Quantitative Amino Acid Analysis in Bacterial Cells

During the exponential phase (7.5–8 h), an appropriate amount of culture [OD₆₀₀ × culture volume (ml) = 8] was collected via centrifugation. The cells were washed with 2 ml of 0.2 mol l⁻¹ HCl to remove the CaCO₃ and then further washed with 2 ml saline. The cells were hydrolyzed with 2 ml of 6 mol l⁻¹ HCl at 105°C for 18 h. The samples were filtered through a Cosmonice filter W (0.45 µm; Nacalai Tesque, Kyoto, Japan), mixed with 10 µl of 600 µM cycloleucine, dried, and dissolved in 50 µl acetonitrile. After adding 50 µl of N-(tert-tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide containing 1% tert-butyldimethylchlorosilane, the samples were incubated at 105°C for 1 h for derivatization. mass isotopomer distributions (MIDs) of proteinogenic amino acids were measured using a gas chromatograph/mass spectrometer (Agilent 7890A GC and 5975C Mass Selective Detector; Agilent Technologies, Santa Clara, USA) with a DB-5MS+DG column (Agilent Technologies). The detailed method is described elsewhere.³⁴ (link)