High-Coverage Immunopeptidomics Using timsTOF Mass Spectrometers with Thunder-DDA-PASEF Boosted by MS²Rescore

Major histocompatibility complex (MHC, or human leukocyte antigen, HLA) peptide ligands can be exploited to develop immunotherapies targeting immunogenic disease-specific immunopeptides, such as virus- or cancer mutation-derived peptides. Liquid chromatography coupled with mass spectrometry (LC-MS)-based immunopeptidomics is the gold standard for identifying MHC ligands. We previously optimized a workflow enabling the identification of more than 10,000 MHC class I ligands per cell line. This process comprises three major steps: (I) a high-recovery immunopeptidome enrichment, (II) an optimized MS acquisition in the timsTOF Pro called Thunder-Data-Dependent Acquisition with Parallel Accumulation-SErial Fragmentation (Thunder-DDA-PASEF), and (III) peptide identification using PEAKS XPro boosted by MS²Rescore data-driven rescoring. Here, we describe our workflow for deep-coverage immunopeptidomics step-by-step, from sample preparation to data analysis and validation.