Issue 16, 2017

Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation

Abstract

Hydrogen Deuterium Exchange (HDX) Mass Spectrometry (MS) is a sensitive analytical technique that provides information on protein conformation and dynamics in solution. It is commonly used in the study of protein–ligand and protein–protein interactions and more recently in the pharmaceutical industry for epitope mapping, screening drug candidates and in the comparison of biopharmaceuticals to biosimilars. HDX-MS monitors the exchange of protein backbone hydrogen atoms with deuterium in solution. Recent advancements in HDX automation and data analysis, have taken the emphasis off developing a fundamental understanding of HDX, which is still lacking. This tutorial review will cover the different mechanisms of exchange and how the exchange reaction is affected by various factors. We also explore the basis of data analysis and the difficulties that often arise in the interpretation of site-specific and segment-averaged HDX data, such as overlapping isotopic distributions and correct identification of peptides. Initial data analysis generates a list of peptides and the deuterium incorporation of each peptide at each labeling time point, i.e., a set of deuterium uptake profiles. Data interpretation and error analysis is subsequently required to ensure that deuterium uptake profiles accurately reflect conformational dynamics in solution. Finally, this review will also discuss the different ways in which HDX data can be represented and how the data can be interpreted.

Graphical abstract: Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation

Article information

Article type
Tutorial Review
Submitted
25 Apr 2017
Accepted
07 Jul 2017
First published
13 Jul 2017

Analyst, 2017,142, 2874-2886

Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation

K. A. Brown and D. J. Wilson, Analyst, 2017, 142, 2874 DOI: 10.1039/C7AN00662D

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements