Evaluation of Sample Preparation Methods for the Analysis of Reef-Building Corals Using 1H-NMR-Based Metabolomics

Abstract

The field of metabolomics generally lacks standardized methods for the preparation of samples prior to analysis. This is especially true for metabolomics of reef-building corals, where the handful of studies that were published employ a range of sample preparation protocols. The utilization of metabolomics may prove essential in understanding coral biology in the face of increasing environmental threats, and an optimized method for preparing coral samples for metabolomics analysis would aid this cause. The current study evaluates three important steps during sample processing of stony corals: (i) metabolite extraction, (ii) metabolism preservation, and (iii) subsampling. Results indicate that a modified Bligh and Dyer extraction is more reproducible across multiple coral species compared to methyl tert-butyl ether and methanol extractions, while a methanol extraction is superior for feature detection. Additionally, few differences were detected between spectra from frozen or lyophilized coral samples. Finally, extraction of entire coral nubbins increased feature detection, but decreased throughput and was more susceptible to subsampling error compared to a novel tissue powder subsampling method. Overall, we recommend the use of a modified Bligh and Dyer extraction, lyophilized samples, and the analysis of brushed tissue powder for the preparation of reef-building coral samples for 1H NMR metabolomics.

Revolutionizing Coral Research: The Power of Holo-Omics

“…Standardization is necessary for metabolomics due to the impact of extraction protocols on detected molecules. While some metabolites are prone to degradation, others are not efficiently extracted [76,78]. A significant challenge in the study of the holobiont is connecting metabolites with their respective producing organisms.…”

Methodological Developments for Metabolic NMR Spectroscopy from Cultured Cells to Tissue Extracts: Achievements, Progress and Pitfalls

“…Although reports on these applications are much sparser than publications on oncological or neurological applications, they will be briefly presented in Supplementary Materials (Section SI 1.4). These investigations concern studies of blood vessel tissue, skin, ascites cells, spermatozoa, serum lipids and milk lipids (mammalian origin) [84][85][86][87][88][89][90][91]; furthermore, measurements of mussels, corals, and plants (non-mammalian origin), as well as microorganisms (bacteria, yeast and other fungi, parasites) [92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107].…”

The coral microbiome: towards an understanding of the molecular mechanisms of coral–microbiota interactions

“…Further, extraction protocols, e.g. differences in solvent mixtures, disruption techniques, or even cooling, have an influence on detected molecules as some metabolites are prone to degradation (Lu et al 2020 ), while others are not efficiently extracted (Andersson et al 2019 ), which calls for standardization. In the context of the holobiont, a major challenge is linking metabolites with their producing organism.…”