen Deuterium ◽  
3D Printed ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

This tutorial provides mechanical drawings, electrical schematics, parts lists, stereolithography (STL) files for producing three-dimensional (3D)-printed parts, initial graphics exchange specification (IGS) files for automated machining, and instructions necessary for construction of a dual protease column, subzero, liquid chromatography system for hydrogen-deuterium exchange mass spectrometry (HDX-MS). Electro-mechanical schematics for construction of two multi-zone temperature controllers that regulate to ±0.05 oC are also included in this tutorial.

scholarly journals Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Structure of Herceptin®

2018 ◽  
Author(s):  
Rosie Upton ◽  
Lukasz G. Migas ◽  
Kamila J. Pacholarz ◽  
Richard G. Beniston ◽  
David Firth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Ion Mobility Mass Spectrometry ◽  
Data Sets ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Interactive Data ◽  
Hdx Ms

<p>To consider the measurable variations in biopharmaceuticals we use mass spectrometry and systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry analyses. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5 – 25 %. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data supports this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together these findings, and the supplied interactive data sets could be used to provide acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs. </p>

scholarly journals Dataset from HDX-MS Studies of IgG1 Glycoforms and Their Interactions with the FcγRIa (CD64) Receptor

2021 ◽  
Vol 126 ◽  
Author(s):  
Kyle W. Anderson ◽  
Kerry Scott ◽  
Ioannis L. Karageorgos ◽  
Elyssia S. Gallagher ◽  
Tayi Venkata S. ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Neutrophils ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Affinity Receptor ◽  
Hdx Ms

This document presents hydrogen-deuterium exchange mass spectrometry (HDX-MS) data from measurements of three purified IgG1 glycoform samples, predominantly G0F, G2F, and SAF, in isolation and in complexation with the high-affinity receptor, FcγRIa (CD64). The IgG1 antibody used in this study, aIL8hFc, is a murine-human chimeric IgG1, which inhibits IL-8 binding to human neutrophils.

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