Untargeted Metabolomics of Epimastigote Forms of <em>Trypanosoma cruzi</em>

<i>Trypanosoma cruzi</i>, the causative agent of Chagas disease, faces significant metabolic challenges due to fluctuating nutrient availability and oxidative stress within its insect vector. Metabolomic techniques, such as gas chromatography-mass spectrometry (GC-MS), have been widely used to study the adaptive mechanisms of the parasite. This article describes a standardized method for the untargeted metabolomics analysis of <i>T. cruzi</i> epimastigote, covering parasite cultivation, sample deproteinization with methanol, metabolite extraction, derivatization with BSTFA, and GC-MS analysis. To ensure robustness and reproducibility, statistical analysis uses univariate tests, as well as multivariate approaches such as principal component analysis (PCA) and partial least squares (PLS) regression. The protocol offers a reliable and sensitive method to study metabolic responses in <i>T. cruzi</i> under environmental stress, with low biological variability and high reproducibility. Key features • GC-MS was used to conduct a standardized metabolomics investigation of <i>Trypanosoma cruzi</i> epimastigote, assuring reproducibility and minimum biological variability. • Includes sample deproteinization, metabolite extraction, and derivatization with BSTFA for accurate metabolite profiling under different biological conditions. • Employs robust statistical approaches (PCA, PLS) to investigate differences among experimental groups and detect significant alterations in metabolism. • Internal standards and multiple replicates ensure high sensitivity and repeatability, which is excellent for investigating metabolic processes in protozoan parasites.