A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates
Fig 2
Overall scheme of LC-MS/MS RNR activity assay.
Either aerobically or anaerobically, a mastermix is created and a zero point is taken prior to initiation with beta (aerobic assays) or nucleotides (anaerobic assays). The reaction is allowed to proceed for 120 s with time points taken every 30 s. Inactivation of the reaction occurs by heating the sample to 95°C in a thermocycler or heat block. After inactivation, anaerobic assays are removed from the box and all assays have 1 μL of calf intestinal phosphatase (CIP) added to dephosphorylate the nucleotides at 37°C for 2 hours. The samples are then filtered through a 0.2 μm filter and transferred to mass spectrometry vials for analysis via LC-MS/MS.