Human leukocyte antigen (HLA) class I immunopeptidomes represent crucial targets for immunotherapy development. However, their identification remains challenging due to low abundance, high dynamic range, and complex physicochemical properties. Current mass spectrometry workflows primarily rely on solely collision-based fragmentation methods, such as collision-induced dissociation (CID) or higher-energy collision-induced dissociation (HCD), which are susceptible to sequence-specific fragmentation biases and often generate a limited proportion of informative MS2 spectra. Consequently, these methods may fail to detect clinically relevant peptides, such as HLA-A*02:01-bound peptides that are central to many immunotherapy strategies. These limitations can compromise peptide sequence assignments in database searches, hinder the identification of post-translational modifications (PTMs), and reduce the accuracy of de novo sequencing approaches.
To address these challenges, Kessler et al. evaluated the Orbitrap Excedion Pro, a novel hybrid mass spectrometer featuring enhanced electron-transfer/higher-energy collision dissociation (EThcD) capabilities. This system employs dual fragmentation, generating both ETD-induced (c/z) and HCD-induced (b/y) fragment ion series within a single MS2 spectrum, enabling comprehensive immunopeptidome analysis. Their workflow incorporated an Aurora Ultimate™ 25 cm×75 µm C18 UHPLC column on a Vanquish Neo UHPLC system coupled with the Orbitrap Excedion Pro mass spectrometer.
This exploratory study demonstrated that EThcD fragmentation achieved 9.3-16.4% higher identification rates compared to HCD alone across multiple cell lines, with median sequence coverage increasing by 12.5-20% across different HLA alleles. The dual b/y and c/z ion series provided by EThcD enabled identification of 15-37% more unique peptides per HLA allele, particularly benefiting the detection of peptides containing internal arginine residues, which are poorly fragmented by HCD.
The researchers further revealed that arginine-rich immunopeptides, especially those presented by HLA-B*07:02, are systematically missed by collision-based methods, potentially leading to overlooked therapeutic targets. This technology addresses a critical gap in immunopeptidome coverage, enabling more comprehensive identification of clinically relevant antigens for next-generation immunotherapies.
Publication
bioRxiv
Authors
Amy L. Kessler, Kyle L. Fort, Hanno C. Resemann, Peter Krüger, Cong Wang, Heiner Koch, Jan-Peter Hauschild, Fabio Marino, Albert J.R. Heck
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