Quantification of Free Amino Acids and Nucleotides

Free amino acids were extracted from 1 × 10⁶ cells with 0.2 ml of 0.1 M HCl in an ultrasonic ice-bath for 10 min. The resulting extracts were centrifuged for 10 min at 4°C and 16.400 g to remove cell debris. Amino acids were derivatized with AccQ-Tag reagent (Waters) and determined as described in [44 (link)]. For determination of nucleotide concentrations, the extracts were derivatized with chloroacetaldehyde as described in [45 (link)] and separated by reversed phase chromatography on an Acquity HSS T3 column (100 mm × 2.1 mm, 1.7 μm, Waters) connected to an Acquity H-class UPLC system. Prior separation, the column was heated to 43°C and equilibrated with 5 column volumes of buffer A (5.7 mM TBAS, 30.5 mM KH₂PO₄ pH 5.8) at a flow rate of 0.6 ml min-1. Separation of adenosine derivates was achieved by increasing the concentration of buffer B (2/3 acetonitrile in 1/3 buffer A) in buffer A as follows: 1 min 1% B, 2 min 8% B, 3.2 min 14% B, 9.5 min 50% B, and return to 1% B in 1.5 min. The separated derivates were detected by fluorescence (Acquity FLR detector, Waters, excitation: 280 nm, emission: 410 nm) and quantified using ultrapure standards (Sigma). Data acquisition and processing was performed with the Empower3 software suite (Waters).