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Human leukocyte antigens (HLA) display peptides largely from intracellular proteins on the surface of cells in major histocompatibility complex (MHC)-peptide complexes. These complexes provide a biological window into the cell, and peptides derived from disease-associated antigens can serve as biomarkers and therapeutic targets. Thus, proper identification of peptides and the corresponding presenting HLA allele in disease phenotypes is important for the design and execution of therapeutic strategies using engineered T-cell receptors or antibodies. Yet, current mass spectrometry methods for profiling the immunopeptidome typically require large and complex sample inputs, complicating the study of several disease phenotypes and lowering the confidence of both peptide and allele identification. Here, we describe a novel secreted HLA (sHLA) Fc-fusion construct that allows for simple peptide identification from single HLA alleles in two important disease models: hypoxic pancreatic ductal adenocarcinoma (PDAC) and cellular senescence. We identify hypoxia and senescence-associated peptides that could act as future targets for immunotherapy. More generally, the method streamlines the time between sample preparation and injection from days to hours, yielding allele-restricted target identification in a temporally controlled manner. Overall, this method identified >30,000 unique HLA-associated peptides across two different HLA alleles and seven cell lines. Notably, ∼9,300 of these unique HLA-associated peptides had previously not been identified in the Immune Epitope Database. We believe the sHLA Fc-fusion capture technology will accelerate the study of the immunopeptidome as therapeutic interest in HLA-peptide complexes increases in cancer and beyond.