Multi-Enzyme Data-Independent Acquisition Mass Spectrometry for Proteoform-Aware Single-shot Proteomics
- 1Division of Infection Medicine, Department of Clinical Sciences, Lund University, Klinikgatan 32, Lund, Sweden
Abstract
Biological regulation does not operate at the level of the protein but across ensembles of protein variants shaped by alternative splicing, post-translational modification and endogenous proteolytic processing. Yet most conventional bottom-up discovery proteomics workflows remain optimized for a canonical protein census, leaving much of the sequence-level variation invisible. Multi-enzyme digestion has been recognized as an effective strategy to expand sequence coverage and proteoform sensitivity, but existing implementations rely on resource-heavy per-enzyme liquid chromatography coupled to mass spectrometry (LC-MS) analysis, dedicated measurements for spectral library construction, and semi-manual reconciliation across multiple software tools, restricting the use to specialized laboratories and limited sample cohorts. Here we introduce mE-DIA, a multi-enzyme, proteoform-resolved data independent acquisition (DIA)-MS workflow that multiplexes complementary protease digested samples into a single DIA injection and processes them through a library-free pipeline within a single analysis engine, without supplemental data dependent acquisition (DDA)-MS measurements. The mE-DIA Pipeline graphical user interface orchestrates the analysis, and a dedicated proteoform inference algorithm and the interactive mE-DIA Peptide Mapper viewer make the sequence-level evidence explorable. Comparing the workflow to prevailing DDA-library-based multi-enzyme DIA on the same raw data and within shared search space showed increased identified protein groups by 30 % and protein groups quantified at [≤] 20 % CV by 47 %. A three-enzyme multiplex of the human pathogen Streptococcus pyogenes resolved 990 protein-backbone variants among 309 canonical protein entries, 1.8-fold more than the most productive single-enzyme analysis, including an H69 C-terminal proteoform of the ribosomal protein uS19 that the trypsin-only workflow did not resolve. By consolidating multi-enzyme acquisition, analysis and proteoform interpretation into one accessible toolset, mE-DIA brings sub-gene-resolved evidence within reach of routine single-shot discovery DIA proteomics.
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BibTeX
@misc{heusel2026multienzyme,
title = {{Multi-Enzyme Data-Independent Acquisition Mass Spectrometry for Proteoform-Aware Single-shot Proteomics}},
author = {M. Heusel and L. Happonen and C. A. Q. Karlsson and E. Ahrman and J. Malmstrom},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.06.764873},
url = {https://www.biorxiv.org/content/10.64898/2026.11.06.764873v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Multi-Enzyme Data-Independent Acquisition Mass Spectrometry for Proteoform-Aware Single-shot Proteomics
AU - M. Heusel
AU - L. Happonen
AU - C. A. Q. Karlsson
AU - E. Ahrman
AU - J. Malmstrom
PY - 2026
DA - 2026/11/06
PB - bioRxiv
DO - 10.64898/2026.11.06.764873
UR - https://www.biorxiv.org/content/10.64898/2026.11.06.764873v1
AB - Biological regulation does not operate at the level of the protein but across ensembles of protein variants shaped by alternative splicing, post-translational modification and endogenous proteolytic processing. Yet most conventional bottom-up discovery proteomics workflows remain optimized for a canonical protein census, leaving much of the sequence-level variation invisible. Multi-enzyme digestion has been recognized as an effective strategy to expand sequence coverage and proteoform sensitivity, but existing implementations rely on resource-heavy per-enzyme liquid chromatography coupled to mass spectrometry (LC-MS) analysis, dedicated measurements for spectral library construction, and semi-manual reconciliation across multiple software tools, restricting the use to specialized laboratories and limited sample cohorts. Here we introduce mE-DIA, a multi-enzyme, proteoform-resolved data independent acquisition (DIA)-MS workflow that multiplexes complementary protease digested samples into a single DIA injection and processes them through a library-free pipeline within a single analysis engine, without supplemental data dependent acquisition (DDA)-MS measurements. The mE-DIA Pipeline graphical user interface orchestrates the analysis, and a dedicated proteoform inference algorithm and the interactive mE-DIA Peptide Mapper viewer make the sequence-level evidence explorable. Comparing the workflow to prevailing DDA-library-based multi-enzyme DIA on the same raw data and within shared search space showed increased identified protein groups by 30 % and protein groups quantified at [≤] 20 % CV by 47 %. A three-enzyme multiplex of the human pathogen Streptococcus pyogenes resolved 990 protein-backbone variants among 309 canonical protein entries, 1.8-fold more than the most productive single-enzyme analysis, including an H69 C-terminal proteoform of the ribosomal protein uS19 that the trypsin-only workflow did not resolve. By consolidating multi-enzyme acquisition, analysis and proteoform interpretation into one accessible toolset, mE-DIA brings sub-gene-resolved evidence within reach of routine single-shot discovery DIA proteomics.
ER -