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 described68 (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.
Accurate mass 6230 tof
Manufactured by Agilent Technologies
Sourced in United States
The Accurate Mass 6230 TOF is a time-of-flight mass spectrometer designed for accurate mass measurements. It provides high-resolution mass analysis with precise mass accuracy.
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Lab products found in correlation
Agilent 1290 liquid chromatography system, by Agilent Technologies (2 mentions)
Agilent qualitative analysis b 08, by Agilent Technologies (2 mentions)
Qualitative analysis b 07, by Agilent Technologies (1 mentions)
Profinder b 06, by Agilent Technologies (1 mentions)
1290 infinity lc system, by Agilent Technologies (1 mentions)
Zorbax rrhd extend c18 column, by Agilent Technologies (1 mentions)
4 protocols using accurate mass 6230 tof
1
Metabolomics Analysis of Mycobacterial Strains
2
Quantification of Intracellular Rifabutin in A. baumannii
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Extractions and measurements were done as previously described 33 (link),34 (link). Log-phase A. baumannii culture was incubated 0.79 or 0.38 μg/mL RBT in the presence or absence of amino acid mixture at 37 °C. Bacteria were harvested 0, 1, 8, and 24 hours and CFUs were determined by plating serial dilutions on agar media. The cell free supernatant was collected by filtration through a 0.22 μm filter. RBT were extracted by adding LC-MS grade acetonitrile:methanol:water (40:40:20) solution that was precooled to − 40 °C. Liquid chromatography mass spectrometry (LC-MS) differentiation and detection of RBT was performed using a Cogent Diamond Hydride Type C column (Microsolve Technologies) with an Agilent Accurate Mass 6230 TOF coupled with an Agilent 1290 Liquid Chromatography system as previously published.33 (link),35 (link) An isocratic pump was used for continuous infusion of a reference mass solution to allow mass axis calibration. Detected RBT ion was validated based on unique accurate mass-retention time identifiers for masses. RBT level was analyzed using Agilent Qualitative Analysis B.08.00 (Agilent Technologies) with a mass tolerance of <0.005 Da. The intracellular RBT was calculated as the [RBT]drug only control – [RBT]filtrate. 3 biological replicates were tested per group.
3
Quantification of Intracellular Rifabutin in A. baumannii
4
LC-MS Metabolite Profiling Protocol
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LC-MS analysis was conducted using an Agilent 1290 Infinity LC system containing a Zorbax RRHD Extend C18 column (2.1 mm × 150 mm; Agilent) coupled to an Agilent Accurate Mass 6230 TOF as previously described [41 (link)]. The mobile phase consisted of solvent A (5 mM tributylamine (TBA), 5.5 mM acetic acid in 97% ddH2O 3% methanol) and solvent B (5 mM TBA, 5.5 mM acetic acid in methanol) run at a flow rate of 0.25 mL/min with the following gradient: 0–3.5 min, 0% B; 4–7.5 min, 30% B; 7.5–8 min, 35% B; and 12–16 min, 99% B; followed by a 5 min equilibration period at 0% solvent B prior to injection of the next sample. Dynamic mass axis calibration was accomplished by continuous infusion of a reference mass solution. ESI capillary and fragmentor voltages were set at 4000 V and 125 V respectively. The nebulizer pressure was set to 45 psig and nitrogen drying gas was set to a flow rate of 8 L/min. The drying gas temperature was maintained at 325°C. The MS acquisition rate was 1.5 spectra/ sec and m/z data ranging from 50–1100 was stored. Data was analyzed using Profinder B.08.00 software, and ions were assigned as specific metabolites based on mass accuracy within 5 parts per million (ppm) and retention times within 1 min of those determined for chemical standards.
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