Distinguishing Ile/Leu Variant Ligands in the Immunopeptidome Using Hybrid EAD + CID Fragmentation (ExCID)

Area of Science:

• Proteomics
• Mass Spectrometry
• Immunology

Background:

• Immunopeptidomics analyzes human leukocyte antigen (HLA)-bound peptides for T-cell target discovery.
• Tandem mass spectrometry (MS/MS) using collision-induced dissociation (CID) faces challenges with immunopeptides due to diverse termini and short lengths.
• Differentiating isobaric amino acids like isoleucine and leucine is difficult with conventional CID.

Purpose of the Study:

• To evaluate Electron-Activated Dissociation (EAD) and its hybrid mode (ExCID) for characterizing isoleucine/leucine variants in immunopeptidomics.
• To optimize ExCID parameters for improved fragmentation and peptide identification in HLA class I and II studies.

Main Methods:

• Utilized EAD and ExCID fragmentation on a SCIEX ZenoTOF 7600+ system.
• Tested synthetic Ile/Leu-substituted peptides to demonstrate variant-distinguishing ion generation.
• Optimized kinetic energy (KE) and reaction time for hybrid ExCID in discovery immunopeptidomics.
• Applied optimized ExCID to HLA peptide samples from C1R-A*02:01 and Panc1 cell lines.

Main Results:

• EAD and ExCID efficiently generated variant-distinguishing w- and d-type satellite ions.
• ExCID successfully differentiated Ile/Leu sequence variants, while CID alone could not.
• Optimized ExCID improved fragmentation and peptide identification for both HLA class I and II.
• ExCID enhanced MS/MS fragment coverage and enriched product spectra with diagnostic ions.

Conclusions:

• The hybrid ExCID mode is a versatile and tunable method for differentiating Ile/Leu variants in immunopeptidomics.
• ExCID offers significant advantages over CID for characterizing immunopeptides, improving T-cell target discovery.
• This methodology enhances the characterization of complex HLA peptide samples.