DOI: 10.5061/dryad.pg4f4qs39
title: Targeted metabolomics data for riboflavin, FMN and FAD in <em>Mycobacteria tuberculosis</em> and <em>Mycolicibacterium smegmatis</em>

description: Riboflavin biosynthesis is essential for most microorganisms, yet its
 production is tightly regulated due to the potential toxic effects of
 riboflavin and its derivatives. While regulatory mechanisms for this
 process have been described in other organisms, none have been identified
 in mycobacteria. Uncovering such a mechanism will be critical to fully
 exploit the riboflavin biosynthetic pathway as a pharmacological target.
 We previously observed that Mycobacterium tuberculosis (Mtb) and
 Mycolicibacterium smegmatis (Msm) can grow successfully under elevated
 riboflavin levels, prompting an investigation into how mycobacteria
 tolerate high intracellular riboflavin. Using a bioinformatic approach of
 proteomic data, we explored the global proteomic response of Mtb and Msm
 to elevated riboflavin. Our results revealed increased abundance of known
 flavin sequesters in response to high riboflavin levels, suggesting a role
 in flavin homeostasis. Using a transcription regulatory network analysis,
 we observed the employment of similar regulatory networks, most notably
 DosR, in both Mtb and Msm in response to elevated riboflavin. We also
 identified a potential link between the riboflavin-induced response and
 isoniazid resistance mechanisms, warranting further investigation.
 Overall, this study provides evidence for the involvement of flavin
 sequesters in maintaining flavin homeostasis and highlights the potential
 regulatory role of DosR in this process.
description: # Targeted metabolomics data for riboflavin, FMN, and FAD in *Mycobacteria
 tuberculosis* and *Mycolicibacterium smegmatis* Dataset DOI:
 [10.5061/dryad.pg4f4qs39](https://doi.org/10.5061/dryad.pg4f4qs39) ##
 Description of the data and file structure This dataset contains raw
 targeted MS/MS data from wild-type *Mycobacterium tuberculosis* and
 *Mycolicibacterium smegmatis* treated with or without exogenous
 riboflavin, focusing on terminal metabolites of the riboflavin
 biosynthetic pathway. To assess how riboflavin availability influences the
 synthesis of FMN and FAD, a targeted high-resolution mass spectrometry
 (HRMS) assay was developed for riboflavin, flavin mononucleotide (FMN),
 and flavin adenine dinucleotide (FAD). The dataset includes qualitative
 and quantitative LC-MS/MS analyses and provides evidence for how
 mycobacteria regulate FMN and FAD synthesis from available riboflavin,
 contributing to key findings on flavin homeostasis in mycobacteria. All
 LC-MS/MS data are provided in original formats, enabling 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 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,e s the nd supernatant
 was collected for metabolomics. Supernatants were then stored at -20ºC
 until further processed. **Liquid Chromatography -MS/MS analysis** The
 supernatant collected for metabolomics was dried in a Savant and its
 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 an Agilent 6546
 quadrupole time-of-flight MS system. 2 µl of samples were loaded per
 injection. Reverse phase UHPLC was performed using InfinityLab Poroshell
 120 EC-C18 Column (Agilent, Part Number: 693975-902) (2.1 x 100 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 0.32 µl/min using a linear gradient of 2% solvent B held for 3 minutes,
 and then to 50% solvent B over a duration of 15 minutes, then 50% solvent
 B to 99% solvent B over 7 minutes. 99% solvent B was held for 6.5 minutes
 and then returned to 2% solvent B for 1.5 minutes and held for 5 minutes
 for equilibration. The sample eluted within the first minute was diverted
 to waste to prevent clogging. Mass spectra and tandem mass spectra were
 carried out using the Targeted MS/MS acquisition method in positive ion
 mode. Column temperature was set at 45 ºC. Source was operated with the
 following settings: 3500 capillary voltage, 10.0 l/min of dry gas at 40
 psi, and 320°C. Post MS-inlet capillary, fragmentor was set to 120 V,
 nozzle voltage was set to 1000 V, and skimmer voltage was set to 40 V. The
 peak-to-peak voltage of the ion transfer octupole was set at 750 V. The
 mass analyzers for MS and MS/MS were set to scan between m/z 100 to 1700
 at a scan rate of 4 spectra/sec and an isolation width of H1.3 amu. 3
 precursors were selected per cycle with an absolute threshold of 200 and a
 relative threshold of 0.01%. Mass error tolerance was set to ± 20 ppm, and
 retention time exclusion tolerance was set to ± 0.2 minutes. Collision
 energy was alternated between 15 and 30 volts during the cycle. ### Files
 and variables File:250912_NO_FMN_FAD_Msm_Mtb.zip Description: Contains .d
 raw HRMS data for the aforementioned metabolites in Mycobacterium
 tuberculosis and Mycolicibacterium smegmatis File:
 HRMS_File_Annotation.xlsx Description: Contains annotation information for
 all .d raw files. (Strain, Biological Replicate and Raw File Name,
 Treatment) Naming Convention for files 1. Strain (Msm: Mycolicibacterium
 smegmatis, Mtb: Mycobacterium tuberculosis OR Blank)-Strain (Wild Type
 (WT)) 2. Treated with Riboflavin (RF) 3. Biological Replicate (1, 2,
 3)-NA: not applicable for blank 4. Type (test or blank) 5. File name (raw
 file names included) e.g., For file name
 “250828_RibHexcl_038_Msm_RF_WT2.d" Sample: Date of Collection:
 08/28/25; Sample Table: Ri.bHexcl; Queue Number: 38;
 Organism: Mycolicibacterium smegmatis; Treated with riboflavin; Strain:
 Wild Type; Biological Replicate: 2 ## 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. ## Access information Other
 publicly accessible locations of the data: * N/A Data was derived from the
 following sources: * N/A