DOI: 10.5061/dryad.z08kprrr1
title: Targeted metabolomics data for dimethyl-ribityl lumazine (DMRL) and riboflavin in Mycobacterium tuberculosis and Mycolicibacterium smegmatis mutants of the riboflavin biosynthetic pathway

description: This dataset contains raw mutliple reaction monitoring (MRM) data for
 intermediate metabolites of the riboflavin biosynthetic pathway (RBP) in
 wild-type and riboflavin biosynthetic pathway gene deletion mutants
 of Mycobacterium
 tuberculosis and Mycolicibacterium smegmatis. These
 strains were generated to investigate the source of metabolic antigens
 recognized by mucosal-associated invariant T (MAIT) cells, which detect
 intermediates from the riboflavin biosynthetic pathway. To determine the
 impact of these gene deletions on the production of riboflavin and its
 precursors, we developed an MRM assay for DMRL and Riboflavin. This
 dataset provides evidence of how these disruption impact the biosynthesis
 of DMRL and Riboflavin which has enables a better understanding of the
 source of MR1 antigen in mycobacteria.
description: # MRM assay - Mycobacterium tuberculosis (DMRL and Riboflavin) Dataset
 DOI:
 [https://doi.org/10.5061/dryad.z08kprrr1](https://doi.org/10.5061/dryad.z08kprrr1) ## Description of the data This dataset contains data that contributes to key finding on the study of metabolic antigens recognized by MR1 restricted T cells in Mycobacteria. The data provided includes targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of riboflavin biosynthetic pathway mutants of Mycobacterium tuberculosis and Mycolicibacterium smegmatis. The dataset provides qualitative and quantitative analysis of Dimethyl-ribityl Lumazine and Riboflavin in the generated mutants. All LC-MS/MS data have been provided in the original format allowing processing with free and accessible tools.  #### Growth and preparation of Mtb and Msm for metabolomics Glycerol stocks of Mtb and *Msm* strains were used to start cultures on 7H11 agar plates supplemented with the appropriate antibiotics with or without riboflavin. Plates were incubated for 2 weeks for Mtb and Msm for 4 to 5 days at 37ºC. Cells were scraped from plates into 7H9 media supplemented with OADC with or without riboflavin and grown at 37ºC until mid-log phase. Cells were collected via centrifugation, washed with mass spectrometry grade water and resuspended in Acetonitrile/Methanol/Water (2:2:1) and then transferred to screw-cap homogenization microtubes prefilled with 0.1 mm zircon beads. The cells were homogenized using the FastPrep-24 bead beater (MP Biomedical, SKU:116004500) at 6 m/s for 30 seconds (10 rounds), cooling on ice for 2 minutes between cycles. Homogenized cells were collected and spun down at 16,000 g for 20 minutes and supernatant collected for metabolomics. Supernatant were then stored at -20ºC until further processed. #### **Liquid Chromatography -MS/MS analysis** Supernatant collected for metabolomics was dried in savant and their weight determined. Dried samples were then resuspended in 3% Acetonitrile in Water (0.1% Formic Acid) to make a final concentration of 2 mg/ml for Mtb and 10 mg/ml for Msm. Samples were analyzed using the Agilent 1290 Infinity III Liquid Chromatography System attached to a 6470 Triple Quadrupole MS system. 10 µl of samples were loaded per injection. Reverse phase UHPLC was performed using InfinityLab Poroshell 120 EC-C18 Column (Agilent, Part Number: 693975-902) (4.6 x 150 mm, 2.7 µm). Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile. LC separation was carried out at a flow rate of 0.3 µL/min using a linear gradient of 2% solvent B held for 1.5 minutes and then to 20% solvent B over a duration 5 minutes, then 20% solvent B to 99 % solvent B for 3 minutes. 99% solvent B was held for 0.5 minutes and then returned to 2% solvent B for 3 minutes for equilibration. Mass spectra and tandem mass spectra were carried out using the dynamic multiple reaction monitoring (DMRM) acquisition method in positive ion mode. Column temperature was set at 45 ºC. Source was operated with the following settings: 10 l/min of gas flow at 325ºC. Nebulizer was set at 20 psi. Sheath gas temperature was set at 350ºC with flow rate of 10 l/min. Capillary voltage was set at 3500 V and nozzle voltage set at 0 V. ## Files and variables #### File: Mtuberculosis\_MRM.zip **Description:** Contains .d raw MRM data for the aforementioned metabolites in Mycobacterium tuberculosis #### File: Msmegmatis\_MRM\_Analysis.zip **Description:** Contains .d raw MRM data for the aforementioned metabolites in Mycolicibacterium smegmatis #### File: MRM\_File\_Annotation.xlsx **Description:** Contains annotation information for all .d raw files. (Strain, Biological Replicate and Raw File Name) #### Naming Convention for files (Msmegmatis\_MRM\_Analysis.zip) 1. #### Sample (Msm: *Mycolicibacterium smegmatis* OR Blank) 2. Strain (WT: Wild Type, WTRF: Wild Type Grown with Riboflavin, A2: *ΔribA2,* A2A2*: ΔribA2::ribA2,* C: *ΔribC,* CC:* ΔribC::ribC,* FBIC:*  ΔfbiC,* FBIC_FBIC:*  ΔfbiC::fbiC,* G:*  ΔribG,* GG: * ΔribG::ribG,* H:*  ΔribH1,* HH:*  ΔribH1::ribH1,* H1H2:*  ΔribH2 ΔribH1, *H1H2H1H2*: ΔribH2 ΔribH1::ribH2 ribH1, *H1H2H2: ΔribH2 ΔribH1::ribH2,* H2: * ΔribH2, H2_H2:*  ΔribH2::ribH2*) 3. Biological Replicate (A, B, C) 4. Date of Collection  e.g. For file name “Msm_WTRF_B_020325" Sample: *Mycolicibacterium smegmatis*; Strain: Wild Type Grown with Riboflavin; Biological Replicate: B; Date of Collection: 02/03/25 #### Naming Convention for files (Mtuberculosis\_MRM.zip) 1. Sample (Mtb: *Mycobacterium tuberculosis* OR Blank) 2. Strain (WT: Wild Type, WT_RF*:* Wild Type Grown with Riboflavin*,* A2KO: *ΔribA2,* A2A2: *ΔribA2::ribA2,* CKO*: ΔribC,* CC:* ΔribC::ribC,* HKO:*  ΔribH,* H_H:*  ΔribH::ribH*) 3. Biological Replicate (A, B, C) 4. Project Name (MRM_Flavins) 5. Volume Injected for Analysis (10ul: 10 microliters) 6. Date of Collection  e.g. For file name “Mtb_WT_RF_B_MRM_Flavins_10ul_100924" Sample: *Mycobacterium tuberculosis*; Strain: Wild Type Grown with Riboflavin; Biological Replicate: B; Project Name: MRM Flavins; Volume Injected for Analysis: 10 microliters, Date of Collection: 10/09/24 ## Code/software Primary Software: Agilent Mass Hunter Qualitative Analysis Software V10.0 can be used to process and visualize .d files provided. Alternative Software: Skyline (Metabolite interface) is a free and open software that can be used to process and visualize .d files provided.