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

## TL;DR

This paper introduces a protocol combining mass spectrometry and AI to map peptides generated by proteasomes that bind to HLA class I molecules, important for immune responses.

## Contribution

The novel contribution is a hybrid protocol integrating mass spectrometry and AI to map proteasome-generated HLA class I epitopes.

## Key findings

- The protocol enables in vitro processing of antigens by proteasomes and selection of HLA class I alleles.
- AI is applied to predict potential HLA class I peptide binders and identify physiologically relevant epitope pools.

## 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

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.