Untargeted Metabolomics of Epimastigote Forms of Trypanosoma cruzi.

Trypanosoma cruzi, 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 T. cruzi 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 T. cruzi under environmental stress, with low biological variability and high reproducibility. Key features • GC-MS was used to conduct a standardized metabolomics investigation of Trypanosoma cruzi 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.