HXMS: a standardized file format for HX/MS data.

Motivation: Hydrogen-deuterium exchange/mass spectrometry (HX/MS) is a rapidly expanding technique used to investigate protein conformational ensembles. The growing popularity and utility of HX/MS has driven the development of diverse instrumentation and software, resulting in inconsistent, non-standardized data analysis and representation. Most HX/MS data formats also employ only centroid-level representations of the data rather than full isotopic mass spectra, reducing the information content of the data and limiting downstream quantitative analysis. Results: Inspired by reliable protein structure and genomics data formats, we present HXMS, a unified, lightweight, scalable, and human-readable file format for HX/MS data. The HXMS format preserves the isotopic mass envelopes for all peptides, captures the full experimental time-course including the fully deuterated control samples, and contains all other key information. It supports multimodal distributions, post-translational modifications (PTMs), and experimental replicates. To promote compatibility with existing HX/MS workflows, we also developed PFLink, a Python package that converts exported data files from commonly used HX/MS analysis software packages to the HXMS format. PFLink and the HXMS format will enable more quantitative, higher-resolution data processing, improved data sharing and storage among HX/MS practitioners, future machine learning applications, and further developments in HX/MS analysis. Availability and implementation: PFLink is publicly available to install locally on HuggingFace, alongside documentation, or use online at HuggingFace (https://huggingface.co/spaces/glasgow-lab/PFLink). We also included a generic unfilled PFlink custom CSV file that users may populate with key experimental conditions and results, which can then be read and converted into the HXMS format.