The freeze-dried samples were dissolved in 30 µL of ultrapure water for RP-IP-LC/QqQ-MS analysis using a Shimadzu Nexera UHPLC system coupled with LCMS 8030 plus (Shimadzu Co., Japan) with an L-column 2 ODS (150 mm × 2.1 mm, 3 μm, Chemicals Evaluation and Research Institute, Japan) as previously described [17 (link)]. Analysis was performed using 10 mM tributylamine-15 mM acetic acid in water as mobile phase A and methanol as mobile phase B. The gradient concentration of the mobile phase with flow rate of 0.2 mL/min were as follows: start from 0% B at 0–1 min, increased to 15% B at 1.0–1.5 min, then held until 3.0 min, then increased to 50 and 100% B from 3.0 to 8.0 min, and 8.0 to 10.0 min, respectively; held 100% B until 11 min, decreased to 0% B from 11 to 11.50 min, and at last held 0% B until 17 min. The column oven temperature was 45 °C and the analysis mode was in negative ion mode. The mass spectrometer conditions were set at the following conditions: the desolvation line temperature was 250 °C, the nebulizer gas flow was 2 L/min, the drying gas flow was 15 L/min, and the heat block temperature was 400 °C and analysis mode was multiple reaction monitoring (MRM) mode (Additional file 1 : Table S2).
Lcms 8030 plus
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Sourced in Japan
The LCMS 8030 plus is a liquid chromatography-mass spectrometry (LC-MS) system designed for high-performance analysis. It features a triple quadrupole mass analyzer and can perform quantitative and qualitative analysis of a wide range of analytes in complex matrices.
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7 protocols using lcms 8030 plus
1
RP-IP-LC/QqQ-MS Analysis of Freeze-Dried Samples
2
Targeted Metabolomic Analysis of Cellular Extracts
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Sample preparation and metabolite extraction were performed according to Izumi et al. [58 ]. For sample preparation, culture broth was filtered under vacuum suction. Filter-bound cells were frozen in liquid nitrogen to quench metabolism. Cell metabolites were extracted using methanol–water–chloroform (5:2:2) extraction, polar and nonpolar phase metabolites were separated and finally lyophilized before analysis. A detailed description is given in [58 ] and Additional file 6.5 . Cell extracts were analyzed according to Huang et al. [59 (link)] by (1) pentafluorophenylpropyl (PFPP) stationary phase liquid chromatography (Discovery HS F5, 150 mm × 2.1 mm, particle size 3 µm, Sigma-Aldrich Corp., Germany) coupled with electrospray ionization (ESI) in positive and negative modes; and (2) reversed phase ion pairing liquid chromatography with a C18 column (CERI L-column 2 ODS, 150 mm × 2.1 mm, particle size 3 μm, Chemicals Evaluation and Research Institute, Kyoto, Japan) coupled with ESI in negative mode, to a triple-quadrupole mass spectrometer (LCMS 8030 plus; Shimadzu, Japan). A detailed description of mobile phases, concentration gradients, flow rates, injection volumes, column oven temperatures, as well as MS parameters is given in Additional file 6.5 .
3
Ion-pair LC/MS/MS Analysis of Metabolites
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Ion-pair LC/MS/MS analysis was performed as previous reported (Ohtake et al., 2017 (link)). Briefly, a Shimadzu Nexera UHPLC system coupled with LCMS 8030 Plus (Shimadzu) using a CERI L-column 2 ODS (150 mm × 2.1 mm, particle size 3 μm, Chemicals Evaluation and Research Institute, Tokyo, Japan) was used. The mobile phase (A) was 10 mM tributylamine and 15 mM acetate in water and mobile phase (B) was methanol. The raw data files were converted into *.abf file then imported into MRMPROBS (Tsugawa et al., 2013 (link)) to create data matrix.
4
Quantification of Acyl-CoAs and Pyruvate
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The freeze-dried samples were dissolved in 30 μL of ultrapure water for IP-LC/QqQ-MS analysis using a Shimadzu Nexera UHPLC system coupled with LCMS 8030 plus (Shimadzu Co., Japan). The protocol for LC/QqQ-MS analysis was based on Dempo et al. (2014 (link)) using an L-column 2 ODS (150 mm × 2.1 mm, 3 μm, Chemicals Evaluation and Research Institute, Japan). The mobile phase A used was 10 mM tributylamine and 15 mM acetic acid in water, while mobile phase B was methanol. The flow rate was 0.2 mL/min and column oven temperature was 40 °C. Gradient curves were as follows: for acyl-CoAs, 0 % B at 0, 50 % B at 1, 60 % B at 10, 90 % B at 11–13, 0 % B at 14-20 min, and for pyruvate, 0 % B at 0, 15 % B at 2, 25 % B at 7, 50 % B at 9, 100 % B at 11–13, 0 % B at 13.01–18 min. The analysis mode was negative ion mode. Probe position was +1.5 mm, desolvation line temperature was 250 °C, nebulizer gas flow was 2 L/min, drying gas flow was 15 L/min, and heat block temperature was 400 °C. The IP-LC/QqQ-MS analysis was performed with multiple reaction monitoring (MRM). Analytical parameters for the quantitative analysis and turnover analysis are listed in Table S2.
5
13C-labeling Kinetics in Metabolite Profiling
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After NaH 13 CO3 was added, 10 mL of culture was filtered at each time point (0, 1, 2, 3, 4, 5, 10, 15, 30, and 60 min) using 0.2 μm pore size Omnipore filter disks (Merck KGaA).
The harvested cells on the filter were immediately immersed in pre-chilled methanol containing 5 μM (+)-10-camphorsulfonate to quench metabolic activity. Quenching was performed for less than 15 sec. The mixture was stored at -80°C until extraction.
Intracellular metabolites in the mixture were extracted using chloroform and Milli-Q water. The supernatant containing intracellular metabolites was collected and concentrated using a centrifugal concentrator SpeedVac SPD1010 (Thermo Fisher Scientific, Waltham, MA, USA). The dried samples were resuspended in 100 μL Milli-Q water for LC-MS/MS analysis. The resuspended solutions were used to measure 13 Clabeling patterns and pool sizes of metabolites using a Shimadzu Nexera UPLC system coupled with LCMS 8030 Plus (Shimadzu, Kyoto, Japan). The column used was a ProteCol-P C18 HQ103 (2.1 mm inside diameter × 150 mm, particle size of 3 μm; SGE Analytical Science, Melbourne, Australia). The chromatographic conditions were as described in a previous study (Dempo et al., 2014) . All data acquisition and analyses were performed using LabSolutions version 5.60 (Shimadzu).
The harvested cells on the filter were immediately immersed in pre-chilled methanol containing 5 μM (+)-10-camphorsulfonate to quench metabolic activity. Quenching was performed for less than 15 sec. The mixture was stored at -80°C until extraction.
Intracellular metabolites in the mixture were extracted using chloroform and Milli-Q water. The supernatant containing intracellular metabolites was collected and concentrated using a centrifugal concentrator SpeedVac SPD1010 (Thermo Fisher Scientific, Waltham, MA, USA). The dried samples were resuspended in 100 μL Milli-Q water for LC-MS/MS analysis. The resuspended solutions were used to measure 13 Clabeling patterns and pool sizes of metabolites using a Shimadzu Nexera UPLC system coupled with LCMS 8030 Plus (Shimadzu, Kyoto, Japan). The column used was a ProteCol-P C18 HQ103 (2.1 mm inside diameter × 150 mm, particle size of 3 μm; SGE Analytical Science, Melbourne, Australia). The chromatographic conditions were as described in a previous study (Dempo et al., 2014) . All data acquisition and analyses were performed using LabSolutions version 5.60 (Shimadzu).
6
Quantifying Intracellular Reactive Sulfur Species
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Intracellular levels of reactive sulfur species (RSS) in OVISE, OVTOKO and OVCAR3 were assessed using monobromobimane (mBBr). Cells were washed with 5% mannitol twice, and then plunged into 500 μl methanol containing 100 nM of D-camphor-10-sulfonic Acid (CSA) as the internal standard. Cell extracts were thoroughly mixed with 500 μl deionized water and 1 ml chloroform, followed by centrifuging at 12,000 g for 15 min at 4oC. The upper aqueous phase was filtered through a centrifugal filter (Ultrafree-MC, 5-kDa cutoff; Human Metabolome Technologies, Tsuruoka, Japan) to remove protein precipitates. After the lyophilization of filtrates, the precipitates were dissolved in 50 μl deionized water. The samples were incubated with 2 mM mBBr on ice for 5 min. Levels of mBBr derivatives were determined by LCMS-8030plus (Shimadzu, Kyoto, Japan). Obtained data were normalized by cellular protein concentrations [4 (link),7 (link),16 (link)]. When necessary, GSH, GSSG, hypotaurine were determined by LC-MS according to our previous method [4 (link)].
7
Ion-pair LC/MS/MS Analysis of Metabolites
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The samples were analyzed based on the method adopted from Dempo et al. [32 (link)]. Briefly, ion-pair reversed phase LC/MS/MS was carried out using a Nexera UHPLC system (Shimadzu, Kyoto, Japan) coupled with LCMS 8030 Plus (Shimadzu) for the time course analysis and coupled with LCMS 8050 (Shimadzu) for analyses of multiple strains. The column was a CERI (Chemicals Evaluation and Research Institute, Tokyo, Japan) L-column 2 metal-free ODS (150 mm 2.1 mm, particle size 3 µm). Mobile phase (A) was 10 mM tributylamine and 15 mM acetate in ultra-pure water, and mobile phase (B) was pure methanol. The flow rate was set at 0.2 mL/min, and the column oven temperature was set at 45 °C. The concentration of mobile phase (B) was programmed to increase from 0% to 15%, 50%, and 100% from 1.0 to 1.5 min, 3.0 to 8.0 min, and 8.0 to 10.0 min, respectively, held until 11.5 min, then decreased to 0% from 11.5 min and held at 0% for 20 min. The analysis mode was set to negative ion detection mode. The injection volume was 3 µL, desolvation line temperature was set at 250 °C, probe position was +1.5 mm, heat block temperature was set at 400 °C, nebulizer gas flow was set at 2 L/min, and drying gas flow was set at 15 L/min.
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