Secreted HLA-Fc fusion profiles immunopeptidome in hypoxic PDAC and cellular senescence.

Human leukocyte antigens (HLA) present peptides largely from intracellular proteins on cell surfaces. As these complexes can serve as biomarkers in disease, proper identification of peptides derived from disease-associated antigens and the corresponding presenting HLA is important for the design and execution of therapeutic strategies. Yet, current mass spectrometry methods for immunopeptidomic profiling require large and complex sample inputs, hindering the study of certain disease phenotypes and lowering confidence in peptide and allele identification. Here, we describe a secreted HLA (sHLA)-Fc fusion construct for simple single HLA allele profiling in hypoxic pancreatic ductal adenocarcinoma (PDAC) and cellular senescence. This method streamlines sample preparation, enables temporal control, and provides allele-restricted target identification. Over 30,000 unique HLA-associated peptides were identified across 2 different HLA alleles and 7 cell lines, with ∼9,300 peptides newly discovered. The sHLA-Fc fusion capture technology holds the potential to expedite immunopeptidomics and advance therapeutic interest in HLA-peptide complexes.