# Protocol for MR1-ligand cross-linking to discover metabolite antigens by proteomics

## TL;DR

This paper introduces a method to identify MR1-bound metabolite ligands using cross-linking and proteomics techniques.

## Contribution

A novel protocol is presented for discovering MR1 ligands through covalent cross-linking and mass spectrometry.

## Key findings

- MR1-ligand complexes can be enriched using MR1-expressing A549 cells.
- Ligands are covalently cross-linked to MR1 lysine 43 via reductive amination.
- Candidate ligands are validated using MR1 surface upregulation and TCR functional assays.

## Abstract

Here, we present a protocol for the discovery of MR1-bound metabolite ligands by stabilizing Schiff base interactions between MR1 and its ligands. We describe steps for enriching MR1-ligand complexes using MR1-expressing A549 cells and covalently cross-linking ligands to the MR1 lysine 43 residue via reductive amination. We then detail procedures for identifying ligand-modified MR1 peptides by mass spectrometry and for further validation of candidate ligands using MR1 surface upregulation and TCR functional assays.

For complete details on the use and execution of this protocol, please refer to Schmidlin et al.1

•Protocol for MR1 ligand discovery by crosslinking and proteomics•Affinity enrichment of MR1-ligand complexes from engineered cell lines•Mass spectrometry-based detection of MR1-ligand adducts•Integrated data analysis for ligand candidate selection and biochemical validation

Protocol for MR1 ligand discovery by crosslinking and proteomics

Affinity enrichment of MR1-ligand complexes from engineered cell lines

Mass spectrometry-based detection of MR1-ligand adducts

Integrated data analysis for ligand candidate selection and biochemical validation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for the discovery of MR1-bound metabolite ligands by stabilizing Schiff base interactions between MR1 and its ligands. We describe steps for enriching MR1/ligand complexes using MR1-expressing A549 cells and covalently crosslinking ligands to the MR1 lysine 43 residue via reductive amination. We then detail procedures for identifying ligand-modified MR1 peptides by mass spectrometry and for further validation of candidate ligands using MR1 surface upregulation and TCR functional assays.