Metabolome Analysis of V. parahaemolyticus Infection

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)).