The intracellular extracts were analyzed by ultra high performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) according to Klitgaard et al. [24 (link)]. Liquid chromatography was performed on an Agilent 1290 Infinity LC system with a DAD detector, coupled to an Agilent 6550 iFunnel Q-TOF with an electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA). The separation was performed on a 2.1 mm × 250 mm, 2.7 μm Poroshell 120 Phenyl-Hexyl column (Agilent) at 60 °C with a water-acetonitrile gradient (both buffered with 20 mM formic acid) going from 10 % (v/v) to 100 % acetonitrile in 15 min, followed by 3 min with 100 % acetonitrile. The flow rate was kept constant at 0.35 mL/min throughout the run. The injection volume, depending on the sample concentration, typically varied between 0.1 and 1 μL. Mass spectra were recorded as centroid data for m/z 85–1700 in MS mode and m/z 30–1700 in MS/MS mode, with an acquisition rate of 10 spectra/s. The lock mass solution in 95% acetonitrile was infused in the second sprayer using an extra LC pump at a flow of 10–50 μL/min, and the solution contained 1 μM tributyle amine (Sigma-Aldrich), 10 μM Hexakis(2,2,3,3-tetrafluoropropoxy) phosphazene (Apollo Scientific Ltd., Cheshire, UK), and 1 μM trifluoroacetic acid (Sigma-Aldrich) as lock masses.
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