Comparison of Three Widely Employed Extraction Methods for Metabolomic Analysis of Trypanosoma brucei

Current protocols Pub Date : 2024-05-06 DOI:10.1002/cpz1.1043

Fanta Fall, Lieven Desmet, Lúcia Mamede, Laura Schioppa, Pascal de Tullio, Michel Frédérich, Bernadette Govaerts, Joëlle Quetin-Leclercq

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Abstract

Trypanosoma brucei (Tb) is the causative agent of human African trypanosomiasis (HAT), also known as sleeping sickness, which can be fatal if left untreated. An understanding of the parasite's cellular metabolism is vital for the discovery of new antitrypanosomal drugs and for disease eradication. Metabolomics can be used to analyze numerous metabolic pathways described as essential to Tb. brucei but has some limitations linked to the metabolites’ physicochemical properties and the extraction process. To develop an optimized method for extracting and analyzing Tb. brucei metabolites, we tested the three most commonly used extraction methods, analyzed the extracts by hydrophilic interaction liquid chromatography high-resolution mass spectrometry (HILIC LC-HRMS), and further evaluated the results using quantitative criteria including the number, intensity, reproducibility, and variability of features, as well as qualitative criteria such as the specific coverage of relevant metabolites. Here, we present the resulting protocols for untargeted metabolomic analysis of Tb. brucei using (HILIC LC-HRMS). © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Culture of Trypanosoma brucei brucei parasites

Basic Protocol 2: Preparation of samples for metabolomic analysis of Trypanosoma brucei brucei

Basic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei

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比较三种广泛应用于布氏锥虫代谢组分析的提取方法

布氏锥虫（Tb）是人类非洲锥虫病（HAT）（又称昏睡病）的病原体，如不及时治疗可致人死亡。了解寄生虫的细胞代谢对发现新的抗锥虫药物和根除疾病至关重要。代谢组学可用于分析对布氏杆菌至关重要的多种代谢途径，但也存在一些局限性，这些局限性与代谢物的理化性质和提取过程有关。为了开发一种提取和分析布鲁氏菌代谢物的优化方法，我们测试了三种最常用的提取方法，用亲水相互作用液相色谱高分辨质谱（HILIC LC-HRMS）分析了提取物，并使用定量标准（包括特征的数量、强度、重现性和可变性）以及定性标准（如相关代谢物的特定覆盖范围）进一步评估了结果。在此，我们介绍使用（HILIC LC-HRMS）对布鲁西结核病进行非靶向代谢组学分析的方案。© 2024 Wiley Periodicals LLC.Basic Protocol 1: Culture of Trypanosoma brucei brucei parasitesBasic Protocol 2: Preparation of sample for metabolomic analysis of Trypanosoma brucei bruceiBasic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei

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