Chemical Derivatization Strategy for Extending the Identification of MHC Class I Immunopeptides

Neoantigen-based
therapeutic vaccines have a high potential impact
on tumor eradication and patient survival. Mass spectrometry (MS)-based
immunopeptidomics has the capacity to identify tumor-associated epitopes
and pinpoint mutation-bearing major histocompatibility complex (MHC)-binding
peptides. This approach presents several challenges, including the
identification of low-abundance peptides. In addition, MHC peptides
have much lower MS/MS identification rates than tryptic peptides due
to their shorter sequence and lack of basic amino acid at C-termini.
In this study, we report the development and application of a novel
chemical derivatization strategy that combines the analysis of native,
dimethylated, and alkylamidated peptides by liquid chromatography–tandem
mass spectrometry (LC–MS/MS) to expand the coverage of the
MHC peptidome. The results revealed that dimethylation increases hydrophobicity
and ionization efficiency of MHC class I peptides, while alkylamidation
significantly improves the fragmentation by producing more y-ions
during MS/MS fragmentation. Thus, the combination of dimethylation
and alkylamidation enabled the identification of peptides that could
not be identified from the analysis of their native form. Using this
strategy, we identified 3148 unique MHC I peptides from HCT 116 cell
lines, compared to only 1388 peptides identified in their native form.
Among these, 10 mutation-bearing peptides were identified with high
confidence, indicating that this chemical derivatization strategy
is a promising approach for neoantigen discovery in clinical applications.