{
  "abstract": "Mass spectrometry based immunopeptidomics is limited by peptide identification. HLA class I ligands are non-tryptic, frequently carry low charge states and often fragment incompletely, yet nearly all workflows rely on a single activation method, higher-energy collisional dissociation (HCD). Unlike proteomics, where a protein is inferred from several peptides, in immunopeptidomics the peptide is itself the analyte and the candidate therapeutic target, so a ligand that is not identified is a target that is not found. Here we evaluate whether two alternative activation chemistries, ion trap collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD), recover ligands that HCD does not. To our knowledge this is the first application of either method to HLA class I immunopeptidomics, made practical by the integrated infrared laser of the Orbitrap Tribrid Apex MultiOmics. Four HLA-typed cell lines were analysed in technical triplicate against the canonical human proteome and a Ribo-seq-derived cryptic ORF database. Because fragment-intensity predictors are trained almost exclusively on HCD spectra, AlphaPeptDeep was fine-tuned separately for each method; fine-tuning and Percolator rescoring increased unique peptide identifications in every cell line and method combination (median +17.1% canonical, +54.5% cryptic), and fine-tuned models transferred across cell lines with a median loss of 0.4%, placing the three methods on a common analytical footing. On that footing the three methods yielded immunopeptidomes of similar size and composition, but each recovered peptides the others missed. HCD produced the most complete fragment-ion ladders and recovered more peptides from the basic-anchor alleles A*11:01 and A*33:03, both members of the A3 supertype. CID and IRMPD recovered a larger share of alleles without a basic C-terminal anchor and, for peptides containing no lysine, arginine or histidine, produced higher search scores and more matched b/y ion current in three of four cell lines. Each method therefore contributed exclusive peptides, and at a fixed budget of three LC-MS/MS runs, one run per method yielded on average more unique peptides than triplicate runs of a single method in all four cell lines (+0.4% to +10.9%). Of 239 novel canonical predicted HLA binders in the two cancer cell lines (HCT116 and MDA-MB-231) absent from the Immune Epitope Database, 55% were detected by only one method; nine derive from cancer-associated antigens. CID and IRMPD can therefore provide sequences that may not be identified by HCD alone, or with better spectral quality for a chemically defined subset of ligands. Their value lies alongside HCD rather than in its place, and realising it will require fragment-intensity models and acquisition schemes built for these chemistries rather than adapted from HCD.",
  "abstract_shown": true,
  "affiliations": [
    "Monash University"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 9,
  "authors": [
    {
      "name": "T. C. C. Lim Kam Sian"
    },
    {
      "name": "F. Salvato"
    },
    {
      "name": "N. Selvakumar"
    },
    {
      "name": "T. Shamekhi"
    },
    {
      "name": "J. Hinkle"
    },
    {
      "name": "C. Mullen"
    },
    {
      "name": "G. A. Goncalves"
    },
    {
      "name": "R. B. Schittenhelm"
    },
    {
      "affs": [
        0
      ],
      "name": "P. Faridi"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/1k4jeybrzg71n.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.24.754253",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754253v1.full",
  "id": "1k4jeybrzg71n",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754253v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "immunology"
    ],
    [
      "openalex",
      "1607|Advanced Proteomics Techniques and Applications"
    ]
  ],
  "openalex": "W7214897697",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754253v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-24",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/1k4jeybrzg71n.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:25:29Z",
      "native_id": "10.64898/2026.09.24.754253",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.24.754253v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:immunology",
      "subfield": "immunology"
    }
  ],
  "title": "Beyond HCD: complementary fragmentation chemistries expand HLA class I immunopeptidome discovery",
  "title_text": "Beyond HCD: complementary fragmentation chemistries expand HLA class I immunopeptidome discovery",
  "type": "preprint",
  "updated_at": "2026-10-30T12:25:29Z",
  "url": "https://research.draconitic.com/paper/1k4jeybrzg71n",
  "venue": "bioRxiv",
  "versions": []
}