Extraction efficiency and quantification of dissolved metabolites in targeted marine metabolomics

Abstract

The field of metabolomics seeks to characterize the suite of small molecules that comprise the endproducts of cellular regulation. Metabolomics has been used in biomedical applications as well as environmental studies that explore ecological and biogeochemical questions. We have developed a targeted metabolomics method using electrospray ionization-liquid chromatography tandem mass spectrometry to analyze metabolites dissolved in seawater. Preparation of samples from the marine environment presents challenges because dilute metabolites must be concentrated and desalted. We present the extraction efficiencies of 89 metabolites in our targeted method using solid phase extraction (SPE). In addition, we calculate the limits of detection and quantification for the metabolites in the method and compare the instrument response factors in five different matrices ranging from deionized water to spent medium from cultured marine microbes. High background organic matter content reduces the instrument response factor for only a small group of metabolites, yet enhances the extraction efficiency for other metabolites on the SPE cartridge used here, a modified styrene-divinylbenzene polymer called PPL. Aromatic or larger uncharged compounds, in particular, are reproducibly well retained on the PPL polymer. This method is suitable for the detection of dissolved metabolites in marine samples, with limits of detection ranging from < 1 pM to 2 nM dependent on the dual impacts of seawater matrix on extraction efficiency and on instrument response factors.Metabolomics is an "omics" technique that seeks to measure the small organic biomolecules produced by cells (Oliver et al. 1998;Fiehn 2002). Because these small molecules are the end-products of multiple levels of metabolic regulation, their concentrations provide a temporal snapshot of the metabolic state or phenotype of an organism. In particular, metabolites produced by nonenzymatic reactions, such as those formed by reaction with a radical oxygen species, or whose production is regulated by other small molecules, must be monitored directly because their production cannot be inferred from genomic or proteomic information. Metabolomics can be used as a diagnostic tool, identifying biomarkers of disease within the human metabolome, such as cancers (Armitage and Barbas 2014) and Crohn's Disease (Jansson et al. 2009). Metabolomics has also been applied in a wide range of organisms and environments, examining how metabolite abundances respond to environmental factors. In the oceans, marine metabolites have been a valuable source of new natural products, while other metabolomics applications are still rare but growing. For example, recent marine culture experiments have revealed metabolite production not predicted by genomic information (Baran et al. 2010;Fiore et al. 2015), metabolic shifts in response to a specific metabolite (Johnson et al. 2016), and changes in the quantity and composition of metabolite production during coculturing (Paul et al. 2012). Complementary field ...

Distinct molecular signatures in dissolved organic matter produced by viral lysis of marine cyanobacteria

“…DOM samples were acidified to pH 2 before solid phase extraction. Agilent Bond Elut PPL cartridges (1 g 6 ml) used in the experiment are retentive for nonpolar to moderately polar compounds, but very lowmolecular weight and highly polar compounds (particularly more basic ones; Johnson et al, 2017) are likely to be lost during the desalting necessary to make the DOM sample amenable to electrospray-MS. Cartridges were washed first with 1 column volume of methanol, equilibrated with 1 column volume of ultrapure water, followed by sample loading. After washing with 2 column volumes of 0.01 M HCl, DOM compounds sorbed to the column were eluted with 6 ml methanol.…”

Revealing the ocean metabolome with mass spectrometry

“…Our detection limits were in the nano-molar range and comparable to those of targeted techniques for marine ecosystems that were developed to quantify single compounds from specific molecular classes ( Tables S2; Table S3 ). In contrast to previously published techniques, which require at least an order of magnitude higher sample volumes, SeaMet only requires 0.5 mL to 1 mL of seawater for metabolite detection (17, 33). Using SeaMet, we measured 107 metabolite standards in seawater, representing major metabolite groups involved in primary metabolic pathways ( Table S4 ).…”

“…Moreover, our workflow only requires 0.5 mL to 1 mL of seawater for metabolite detection, at least an order of magnitude less sample volumes than previously published techniques (13,18) . To date, we have successfully measured 107 metabolite standards dissolved in artificial seawater, representing major metabolite groups involved in primary metabolic pathways ( Table S4 ), highlighting that SeaMet can be used for both targeted and untargeted applications.…”

Metabolite composition of sinking particles reflects a changing microbial community and differential metabolite degradation

“…This resulted in flux values (F) of nmol m -2 d -1 for each detected metabolite. After data quality controls, we considered 34 metabolites for further study, of which 27 metabolites occurred in the sinking particle samples (Table 1; (see Johnson et al 2017 for detection limits)).…”