Computational protocol: Untargeted metabolomic analysis of miltefosine action in Leishmania infantum reveals changes to the internal lipid metabolism☆

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Protocol publication

[…] Cells were inoculated at 5 × 106/ml in M199 from a log-phase culture and miltefosine was added at 20.6 μM (IC90). Samples were taken at the time taken for 50% of wild-type cells to die (5 h) as well as time points (0, 1.25, 2.5 and 3.75 h) up to 5 h. All samples were taken in triplicate from independent cultures. Cell metabolism in 4 × 107 cells was quenched by rapid cooling to 4 °C in a dry ice-ethanol bath before centrifugation at 1250g for 5 min to remove medium. Residual medium was removed by washing in 4 °C HEPES–NaCl and cells were lysed and proteins precipitated by shaking in 200 μL 4 °C chloroform:methanol:water (1:3:1) plus standards (theophylline, 5-fluorouridine, N-methyl glucamine, canavanine and piperazine, all at 1 μM) for 1 h. Protein precipitate was removed by centrifugation and a 10 μL aliquot of the supernatant was injected onto a 150 mm × 4.6 mm 5 μm 200 Å ZIC-HILIC HPLC column (SeaQuant, Merck). The column was coupled to an Orbitrap Exactive (Thermo) and masses and retention times were detected using a previously published method (). Data analysis was performed using mzMatch () and Excel (Microsoft) with a VBS macro-based package called IDEOM () for further filtering and metabolite annotation. The independent biological replicates were taken for each time point from separate cultures on different days. These were run on the mass spectrometer in the same run.Five internal standards were used to correct for mass drift and a further mixture of 143 standards was used to create the retention time calculator in IDEOM, allowing accurate mass and retention time identification as well as metabolite validation. Masses were filtered through mzMatch keeping those that were reproducible across all three biological replicates at each time point, had relative standard deviations below 0.5 and had intensities above 3000. Metabolites were further filtered in IDEOM (version 13) to have a retention time error of below 35% and mass errors below three parts per million (ppm) for those metabolites with predicted retention times of below 5% and three ppm for metabolites with a standard. Partial least squares analysis was performed using Metaboanalyst after pre-processing in IDEOM and data filtering using the relative standard deviation (http://www.metaboanalyst.ca/). […]

Pipeline specifications

Software tools Ideom, MetaboAnalyst
Application MS-based untargeted metabolomics
Organisms Leishmania infantum
Diseases Gastrointestinal Diseases, Leishmaniasis
Chemicals Oxygen, Polyamines, Sulfhydryl Compounds