Work

Protocol for hybrid mass spectrometry-AI mapping of proteasome-generated HLA class I epitopes

Year: 2025

Type: article

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 • Steps for obtaining cell lines stably expressing HTBH-tagged proteasomes • Instructions for in vitro processing of antigens by proteasomes • Guidance on applying artificial AI to predict potential HLA class I peptide binders • Procedures for identifying physiologically relevant pools of HLA class I epitopes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104154

Authors: Maxim T. Ri, George A. Saratov, Anna A. Kudriaeva, Alexey A. Belogurov

Institutions: Russian Academy of Sciences, Institute of Bioorganic Chemistry, Ministry of Health of the Russian Federation

Language: English

Topic: vaccines and immunoinformatics approaches

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550