Year: 2025
Type: article
Abstract: Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a powerful tool for studying protein structure and dynamics. As a bottom-up LC-MS technique, its success largely depends on peptide identifications made by peptide mapping prior to HDX measurements. We previously demonstrated that combining peptide mapping results from complementary single- and multipass cyclic ion mobility-mass spectrometry (cIM-MS) experiments, an approach we term "multi-sequence" cIM-MS, can enhance HDX-MS by increasing peptide identifications. However, this approach required labor-intensive, manual handling of the acquired data, including lengthy optimization of drift time (DT) versus DT full width half maximum (FWHM) trendlines during peak detection processing to combat cyclic wrap-around effects. Here, we present MultiPassMerger, an open-source software tool that automates the processing, merging, and filtering of single- and multipass cIM-MS peptide mapping data. MultiPassMerger was validated through re-analysis of several model proteins. Its automated capabilities enabled better optimization of DT versus DT FWHM trendlines on a protein-specific basis, enhancing peptide identification relative to manually optimized trendlines. Beyond automating our previous approach, MultiPassMerger also introduces a novel "multi-trendline" processing method, involving iterative processing using multiple trendlines and merging of results to better sample ions across the DT versus DT FWHM distribution. Using MultiPassMerger with both multi-sequence and multi-trendline strategies increased peptide identifications up to 392% relative to SYNAPT G2-Si with linear ion mobility and 102% relative to use of single-pass cIM-MS alone. Consequently, MultiPassMerger can enhance peptide mapping and makes this approach practical and more accessible to the wider HDX-MS community. MultiPassMerger is available as a downloadable Windows executable at https://politislab.uk/multipassmerger.
Source: Protein Science
DOI: 10.1002/pro.70168
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $4,480
Awards: Capturing eukaryotic transporters in action, ERA CHAIR HOLDER TO ESTABLISH BIOMOLECULAR AND STRUCTURAL MASS SPECTROMETRY FOR THE ANALYSIS OF COMPLEX BIOLOGICAL PATHWAYS, G3904638509, G6371514603, 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
OpenAlex ID: W4410715868