Beyond HCD: complementary fragmentation chemistries expand HLA class I immunopeptidome discovery
- 1Monash University
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.
Related
- Inhibition of cellular NEDDylation partially alters the MHC class I immunopeptidome
- A monoallelic immunopeptidome atlas of 10 prevalent canine DLA-88 alleles
- eHLAstic reveals an extended canonical and non-canonical HLA-E immunopeptidome in human cancers
- Beyond χ2: Ensemble null tests for reliable scientific discovery
- Beyond hepatitis D virus: the discovery of animal deltaviruses, their diversity, helpers and their position in an expanding world of circular RNA replicons
BibTeX
@misc{sian2026beyond,
title = {{Beyond HCD: complementary fragmentation chemistries expand HLA class I immunopeptidome discovery}},
author = {T. C. C. Lim Kam Sian and F. Salvato and N. Selvakumar and T. Shamekhi and J. Hinkle and C. Mullen and G. A. Goncalves and R. B. Schittenhelm and P. Faridi},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.24.754253},
url = {https://www.biorxiv.org/content/10.64898/2026.09.24.754253v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Beyond HCD: complementary fragmentation chemistries expand HLA class I immunopeptidome discovery
AU - T. C. C. Lim Kam Sian
AU - F. Salvato
AU - N. Selvakumar
AU - T. Shamekhi
AU - J. Hinkle
AU - C. Mullen
AU - G. A. Goncalves
AU - R. B. Schittenhelm
AU - P. Faridi
PY - 2026
DA - 2026/09/24
PB - bioRxiv
DO - 10.64898/2026.09.24.754253
UR - https://www.biorxiv.org/content/10.64898/2026.09.24.754253v1
AB - 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.
ER -