Work

Retention Time Interpolation Enhances Peptide Mapping for HDX-MS

Year: 2025

Type: article

Abstract: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for studying protein structural dynamics. A critical step in the HDX-MS workflow is generating a peptide map from nondeuterated samples, which serves as the reference for identifying and monitoring peptides in subsequent deuterium-labeled experiments. Maximizing peptide identifications improves sequence coverage and redundancy, enhancing the information content and spatial resolution of the HDX-MS data. However, peptide identification is often limited by suboptimal peptide separation/fragmentation. In other proteomic workflows, longer liquid chromatography (LC) gradients are commonly used to improve the peptide identification by increasing resolution. However, in HDX-MS workflows, such gradients are generally incompatible due to time constraints imposed by deuterium/hydrogen back-exchange. To address this, we introduce a flexible workflow that uses long-gradients during initial peptide mapping, followed by retention time (RT) interpolation for application in subsequent short-gradient HDX-MS. By performing both long- and short-gradient peptide mapping, we used shared peptides to generate a regression model that predicts short-gradient RTs for all peptides identified in the long-gradient experiment. This enables the use of the richer peptide maps provided by long-gradient chromatography without compromising the deuterium retention. The method is implemented by RTinterpolator, a freely available R script compatible with widely used HDX analysis platforms that rely on reference RT values for peptide monitoring in deuterium-labeled data. By providing predicted RTs aligned to short gradients, RTinterpolator offers a practical, accessible, and instrument-independent way of increasing sequence coverage and redundancy in HDX-MS experiments, particularly for large or complex proteins susceptible to the limitations of short-gradient chromatography.

Source: Journal of the American Society for Mass Spectrometry

DOI: 10.1021/jasms.5c00156

Authors: Damon Griffiths, Juan P. Rincon Pabon, Charlotte Guffick, Argyris Politis

Institutions: University of Manchester, Alexander Fleming Biomedical Sciences Research Center

Language: English

Topic: Mass Spectrometry Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funders: European Commission, Medical Research Council, Engineering and Physical Sciences Research Council, Biotechnology and Biological Sciences Research Council

Awards: Capturing eukaryotic transporters in action, ERA CHAIR HOLDER TO ESTABLISH BIOMOLECULAR AND STRUCTURAL MASS SPECTROMETRY FOR THE ANALYSIS OF COMPLEX BIOLOGICAL PATHWAYS, High resolution cyclic ion mobility HDX mass spectrometry of protein dynamics and function, Mapping membrane protein dynamics in time and space with mass spectrometry, Structure, Dynamics and Activity of the Bacterial Secretosome