diaPASEF Analysis for HLA-I Peptides Enables Quantification of Common Cancer Neoantigens

Area of Science:

• Immunology
• Proteomics
• Analytical Chemistry

Background:

• Human leukocyte antigen class I (HLA-I) molecules present peptides to T cells, crucial for immune surveillance.
• Identifying and quantifying low-abundance HLA-I peptides, especially tumor neoantigens, remains a significant challenge in mass spectrometry.
• Existing methods require enhanced sensitivity for comprehensive immunopeptidome analysis.

Purpose of the Study:

• To evaluate data-independent acquisition (DIA) combined with parallel accumulation serial fragmentation ion mobility (diaPASEF) for high-sensitivity HLA-I peptide identification and quantification.
• To assess the performance of diaPASEF for detecting low-abundance peptides, including neoantigens, from limited sample amounts.
• To optimize a streamlined diaPASEF workflow for immunopeptidome analysis.

Main Methods:

• Implementation of a streamlined diaPASEF workflow for HLA-I peptide analysis.
• Utilized HLA binder-specific in silico predicted spectral libraries to improve peptide identification.
• Applied SILAC-DIA for quantitative analysis of HLA-I peptides and verified neoantigen quantification using spiked synthetic peptides.

Main Results:

• Identified 11,412 unique peptides and 3426 8-11mer peptides from as few as 500,000 cells with high reproducibility.
• Achieved high quantitative accuracy up to 5-fold dilution using SILAC-DIA.
• Successfully identified and quantified 13 shared neoantigens in a C1R cell line model.

Conclusions:

• The diaPASEF workflow significantly increases HLA-I peptidome coverage, particularly for low sample amounts.
• DIA's sensitivity and quantitative precision enable the detection and quantification of low-abundance peptides like neoantigens.
• This approach facilitates the analysis of tumor-specific epitopes and advances immunopeptidome research.