Profinder b 06

Liquid chromatography mass spectrometry (LC-MS) differentiation and detection of WT Mtb, ICL knock-down, BCG, M. bovis, and ΔRD1 metabolites were performed with an Agilent Accurate Mass 6230 TOF coupled with an Agilent 1290 Liquid Chromatography system using a Cogent Diamond Hydride Type C column (Microsolve Technologies, Long Branch, NJ, USA) using solvents and configuration as previously described^{68 (link)}. An isocratic pump was used for continuous infusion of a reference mass solution to allow mass axis calibration. Detected ions were classified as metabolites based on unique accurate mass-retention time identifiers for masses showing the expected distribution of accompanying isotopologues. Metabolites were analyzed using Agilent Qualitative Analysis B.07.00 and Profinder B.06.00 software (Agilent Technologies, Santa Clara, CA, USA) with a mass tolerance of <0.005 Da. Standards of authentic chemicals of known amounts were mixed with bacterial lysates and analyzed to generate the standard curves used to quantify metabolite levels. All data obtained by metabolomics were the average of at least two independent triplicates for each condition tested.

Data were acquired with MassHunter Workstation LC/MS Data Acquisition software B 05.00 (Agilent Technologies, Waldbronn, Germany), and was subsequently analyzed with Profinder B.06.00 (Agilent Technologies) extracting the so-called ‘features’ by their RT, molecular mass, and their peak intensity in various extracts. This was performed in a combination of the three injections of each sample after removing the features found in the corresponding blank samples. The parameters are set to a peak filter of 1000 counts peak height, ion species to ‘positive ions’ with H⁺, Na⁺, K⁺, and NH₄⁺, ‘charge state’ to 1, the ‘expected RT’ to ±3.00 min, and the mass to ±10 ppm. The extracted ion chromatograms (EICs) were smoothed with a Gaussian function using 9 points function width and 5000 points Gaussian width. This limits the result finally to 2000 compound groups. The detailed workflow was discussed in Wahman et al., 2022 [20 (link)].

LC-MS differentiation and detection of Erdman, CDC1551, and mutant strains were performed with an Agilent Accurate mass 6230 time of flight (TOF) device coupled with an Agilent 1290 liquid chromatography system using solvents and configuration as previously described (11 (link), 13 (link)). An isocratic pump was used for continuous infusion of a reference mass solution to allow mass axis calibration. Detected ions were classified as metabolites based on unique accurate mass-retention time identifiers for masses showing the expected distribution of accompanying isotopologues. Metabolites were analyzed using Agilent Qualitative Analysis B.07.00 and Profinder B.06.00 software (Agilent Technologies, Santa Clara, CA, USA) with a mass tolerance of <0.005 Da. Standards of authentic chemicals of known amounts were mixed with bacterial lysates and analyzed to generate the standard curves used to quantify metabolite levels.