Agilent ce capillary electrophoresis system

The kidneys were subjected to metabolomic analysis as described previously (Kato et al., 2010 (link)) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) (Soga et al., 2003 (link)). Briefly, frozen kidney tissues were immediately plunged into methanol (0.5 ml) containing internal standards (300 μM each of methionine sulfate for cations and MES for anions) and homogenized for 3 min to inactivate enzymes. 200 μl of deionized water and 500 μl of chloroform were added, and the mixture was thoroughly mixed. The solution was centrifuged at 15,000 rpm for 15 min at 4°C and the 600 μl upper aqueous layer was filtered centrifugally through a Milipore-5 kDa cutoff filter to remove proteins. All CE-TOFMS was performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Santa Clara, CA). We measured the levels of metabolites in glycolysis and the tricarboxylic acid (TCA) cycle, and amino acids and their derivatives.

The cells were transfected with a plasmid for Mrs2 knockdown by electroporation using Neon (Life Technologies). The cells were plated on a 100 mm dish, and grown for 3 days at 37 °C in a humidified atmosphere containing 5% CO₂. Culture medium was changed every day. Procedure for sample preparation of metabolome analysis was previously described37 (link). Briefly, after washing cells twice with ice-cold 5% mannitol, metabolites were extracted by 1 mL ice-cold methanol containing internal standards (25 μM each of methionine sulfone (MetSul; Wako, Osaka, Japan), 2-(N-morpholino)ethanesulfonic acid (MES; wako), D-Camphor-10-sulfonic acid (CSA; Wako). 400 μL of collected extracts were transferred into another tube, mixed with 400 μL chloroform and 200 μL Milli-Q water, and centrifuged at 10,000 × g for 3 min at 4 °C. A 400 μL aliquot of the aqueous layer was centrifugally filtered through a 5 kDa cutoff membrane (UltrafreeMC-PLHCC for Metabolome Analysis; Human Metabolome Technologies, Yamagata, Japan) to remove proteins from samples, followed by the centrifugal-concentration at 42 °C. CE-MS experiments were performed using Agilent CE Capillary Electrophoresis System.

Cells were collected for metabolome analysis at the indicated infection times. Approximately 2.5 × 10⁶ cells were infected with each V. parahaemolyticus strain at each given time point at an MOI of 50:1. The supernatant was removed, and cells were washed twice with 5 ml of cold 5% mannitol solution. Metabolic activity was rapidly quenched by adding 0.5 ml methanol containing internal standards (100 μM methionine sulfone and camphor 10-sulfonic acid). Intracellular metabolites were extracted using a solvent extraction method by mixing homogenates with 400 μl chloroform and 200 μl Milli-Q water. The mixture was centrifuged (5,000 rpm, 4°C, 5 min). Subsequently, the aqueous layer was filtered using 5-kDa-cutoff filters (Millipore, Bedford, MA) and centrifuged (10,000 rpm, 4°C, 6 h). The filtrate was dried using a vacuum evaporator (4,000 rpm, 4°C, 4 h) and reconstituted in 50 μl Milli-Q water containing spiked internal standards (25 mM [each] 3-aminopyrrolidine and trimesic acid) before analysis.
CE-TOF/MS was performed using an Agilent CE capillary electrophoresis system coupled with an Agilent 6210 time of flight mass spectrometer (Agilent Technologies, Palo Alto, CA) by Human Metabolome Technologies, Inc. (HMT; Tsuruoka, Japan), as described previously (52 (link)).

All CE-TOF-MS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), Agilent G1969A and G6220A Accurate-Mass TOF LC-MS system (Agilent Technologies, Palo Alto, CA, USA), Agilent 1100 and 1200 series isocratic high-performance LC pumps, G1603A Agilent CE-MS adapter, and Agilent CE electrospray ionization (ESI)-MS sprayer kit (G1600AX and G7100A). An Agilent G1607-60001 platinum ESI needle was used for anion analysis. The Agilent ChemStation software (ver. A.10.02, B.02.01.SR1, and B.03.02, C.01.07.SE1, Agilent Technologies, Waldbronn, Germany) for CE and the Agilent MassHunter software (ver. B.02.00, Agilent Technologies, Palo Alto, CA, USA) was used for the system control and data acquisition.

The metabolome profiles were evaluated by extracting metabolites from frozen plasma and liver samples by adding 400 μL of methanol including the internal standards (20 μM each of methionine sulfone and D-camphor-10-sulfonic acid (CSA)) was added to the 40 μL of samples. Next, this mixture was then mixed with 120 μL of ultrapure water and 400 μL of chloroform before centrifuging at 10,000 × g for 3 min at 4 °C. Thereafter, proteins and lipids were removed by transferring the aqueous layer to a centrifugal filter tube (UltrafreeMC-PLHCC 250/pk for Metabolome Analysis, Human Metabolome Technologies). The filtrate was centrifugally concentrated and dissolved in 20 μL of ultrapure water that contained reference compounds (200 μM each of 3-aminopyrrolidine and trimesic acid) immediately before CE-TOFMS analysis^{80 (link)}. All CE-TOFMS experiments were performed using the Agilent CE capillary electrophoresis system (Agilent Technologies). Annotation tables were produced from the measurement of standard compounds that were aligned with the datasets according to similar value and normalized migration time. Peak areas were then normalized against those of the internal standards methionine sulfone or CSA for cationic and anionic metabolites, respectively. The concentrations of each metabolite were calculated based on their relative peak areas and concentrations of standard compounds.

CE-time-of-flight mass spectrometry (TOFMS) was carried out using an Agilent CE Capillary Electrophoresis system equipped with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies, Waldbronn, Germany). The metabolites were analyzed by using a fused silica capillary (50 μm i.d. × 80 cm total length), with commercial electrophoresis buffer (solution ID: H3301-1001 for cation analysis and H3302-1021 for anion analysis, Human Metabolome Technologies, Inc., Tsuruoka, Japan) as the electrolyte. The sample was injected at a pressure of 50 mbar for 10 s (approximately 10 nL) in cation analysis and 25 s (approximately 25 nL) in anion analysis. The spectrometer was scanned from m/z 50 to 1,000. Other conditions were as described previously [18 (link)-20 (link)].