Over a decade ago, the first bona-fide MR1 ligand, 6-formylpterin, was characterised. 10 years on, and the list of known MR1-binding metabolites remains limited. Elucidating MR1’s ligandome will increase our understanding of MR1 biology in many fields, including uncovering antigens which stimulate MR1-restricted T cell populations against cancer and bacterial infection, as well as characterising the pathway for metabolite loading and MR1 cell surface expression. Known MR1 ligands include metabolites with varying structures, stabilities, and chemical properties which are presented at low copy numbers on the cell surface. To enable identification of low abundance MR1-bound ligands, we have developed a method to stabilise short-lived metabolites prior to detection.
Here, we present a liquid phase extraction method capable of isolating, stabilising, and detecting a range of metabolites refolded with MR1 in a single sample. The ligands were selected based on their varying chemical properties, half-lives, and structures to develop a robust and sensitive protocol. A liquid chromatography coupled to mass spectrometry (LC-MS) detection set up was optimised for the simultaneous detection of each metabolite and their corresponding independent recovery efficiencies. Different conditions were tested alongside the liquid phase extraction for their effect on ligand recovery and we report their extraction efficiencies. This workflow will be adapted for future untargeted metabolomic ligand discovery studies.