Environmental metabolomics: Analytical strategies

Highlights

• Presents targeted and untargeted metabolomics methods for marine microbes

• Describes methods for sampling, metabolite extraction, and instrumental analysis

• Assesses both intracellular and extracellular metabolites

• Metabolomics provides insight into microbe-DOM interactions.

Abstract

Microbial metabolism plays a primary role in shaping the marine carbon cycle through processes of carbon fixation and remineralization. Many metabolic intermediates pass through the reservoir of marine dissolved organic matter (DOM), as compounds move among microbes as part of complex ecological networks of interactions. Environmental metabolomics can be used to identify and quantify these compounds, and thus will provide insight into the chemical underpinnings of microbial networks at the foundation of global biogeochemical cycles. Here we present methods for metabolite profiling (untargeted metabolomics) and for relative quantification (targeted metabolomics) of intracellular and extracellular metabolites from marine microbes. We describe our approach to method development with regard to metabolite extraction and instrumental analysis, culminating in the methods currently in use in our laboratory.

Introduction

Marine microbes play a key role in global biogeochemical cycles through their metabolic interactions with dissolved organic matter (DOM) in the ocean. The suite of compounds produced by a microbe reflects its metabolic state, which is a function of its genetic potential and its environment. Interactions with neighboring organisms, the availability of nutrients, and environmental conditions all exert an influence on the expressed metabolic profile and hence on microbial contributions to the DOM pool (Carlson, 2002, Nagata, 2000). Heterotrophic and many autotrophic microorganisms also rely on the low molecular weight (< 600 Da) fraction of DOM for their growth and metabolism (reviewed in Kujawinski (2011)). Identifying metabolites that pass through the pool of DOM and that interconnect microbial species is an important step in understanding the impact of microbes on the global carbon cycle. New insights into microbe-DOM interactions within the marine ecosystem can be gained through the use of tools from the field of metabolomics.

Metabolomics is the study of the low-molecular weight metabolites within a biological system (e.g. cells, tissues, or biofluid) (Viant, 2007). The purpose of environmental metabolomics is the characterization of an organism's metabolic response to natural or anthropogenic stressors in its environment (Viant, 2007). Metabolomics experiments generally fall into two categories, untargeted and targeted. Untargeted metabolomics seeks to detect and describe as much of the metabolome as possible without bias (Patti et al., 2012). By contrast, targeted metabolomics studies are typically focused on quantifying a specified set of compounds through the use of authentic standards (Lu et al., 2008). The suite of target compounds is often derived from one or more metabolic pathways of the biological system of interest (Patti et al., 2012).

Metabolomics methods can be applied to the study of microbial communities in their environment, including in marine systems. Here, untargeted metabolomics enables the discovery of new or unexpected metabolites, thereby expanding the metabolic pathways that can be queried in microbial consortia. For example, analysis of metabolites exuded by microorganisms into the surrounding dissolved phase (i.e. extracellular metabolites) can be used to elucidate cooperative relationships among microbial consortia members. Targeted metabolomics provides an important quantitative complement to untargeted methods; and in environmental applications, many of these targets are not currently studied in standard organic geochemistry investigations. For example, quantitative profiles of intracellular metabolites from targeted metabolomics can provide a snapshot of how microorganisms respond to environmental stress, such as nutrient limitation.

The application of metabolomics methods to the marine environment is, however, still in its infancy. In contrast to the large number of metabolomics studies in plants (Quanbeck et al., 2012, Tolstikov and Fiehn, 2002), human systems (Dunn et al., 2008, Kind et al., 2007, Want et al., 2010, Wikoff et al., 2009), and model microorganisms (e.g. yeast or Escherichia coli, Bajad et al., 2006, Lu et al., 2010, Rabinowitz and Kimball, 2007, Yanes et al., 2011), fewer metabolomics profiles have been collected for marine microbes. Studies to date include measurements of specific metabolite classes in seawater, such as B vitamins (Heal et al., 2014, Sañudo-Wilhelmy et al., 2012), as well as broader assessments of primary and secondary metabolites within marine microbial cultures (Baran et al., 2010, Bennette et al., 2011, Fiore et al., in press, Longnecker et al., 2015, Paul et al., 2009, Romano et al., 2014).

Marine environmental metabolomics follows in the footsteps of seminal organic geochemistry studies that explored the sources and sinks of biochemicals in the oceans such as amino acids and carbohydrates (Benner, 2002, Lee et al., 2000). These compounds are common to most microbes and participate in numerous metabolic reactions. For example, amino sugars are components of cell walls, peptides and iron-binding ligands (Kaiser and Benner, 2008) and are catabolized by a variety of bacteria in the ocean. The ratios of individual molecules and their stereoisomers track the rate of organic matter degradation during transport to the deep ocean (Kaiser and Benner, 2009). Compound-class centric studies, such as these, have provided fundamental insights to specific research questions within biogeochemistry. With the advent of new analytical approaches, environmental metabolomics can significantly broaden the range of metabolites under study by probing many compound classes simultaneously. Importantly, these complex datasets complement other systems biology tools such as meta-genomics, meta-transcriptomics and meta-proteomics, and together these tools enable systems-wide understanding of microbial metabolism and organic carbon cycling in the environment.

A variety of analytical platforms have been used for metabolomics experiments, including liquid (LC) or gas (GC) chromatography coupled with mass spectrometry (MS) (Fiehn, 2008, Lu et al., 2008, Want et al., 2007, Zelena et al., 2009), NMR spectroscopy (Barton et al., 2008), direct infusion MS (Southam et al., 2007), and capillary electrophoresis–MS (Monton and Soga, 2007). Of these, chromatography coupled with MS has emerged as a preferred approach for global metabolic profiling and metabolite quantification (Bennette et al., 2011, Dunn et al., 2011, Patti et al., 2012, Want et al., 2010).

Creating a mass-spectrometry based metabolomics method presents several challenges. Metabolites vary widely in their chemical properties and dynamic range, making the selection of a comprehensive extraction protocol and chromatographic separation technique a considerable task. The choice of ionization method can be an additional source of bias. Ionization techniques such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption/ionization (MALDI) select for certain analyte physical and chemical properties (Nordström et al., 2008). ESI is particularly well suited for polar metabolites that would normally carry a charge in solution; many metabolites of interest, in fact, have ionizable basic or acidic groups. Many neutral compounds, however, will not ionize efficiently (or at all) with ESI. For example, myo-inositol, a small carbohydrate, does not ionize by ESI. Marine microbial samples pose additional analytical challenges due to the high levels of salt in growth media and in the ocean itself. Salt in the sample matrix can lead to ion suppression of mass spectrometry signal during electrospray ionization, and the build-up of salt over time in the mass spectrometer and ESI source can decrease instrument performance and data quality. Metabolomics methods must also be able to make reproducible measurements, and include strategies to monitor that reproducibility. To this end, the particular strengths of the mass analyzer must be well matched to the experimental goals (i.e. quantitative analysis or global profiling).

The purpose of this manuscript is to review our approach over the past five years to the development of methods for marine environmental metabolomics. We present both untargeted and targeted metabolomics methods, in which we extract intracellular and extracellular metabolites from marine microbial samples and measure their composition and relative abundance with ESI coupled to liquid chromatography–mass spectrometry (LC–ESI–MS). Here, we detail our decisions in developing our analytical strategies with regard to experimental design, metabolite extraction, and LC–MS analysis, culminating with the methods currently in use in our laboratory. We address data analysis methods in a companion paper in this issue (Longnecker et al., 2015).