Development of HPLC protocol for isolation of MHC class I related protein 1 (MR1) antigens from mycobacteria

Major histocompatibility class I related protein 1 (MR1) restricted T-cells (MR1T cells) play an important role during Mycobacterium tuberculosis (Mtb) infection. Unlike conventional T-cells that recognize peptides, MR1T cells recognize metabolic antigens. Studies to better understand the role of MR1T cells during Mtb infection depend on the identification of their cognate metabolic antigens. Although we and others have shown the importance of the riboflavin biosynthetic pathway for the production of these MR1 antigens, the identity of these metabolites from Mtb is yet to be determined. The identification of these antigens has been hampered by their relatively low abundance and the complex nature of the metabolome of Mtb. Also, Mtb produces several metabolites that competitively inhibit and antagonize MR1 antigens. Therefore, the discovery of MR1 antigens from Mtb poses a significant challenge. To overcome this challenge, we developed a high-performance liquid chromatography (HPLC) workflow to enrich for MR1 antigens from Mtb. First, by comparing multiple extraction approaches, we found that mechanical disruption by bead beating combined with an acetonitrile:methanol:water solvent system (2,2,1, v/v/v) provided the most efficient and reproducible extraction of MR1 ligands from mycobacteria. Finally, we optimized key chromatographic parameters, including mobile-phase pH, injection solvent, and column chemistry, to ensure stable and reproducible enrichment of MR1 ligands from mycobacterial extracts. Optimal separation was achieved using a HILIC column (BEH Amide) at pH 6, with a polar organic solvent system used for both extraction and sample injection. Under these conditions, we successfully enriched MR1 antigens from Mtb. This study serves as the first comprehensive report describing an enrichment workflow for MR1 antigens from mycobacteria. Further work will focus on identifying these ligands by integrating MR1 tetramer-based loading with LC-MS/MS analysis of the enriched fractions. Collectively, this method provides a practical platform to facilitate discovery of novel MR1 antigens from Mtb and other mycobacterial species.