HXMS: a standardized file format for HX/MS data

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.

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.

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.