Quantifying Isoprenoid Precursors by LC-MS/MS

The 4000QTRAP LC-MS/MS system (AB Sciex) was used in multiple-reaction monitoring (MRM) mode using negative ionization. The detailed instrument configuration and compound-dependent parameters for isoprenoid precursors were as previously described^{13 (link)}. LC separation prior to MRM detection was achieved by ion pair reverse-phase chromatography as described previously^{63 (link)}, with 10 mM tributylammonium acetate (pH 5.1-5.5) used as the ion pair reagent and the following modifications: (1) RP-hydro 100 mm × 2.0 mm, 2.5 μm high performance liquid chromatography column (Phenomenex), (2) flow rate of 0.14 mL/min, (3) solvent A of 10 mM tributylammonium acetate in 5% methanol, (4) binary LC gradient (20% solvent B (100% methanol) from 0 to 2.5 min, 30% B for 12.5 min, 80% B for 5 min, and column equilibration for 5 min), and (5) autosampler injection volume of 20 μL. For deoxyxylulose 5-phosphate (DOXP) and methylerythritol cyclodiphosphate (MEcPP) metabolites, one-way ANOVA was used to test for significance (VassarStats). A t-test was used to test for significance between UNT and 1x RCB-185 cytidine diphosphate methylerythritol (CDP-ME) levels (VassarStats). A significance test was not performed for MEP, as its levels were below the limit of detection (12.5 ng/mL) for RCB-185-treated parasites.