Quantitative Profiling of HLA Class I and II Antigens and Neoantigens in Tissue and PBMC Samples with an Improved Mass Spectrometry Approach

Major histocompatibility complex (MHC) molecules play a pivotal role in orchestrating immune responses by presenting antigenic peptides derived from both self and foreign proteins. In cancer, understanding the repertoire of tumor-associated antigens displayed by MHC molecules is critical for revealing how the immune system detects and responds to malignant cells. The identification of neoantigens—peptides arising from tumor-specific somatic mutations—has become a central focus in immunopeptidomic research.

A major challenge in systematic immunopeptidomics remains the high input material typically required for robust analysis.To address this, we developed a semi-automated workflow (Fig. 1) optimized for the reliable identification and quantification of immunopeptides from limited cell numbers and small tissue biopsies. We further assessed the compatibility of this workflow with allele-specific HLA enrichment. Finally, we tested its performance on peripheral blood mononuclear cells (PBMCs), a clinically relevant and commonly used sample matrix, demonstrating its utility in translational and clinical research settings.