Practical guide for marine exo-metabolomic sample preparation.

Metabolomic strategies are being increasingly applied in marine systems, leading to unprecedented insights into the ocean and its inhabitants. In particular, exo-metabolites, small molecules produced by marine organisms and released into the environment, have received recognition as factors that influence marine microbial communities, global nutrient cycles, and ecosystem function. Studying exo-metabolites in the marine system is challenging due to the low metabolite concentrations, high salt content, and substantial chemical diversity, all of which can interfere with metabolite detection and therefore necessitate specialized, often tedious sample preparation. Advantageously, new marine metabolomic methods have recently emerged that improve the coverage and detection of key metabolite classes. Here, we offer a practical guide for selecting suitable extraction methods based on specific research needs alongside a systematic review of five recently published methods designed for marine exo-metabolomics. Specifically, these workflows enable quantification of various primary metabolites using liquid and gas chromatography coupled to mass spectrometry. The selection process is guided by several key questions that consider both experimental design and the specific research questions. Additionally, we highlight both the practical constraints and aspects of sample handling for each method. This guide aims to support researchers in effectively choosing a method that aligns with their study goals and logistical capabilities, while also highlighting the opportunities for innovation in the field moving forward.