Immunopeptidomics aims to facilitate T-cell target discovery by analyzing human leukocyte antigen (HLA)-bound peptides using tandem mass spectrometry (MS/MS). MS/MS analysis of peptides conventionally relies on collision-induced dissociation (CID) of selected precursor ions. However, CID analysis of immunopeptides, owing to their diverse termini (nontryptic) and relatively short lengths (8- to 12-mer for class I HLA), can be challenging. It is impossible to differentiate isobaric amino acids, such as isoleucine and leucine sequence variants, which are frequent amino acids in the immunopeptidomes of many HLA allotypes. To address this, we leveraged the alternative fragmentation method, Electron-Activated Dissociation (EAD), and its CID-supplemented mode (EAD x CID, or ExCID) on the SCIEX ZenoTOF 7600+ system to characterize Ile/Leu sequence variants in the immunopeptidome. We first used a panel of seven synthetic Ile/Leu-substituted peptides and demonstrated that both EAD and ExCID efficiently generate variant-distinguishing w- and d-type satellite ions. We then determined optimal kinetic energy (KE) and EAD reaction time for utilizing hybrid ExCID ion activation in a discovery immunopeptidomics setting. We demonstrated that Ile/Leu sequence variants present in the immunopeptidome can be distinguished using ExCID but not CID alone. Using this optimized ExCID methodology, we observed improvements in fragmentation and peptide identification for both HLA class I and II studies, allowing us to benefit from EAD while minimizing the compromise to cycle time and identification depth. We further applied the optimal ExCID method to complex HLA peptide samples isolated from two model cell lines, namely, a “monoallelic” HLA-A2 expressing cell line (C1R-A*02:01) and the pancreatic cancer cell line Panc1. The versatile and tunable hybrid ExCID mode improves MS/MS fragment coverage and enriches the product spectra with diagnostic ions, enabling us to differentiate and characterize Ile/Leu sequence variants in immunopeptidomics studies.
Dorvash et al. (Tue,) studied this question.