Quenching and extraction of intracellular metabolites
Approximately 5 mL sample was rapidly taken from fermenter and injected
into 25 mL precooled (–40°C) 60% (v/v) methanol solution to interrupt
the cell metabolism as soon as possible, resulting in a final
concentration of 50% (v/v) methanol. The quenching process lasted 3
min. Cell pellets were then separated using a fast vacuum dependent
filtration (0.45 μm nominal pore size) and washed with 5 mL precooled
(–40°C) 50% (v/v) methanol solution to remove the residual medium
component on the cell surface. Next the filter adhered with cell pellet
was frozen by liquid nitrogen, and rapidly transferred into 9.5 mL
precooled (–40°C) extraction solution composed of 2.5 mL methanol, 5 mL
chloroform, and 2 mL fresh Tricine-EDTA buffer solution (3 mmol/L
Tricine and 3 mmol/L EDTA, pH 7.0, stored at 4°C). The mixture was
vigorously vibrated for 60 min at –40±5°C (dry ice-ethanol bath). The
first aqueous phase was collected by a centrifugation step (12,000 rpm,
5 min, –19°C), more methanol and Tricine-EDTA buffer solution was added
in for a second extraction, and the second aqueous phase was collected
and pooled with the first one. The aqueous phase was stored at −80°C and
evaporated to dryness before use. The dry residue was reconstituted in
100 μL of Mill-Q water for LC-MS/MS analysis.