Improving Spectral Validation Rates in Hydrogen–Deuterium Exchange Data Analysis

Abstract

The data analysis practices associated with hydrogen− deuterium exchange mass spectrometry (HX-MS) lag far behind that of most other MS-based protein analysis tools. A reliance on external tools from other fields and a persistent need for manual data validation restrict this powerful technology to the expert user. Here, we provide an extensive upgrade to the HX data analysis suite available in the Mass Spec Studio in the form of two new apps (HX-PIPE and HX-DEAL), completing a workflow that provides an HXtailored peptide identification capability, accelerated validation routines, automated spectral deconvolution strategies, and a rich set of exportable graphics and statistical reports. With these new tools, we demonstrate that the peptide identifications obtained from undeuterated samples generated at the start of a project contain information that helps predict and control the extent of manual validation required. We also uncover a large fraction of HX-usable peptides that remains unidentified in most experiments. We show that automated spectral deconvolution routines can identify exchange regimes in a project-wide manner, although they remain difficult to accurately assign in all scenarios. Taken together, these new tools provide a robust and complete solution suitable for the analysis of high-complexity HX-MS data.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“… HX-PIPE combines both identification of clean features from the MS1 scans and peptide assignment from MS/MS data to accomplish peptide curation with less need for manual intervention. Reproduced with permission from ref ( 651 ). Copyright 2021 American Chemical Society.…”

“…Therefore, there can potentially be highly useful peptides with clean signal in an HDX-MS data set that simply do not fragment and generate the type and quality MS/MS spectra required by the existing proteomics tools for confident identification. 651 …”

“…As an example, HeXicon uses a random-forest classifier using abundance, m / z values, retention time, and D-distribution to quantitatively select superior peptides . HX-PIPE (a recent tool in MassSpec Studio) combines analysis of resolved features within the MS1 spectra together with identifications from MS/MS data for an initial working set of peptides . The full scans are then further scrutinized to identify and omit any peptides that have potential spectral contamination from nearby overlapping features, thereby offering a rapid tool for curation of a peptide list to only the “clean” peptides (Figure ).…”

“…One current limitation with peptide assignment is that most peptide identification tools have been established for general proteomics and are less well-suited for identification of peptides derived from nonspecific proteases. Therefore, there can potentially be highly useful peptides with clean signal in an HDX-MS data set that simply do not fragment and generate the type and quality MS/MS spectra required by the existing proteomics tools for confident identification …”

Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

“…Full experimental parameters for each batch are outlined in Table S9. Finally, significant changes in deuteration was assigned based on two cutoffs (3 x SD and 1-p = 0.98) using MS Studio 94 .…”