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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.

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

  • 实验方案

  • 发表日期：

    2026年

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  • David Gomez-Zepeda,
  • Julian Beyrle,
  • Annica Preikschat,
  • Arthur Declercq,
  • Yannic Chen,
  • Ralf Gabriels,
  • Lennart Martens,
  • Ute Distler,
  • Stefan Tenzer

摘要

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.