Xevo g2 s q tof uplc instrument

Protein solutions for mass spectrometry (40 μM) were prepared by dilution of purified proteins (500–1000 μM) in 200 μM ammonium acetate buffer, pH 7.0. Liquid chromatography-mass spectrometry (LC–MS) was performed on a Xevo G2-S Q-TOF UPLC instrument (Waters, Elstree UK) coupled to an Acquity UPLC system. Samples were eluted through an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1 × 50 mm) using a mobile phase of Solvent A: water with 0.1% formic acid, and Solvent B: 95% acetonitrile containing 0.01% formic acid. The elution gradient was run using 95% Solution A for 5.21 minutes, 100% Solution B for 1 minute, and 100% Solution A for 1 minute at a flow rate of 0.2 ml min⁻¹ over a total run time of 7.29 minutes. The electrospray source was operated with a capillary voltage of 2.0 kV and a cone voltage of 40 V. Nitrogen was used as the desolvation gas at a total flow of 850 litres hr⁻¹. Data acquisition and processing was performed using Micromass MassLynx v4.1 software with total mass spectra reconstructed from the ion series using the pre-installed MaxEnt algorithm.

Protein stock solutions (∼40 μm) were prepared by dilution of purified CYP126A1 protein (∼500 μm) in 200 mm ammonium acetate buffer, pH 7.0. Samples were buffer-exchanged by size exclusion chromatography using Micro Biospin 6 columns, molecular weight cut-off 6000 (Bio-Rad, Hemel Hempstead, UK). LC-MS was performed on a Xevo G2-S QTof UPLC instrument (Waters, Elstree, UK) coupled to an Acquity UPLC system. Samples were eluted through an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1 × 50 mm) using mobile phases of water with 0.1% (v/v) formic acid (Solvent A) and 95% (v/v) acetonitrile in water, containing 0.01% (v/v) formic acid (Solvent B). A solvent gradient of 95% Solvent A for 5.21 min, 100% Solvent B for 1 min, and 100% Solvent A for 1 min at a flow rate of 0.2 ml min⁻¹ over a total run time of 7.29 min was used to elute samples. The electrospray source was operated with a capillary voltage of 2.0 kV and a cone voltage of 40 V. Nitrogen was used as the desolvation gas at a total flow of 850 liters/h. Data acquisition and processing were performed using Micromass MassLynx version 4.1 software with total mass spectra reconstructed from the ion series using the preinstalled MaxEnt algorithm.