Silicatip

To prepare cytidine diphosphate ribitol (CDP-ribitol) and cytidine diphosphate ribose (CDP-ribose), 2 μM His8-hISPD was incubated at 37°C with either 1 mM ribitol-5-phosphate or 1 mM ribose-5-phoshate as the acceptor substrate in a reaction containing 50 mM Tris-HCl pH 7.4, 1 mM MgCl₂, 1 mM DTT, and 1 mM CTP. Reactions were allowed to incubate for 16–18 hr and were stopped by removal of His8-hISPD by filter centrifugation through a Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane (EMD Millipore). Reaction products were confirmed using a linear ion trap-Fourier transform mass spectrometer (LTQ-Orbitrap Discovery, Thermo-Fisher, San Jose, CA). Reaction products were mixed with an equal volume of 80% acetonitrile and 0.1% formic acid and analyzed by direct infusion in negative ion mode using a nanospray ion source with a fused-silica emitter (360 × 75 × 30 μm, SilicaTip, New Objective) at 1.5 kV capillary voltage, 200°C capillary temperature, and a syringe flow rate of 1 μL/min. All products were confirmed by MS/MS ion trap mass spectrometry (ITMS) acquired at 45% collision-induced dissociation (CID) and 2 m/z isolation width.