Abstract
Environmental metabolomics has become a growing research field to understand biological and biochemical perturbations of organisms in response to various abiotic or biotic stresses. It focuses on the comprehensive and systematic analysis of a biologic system’s metabolome. This allows the recognition of biochemical pathways impacted by a stressor, and the identification of some metabolites as biomarkers of potential perturbations occurring in a body. In this work, we describe the development and optimization of a complete reliable methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) for untargeted metabolomics studies within a fish model species, the three-spined stickleback (Gasterosteus aculeatus). We evaluated the differences and also the complementarities between four different matrices (brain, gills, liver and whole fish) to obtain metabolome information. To this end, we optimized and compared sample preparation and the analytical method, since the type and number of metabolites detected in any matrix are closely related to these latter. For the sample preparation, a solid-liquid extraction was performed on a low quantity of whole fish, liver, brain, or gills tissues using combinations of methanol/water/heptane. Based on the numbers of features observed in LC-HRMS and on the responses of analytical standards representative of different metabolites groups (amino acids, sugars…), we discuss the influence of the nature, volume, and ratio of extraction solvents, the sample weight, and the reconstitution solvent. Moreover, the analytical conditions (LC columns, pH and additive of mobile phases and ionization modes) were also optimized so as to ensure the maximum metabolome coverages. Thus, two complementary chromatographic procedures were combined in order to cover a broader range of metabolites: a reversed phase separation (RPLC) on a C18 column followed by detection with positive ionization mode (ESI+) and a hydrophilic interaction chromatography (HILIC) on a zwitterionic column followed by detection with negative ionization mode (ESI-). This work provides information on brain, gills, liver, vs the whole body contribution to the stickleback metabolome. These information would help to guide ecotoxicological and biomonitoring studies.
Highlights
Since the 1970s, it has been demonstrated that a great number of anthropogenic contaminants are present at low concentrations in the environment
After evaluating several liquid chromatography (LC)-high-resolution mass spectrometry (HRMS) conditions and extraction methods, we propose an analytical workflow based on a single solid-liquid extraction followed by dual hydrophilic interaction chromatography (HILIC)(ESI-)-HRMS/reversed phase liquid chromatography (RPLC)(ESI+)-HRMS analyses, allowing to obtain high coverage of the metabolomes while keeping the analysis time reasonable
This study describes the development of a reliable method for untargeted metabolomics studies, based on a solid-liquid extraction followed by LC-HRMS analysis, to detect the metabolites contained in brain, gills, liver, and whole fish of the three-spined stickleback
Summary
Since the 1970s, it has been demonstrated that a great number of anthropogenic contaminants are present at low concentrations in the environment. They are currently defined as pseudopersistent since they are continually released into the water bodies [1]. This environmental pressure can affect the health of the aquatic ecosystem, causing mortalities, pathological aberrations in the species development, the apparition of high resistance genes, growth retardation, increase of oxidative stress, or effect on reproductive activity [1, 2]. It remains difficult to have a global vision of the disturbance of the organism and to determine the links between the different individual biomarker responses
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