HLA Ligand Atlas DIA: extending the benign immunopeptidomics resource with increased sensitivity through data-independent acquisition mass spectrometry.

The human leukocyte antigen (HLA)-presented peptide repertoire, termed immunopeptidome, plays a crucial role for T-cell mediated immune reactions. Previously, the human immunopeptidome of non-malignant tissues has been mapped in a large-scale study, the HLA Ligand Atlas, via high-resolution data-dependent acquisition (DDA) mass spectrometry. This publicly available and user-friendly web interface (https://hla-ligand-atlas.org) is frequently used as a benign tissue reference in antigen discovery, especially for immunotherapy of cancer. Here, we extend the HLA Ligand Atlas resource with paired data-independent acquisition (DIA) runs for all tissue-subject combinations. This novel dataset comprises 946 DIA HLA class I and II immunopeptidomic runs from 242 non-malignant human samples across 18 subjects and 29 distinct tissues. Together with the published DDA runs, this extends the range and depth of analyses performed on the HLA Ligand Atlas dataset. In a concise analysis, we showcase advantages of DIA over DDA concerning spectral sampling and sensitivity. These findings are attributed to the increased dynamic range in DIA, enabling the identification of peptide transitions with low signal intensities. Moreover, we demonstrate the superior sensitivity by applying an HLA-A*02:01 allotype-specific spectral library search to identify and quantify HLA-presented peptides. We encourage reanalysis of the provided DDA and DIA data in combination as a reference for future research concerning human immunology.