{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Division of Infection Medicine, Department of Clinical Sciences, Lund University, Klinikgatan 32, Lund, Sweden"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 5,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "M. Heusel"
    },
    {
      "name": "L. Happonen"
    },
    {
      "name": "C. A. Q. Karlsson"
    },
    {
      "name": "E. Ahrman"
    },
    {
      "name": "J. Malmstrom"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/axd4w4cm4qq92.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.11.06.764873",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.11.06.764873v1.full",
  "id": "axd4w4cm4qq92",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.11.06.764873v1",
  "language": null,
  "licence": "CC-BY-NC",
  "licence_label": "CC BY-NC",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "systems biology"
    ]
  ],
  "openalex": null,
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.11.06.764873v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-11-06",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/axd4w4cm4qq92.ris",
  "sources": [
    {
      "first_seen": "2026-11-09T12:21:46Z",
      "native_id": "10.64898/2026.11.06.764873",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.11.06.764873v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:systems biology",
      "subfield": "systems-synthetic"
    }
  ],
  "title": "Multi-Enzyme Data-Independent Acquisition Mass Spectrometry for Proteoform-Aware Single-shot Proteomics",
  "title_text": "Multi-Enzyme Data-Independent Acquisition Mass Spectrometry for Proteoform-Aware Single-shot Proteomics",
  "type": "preprint",
  "updated_at": "2026-11-09T12:21:46Z",
  "url": "https://research.draconitic.com/paper/axd4w4cm4qq92",
  "venue": "bioRxiv",
  "versions": []
}