Chromolith performance rp 18e 100 2 mm column

A. thaliana PCOs 1 to 5 were expressed and purified by sequential steps of immobilized nickel affinity and size exclusion chromatography as previously described⁴⁵ . The activities of each AtPCO were examined by incubating 200 µM of peptide corresponding to the first 14 amino acids of the methionine excised N-terminus of VRN2 (VRN2_2–15) or RAP2.2/2.12 (RAP2_2–15) with and without 0.1–0.8 µM enzyme in a bench top thermocycler (Eppendorf) at 5 or 22 °C under aerobic conditions. Time points were taken at regular intervals by quenching the reaction 1 in 10 with 1% formic acid, allowing oxidation to be monitored by ultra-high performance chromatography (UPLC)-mass spectrometry (MS). Turnover was quantified by comparing the areas underneath the product and substrate ions extracted from the total ion current chromatogram. UPLC-MS measurements were obtained using an Acquity UPLC system coupled to a Xevo G2-S Q-ToF mass spectrometer (Waters) operated in positive electrospray mode. Instrument parameters, data acquisition and data processing were controlled by Masslynx 4.1 with source conditions adjusted to maximise sensitivity and minimise fragmentation. Samples were injected on to a Chromolith Performance RP-18e 100-2 mm column (Merck) heated to 40 °C and eluted at 0.3 ml/min using a gradient of 95% deionized water supplement with 0.1% (v/v) formic acid to 95% acetonitrile.

Ultra-high performance chromatography (UPLC)–MS measurements were obtained using an Acquity UPLC system coupled to a Xevo G2-S Q-ToF mass spectrometer (Waters) operated in positive electrospray mode. Instrument parameters, data acquisition and data processing were controlled by Masslynx 4.1. Source conditions were adjusted to maximize sensitivity and minimize fragmentation while Lockspray was employed during analysis to maintain mass accuracy. Two microlitres of each sample was injected on to a Chromolith Performance RP-18e 100-2 mm column (Merck) heated to 40 °C and eluted using a gradient of 95% deionized water supplemented with 0.1% (v/v) formic acid (analytical grade) to 95% acetonitrile (HPLC grade) and a flow rate of 0.3 ml min⁻¹. Fragmentation spectra of substrate and product peptide ions (MS/MS) were obtained using a targeted approach with a typical collision-induced dissociation energy ramp of 30 to 40 eV. Analysis was carried out with the same source settings, flow rate and column elution conditions as above.