Abstract
Proteasomes play a central role in antiviral and antitumor cytotoxic immunity, generating most of the >10,000 unique proteome-derived peptides bound to ∼200,000 human leukocyte antigen (HLA) class I molecules on each human cell. Here, we present a protocol for hybrid mass spectrometry-AI mapping of proteasome-generated HLA class I epitopes. We describe steps for in vitro processing of antigens by proteasomes, HLA class I allele selection, and peptide pool preparation. We then detail procedures for determining HLA class I ligands and population immunity. For complete details on the use and execution of this protocol, please refer to Kudriaeva et al.(1).