A Robust Method for Sample Preparation of Gastrointestinal Stromal Tumour for LC/MS Untargeted Metabolomics

Szymon Macioszek,Patrick Schöffski,Michał J Markuszewski,Agnieszka Wozniak,Julia Jacyna,Danuta Dudzik

doi:10.3390/metabo11080554

Szymon Macioszek, Patrick Schöffski + Show 4 more

Open Access

https://doi.org/10.3390/metabo11080554

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Journal: Metabolites	Publication Date: Aug 21, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: KU Leuven, Gdańsk Medical University

Abstract

Gastrointestinal stromal tumour has already been well explored at the genome level; however, little is known about metabolic processes occurring in the sarcoma. Sample preparation is a crucial step in untargeted metabolomics workflow, highly affecting the metabolome coverage and the quality of the results. In this study, four liquid-liquid extraction methods for the isolation of endogenous compounds from gastrointestinal stromal tumours were compared and evaluated. The protocols covered two-step or stepwise extraction with methyl-tert-butyl ether (MTBE) or dichloromethane. The extracts were subjected to LC-MS analysis by the application of reversed-phase and hydrophilic interaction liquid chromatography to enable the separation and detection of both polar and nonpolar analytes. The extraction methods were compared in terms of efficiency (total number of detected metabolites) and reproducibility. The method was based on the stepwise extraction with MTBE, methanol, and water proved to be the most reproducible, and thus, its robustness to fluctuations in experimental conditions was assessed employing Plackett–Burman design and hierarchical modelling. While most studied factors had no effect on the metabolite abundance, the highest coefficient value was observed for the volume of MTBE added during extraction. Herein, we demonstrate the application and the feasibility of the selected protocol for the analysis of gastrointestinal stromal tumour samples. The method selected could be considered as a reference for the best characterization of underlying molecular changes associated with complex tissue extracts of GIST.

Highlights

Tumour metabolomics provides an invaluable tool for understanding complex diseases, better patient stratification, prognostics, monitoring disease progression, and therapeutic targets development [1,2,3]
More attention should be placed on the selection of the most appropriate and robust sample preparation protocol, in the case of demanding specimens such as tumours tissue
The selected method, based on stepwise extraction with methyl-tert-butyl ether (MTBE), methanol, and water (1.3:1:1.2, v/v) was subjected to robustness testing to maximise the quality of the data obtained during untargeted metabolomics analysis

Summary

Introduction

Tumour metabolomics provides an invaluable tool for understanding complex diseases, better patient stratification, prognostics, monitoring disease progression, and therapeutic targets development [1,2,3]. More attention should be placed on the selection of the most appropriate and robust sample preparation protocol, in the case of demanding specimens such as tumours tissue. This key issue is yet often overlooked and should be rather considered as an essential fit-for-purpose step of the metabolomics workflow. Tumour tissue is a specific sample that is usually limited by its small volume, obtained during biopsy, surgery or originating from an animal model It is characterised by high cellular heterogeneity or contamination, attributed to remaining blood or healthy tissue [7]. Both sample harvesting and preparation are critical steps to retrieve the true biological information that allows drawing an accurate picture of the current metabolic status of the studied system [8]

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