Parasites (5 × 107 cells) collected from cultures grown to mid log phase were harvested and quenched and metabolites extracted in 100 μl of chloroform/methanol/water (1:3:1, by vol) as previously described in [60 (link)]. HPLC using a ZIC-pHILIC column (150 mm × 4.6 mm, 5 μm column, Merck Sequant and a Dionex UltiMate 3000 RSLC system (Thermo, Hemel Hempstead, UK) with metabolite masses identified using a Thermo Orbitrap Exactive (Thermo Fisher Scientific, Hemel Hempstead, UK) operated in polarity switching mode with lock-mass correction applied to enhance calibration stability.
XCMS software [61 (link)] was used for untargeted peak detection and mzMatch.R [62 (link)] for peak matching and annotation of related peaks. The IDEOM software package [63 (link)] was used to identify metabolites either through matching accurate masses and retention times of authentic standards (Metabolomics Standards Initiative confidence level 1) or using predicted retention times using a previously validated model [64 (link)] (Metabolomics Standards Initiative confidence level 2) if authentic standards were not available.
XCMS software [61 (link)] was used for untargeted peak detection and mzMatch.R [62 (link)] for peak matching and annotation of related peaks. The IDEOM software package [63 (link)] was used to identify metabolites either through matching accurate masses and retention times of authentic standards (Metabolomics Standards Initiative confidence level 1) or using predicted retention times using a previously validated model [64 (link)] (Metabolomics Standards Initiative confidence level 2) if authentic standards were not available.
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