Protocols
The following protocols will provide you with a step-by-step instructions that can be adapted to the specifics of your sample types making them compatible for our untargeted tandem mass spectrometry approaches. We are working on additional protocols and we will make them available here.
Solid Phase Extraction (SPE) for Dissolved Organic Matter (DOM)
Authors: Monica Thukral, Irina Koester, Daniel Petras, Ralph Riley Torres, Allegra Aron, Emily Gentry, Xavier Siwe-Noundou, Rosemary Dorrington, Kerry McPhail, Aaron Hartmann, Lihini Aluwihare
General Remarks: Protocol for PPL Solid Phase Extraction (SPE) for sample preparation for LC-MS/MS analysis of DOM. Using a SPE vacuum station, this protocol can be adapted for large (>200 mL) or smaller sample volumes (<200 mL). The general procedure is written for larger volumes with notes embedded with procedural modifications for smaller samples. Examples of recommendations for sample size are 1 L for environmental samples, and 50-100 mL for a laboratory culture. Avoid materials with silicone coating and PEG contaminants (e.g. silicon tubing and sterivex and supor filters).
Extraction Protocol for untargeted LC-MS/MS - Animal tissues
Authors: Mauricio Caraballo, Marwa Tawfik, Janet Tseng, Monica Wu
This protocol details the preparation of animal tissue samples for untargeted metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). It covers sample collection, homogenization, metabolite extraction, and preparation for LC-MS/MS analysis.
Protocol for Experimental Design and PPL Solid Phase Extraction for Chemical Exudates Released by Marine Macrophytes in Preparation for LC-MS/MS Analysis
Authors: Karolina Zabinski, Vivian K. Rojas, Gina Chaput, Emilia Sogin and Mauricio Caraballo
It is increasingly clear that small molecules, or metabolites, are key in understanding ecological and evolutionary processes from elemental cycling to microbial symbioses. However, extracting molecules from marine systems remains a challenge. Here, we build off previous work (see Thukral et al., 2022) that provides methods for isolating dissolved organic matter (DOM) from bulk seawater using Solid-Phase Extraction (SPE). We extend this approach by providing both direct and indirect approaches to extract the chemical exudates released by marine macrophytes and their associated microbiomes using the seagrass, Zostera marina, as our model. In addition to embedding pictures on technical setups and sample preparation for liquid chromatography tandem mass spectrometry (LC-MS/MS), we also provide tips on experimental design for planning field and lab-based experiments. Together, this protocol aims to centralize methods related to (i) planning and implementing experiments and (ii) acquiring the data that identifies the small molecules released by key marine foundation species and their microbial symbionts.
MSCollaboratory
