Data-Driven Optimization of Metabolomics Methods Using Rat Liver Samples

Abstract

The aim of metabolomics is to identify, measure, and interpret complex time-related concentration, activity, and flux of metabolites in cells, tissues, and biofluids. We have used a metabolomics approach to study the biochemical phenotype of mammalian cells which will help in the development of a panel of early stage biomarkers of heat stress tolerance and adaptation. As a first step, a simple and sensitive mass spectrometry experimental workflow has been optimized for the profiling of metabolites in rat tissues. Sample (liver tissue) preparation consisted of a homogenization step in three different buffers, acidification with different strengths of acids, and solid-phase extraction using nine types of cartridges of varying specificities. These led to 18 combinations of acids, cartridges, and buffers for testing for positive and negative ions using mass spectrometry. Results were analyzed and visualized using algorithms written in MATLAB v7.4.0.287. By testing linearity, repeatability, and implementation of univariate and multivariate data analysis, a robust metabolomics platform has been developed. These results will form a basis for future applications in discovering metabolite markers for early diagnosis of heat stress and tissue damage.

Gas Chromatography- Mass Spectrometry Based Metabolomic Approach for Optimization and Toxicity Evaluation of Earthworm Sub-Lethal Responses to Carbofuran

“…Since each extraction solvent has its own chemical and physical properties, it is not easy to generate such an extraction solvent for global metabolomic profiling of earthworms. To select an optimal extraction solvent system for the global metabolomic profiling of earthworm, Metaphire posthuma , we have evaluated five different solvent systems, MCW, 80% MeOH, MIPW, AMW and pure MeOH, which are known to have good extraction efficiency for various tissue and cell metabolome [30], [31], [32], [33], [34], [35]. Out of the five solvents tested, best extraction solvent was selected based on (i) peak intensity of structurally identified metabolites, (ii) multi variant analysis and (iii) distributions over individual metabolite features of coefficient of variation (reproducibility) [30], [34], [36], [73].…”

“…Metabolism quenching, tissue disruption and extraction were the key steps for metabolomic profiling. Various sample preparation protocols have been developed for species specific metabolome profile such as bacterial cells [29], different mammalian cells [30], [31], [32], microorganisms [33], Caenorhabditis elegans [34], ragworms [35], and different human matrices such as blood [36], urine [37], CSF [38], and human faecal water [39]. An optimal extraction method should obey the criterion, which includes extraction of large number of metabolites without affecting their stability, easily adaptable to analytical technique, and should be reliable and reproducible in order to identify metabolomic changes [40], [41].…”

Exhaled biomarkers in lung cancer

“…Most likely, a complete pattern of different molecules (breathomics), rather than individual breath constituents, can provide a relevant ''exhaled biomarker fingerprint'' of lung cancer. New developments in statistical approaches [126][127][128][129][130][131][132][133] and data sharing between electronic noses [134] can facilitate this research area.…”

Systematic variations associated with renal disease uncovered by parallel metabolomics of urine and serum

“…GC/MS is a robust analytical platform for quantification with better sensitivity and resolution than the commonly used NMR approach and better reliability in structure identification of candidate biomarkers than LC/MS [11]. It has been widely applied in metabolomics research of biofluids, feces, and tissue samples [6,12-14]. Nowadays, metabolomics has been successfully used in the fields of physiology, diagnostics, functional genomics, pharmacology, toxicology, and nutrition.…”