Systematic Workflow Optimization for Ultra-Sensitive Targeted Immunopeptidomics

Area of Science:

• Mass spectrometry
• Immunopeptidomics
• Proteomics

Background:

• Epitope detection sensitivity is a major challenge in mass spectrometry (MS)-based immunopeptidomics.
• Conventional discovery workflows (DDA, DIA) often lack the sensitivity for ultra-low abundant targets like viral or neoepitopes.

Purpose of the Study:

• To develop an ultra-sensitive, targeted-first workflow (optiPRM+) for enhanced epitope detection on the Orbitrap Exploris 480 platform.
• To bridge the sensitivity gap in immunopeptidomics by integrating optimized targeted acquisition with untargeted DIA.

Main Methods:

• Empirical characterization of target peptides via direct infusion-MS to optimize fragmentation.
• Inclusion list-driven data-dependent acquisition (iDDA) with ultra-high MS2 resolutions (up to 480,000) and long injection times.
• Optimization of fragmentation energy for non-tryptic peptides based on charge state and basic amino acid count.

Main Results:

• Successfully identified and validated the HLA-A*02:01-restricted epitope TIHDIILECV in Human Papillomavirus type 16 (HPV16).
• Achieved the first MS-based detection of two novel HPV16 viral epitopes: ISEYRHYCY (HLA-A*01:01) and CVYCKQQLLR (HLA-A*11:01).
• Confirmed that these ultra-low abundance peptides were undetectable by conventional untargeted DIA methods.

Conclusions:

• The optiPRM+ workflow significantly improves sensitivity for detecting biologically relevant epitopes.
• A systematically optimized targeted-first approach can uncover critical epitopes missed by standard discovery workflows.
• This method is crucial for identifying viral or mutation-derived neoepitopes in challenging biological contexts.