Immunopeptidomics Workflow for Isolation and LC-MS/MS Analysis of MHC Class I-Bound Peptides Under Hypoxic Conditions.

Immunopeptidomics enables the identification of peptides presented by major histocompatibility complex (MHC) molecules, offering insights into antigen presentation and immune recognition. Understanding these mechanisms in hypoxic conditions is crucial for deciphering immune responses within the tumor microenvironment. Current immunopeptidomics approaches do not capture hypoxia-induced changes in the repertoire of MHC-presented peptides. This protocol describes the isolation of MHC class I-bound peptides from in vitro hypoxia-treated cells, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. It describes optimized steps for cell lysis, immunoaffinity purification, peptide elution, and MS-compatible preparation under controlled low-oxygen conditions. The method is compatible with various quantitative mass spectrometry approaches and can be adapted to different cell types. This workflow provides a reliable and reproducible approach to studying antigen presentation under hypoxic conditions, thereby enhancing physiological relevance and facilitating deeper immunological insights. Key features • Enables isolation of MHC class I-bound peptides from cells cultured under hypoxic conditions. • Designed for low-input samples and optimized for maintaining cell viability during extended hypoxic exposure. • Compatible with label-free LC-MS/MS for detailed immunopeptidome analysis. • Adaptable to all human and murine cell lines commonly used in cancer and immunology research.