Evaluation of sample preparation methods for the analysis of reef-building corals by 1H NMR based metabolomics
Erik R. Andersson, Russell D. Day, Cheryl M. Woodley, Julie M. Loewenstein, Tracey B. Schock
The field of metabolomics generally lacks a standardized method for the preparation of samples prior to analysis. This is especially true for metabolomics of reef-building corals, where the handful of studies that have been 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 sample preparation of coral samples destined for metabolomics analysis would aid this cause. Therefore, 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 (MTBE) and methanol extractions, while a methanol extraction is superior for feature detection. Additionally, few differences are detected between spectra from coral samples that are frozen or lyophilized during sample handling. However, lyophilization allows for more precise control over, and detailed observations of, the coral samples. Finally, extraction of entire coral nubbins increases feature detection, but decreases throughput and is more susceptible to subsampling error compared to a novel tissue powder subsampling method. These results lead to the proposal of a recommended sample preparation workflow optimized to study reef-building corals. Overall, we recommend the use of a modified Bligh and Dyer extraction, lyophilized samples, and analysis of brushed tissue powder for the preparation of reef-building coral samples for 1H NMR metabolomics.