Retention Time Interpolation Enhances Peptide Mapping for HDX-MS

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 <i>RTinterpolator</i>, 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, <i>RTinterpolator</i> 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.