FT-MS Facility Research Highlights
Microbial Metabolomics
Release of ecologically relevant metabolites by the cyanobacterium Synechococcus elongatus CCMP 1631(Cara L. Fiore, Krista Longnecker, Melissa C. Kido Soule, Elizabeth B. Kujawinski, Environmental Microbiology, DOI: 10.1111/1462-2920.12899)
Instruments: FT-ICR MS, TSQ-MS
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Summary: Photoautotrophic plankton in the surface ocean release organic compounds that fuel secondary production by heterotrophic bacteria. Here we show that an abundant marine cyanobacterium, Synechococcus elongatus, contributes a variety of nitrogen-rich and sulfur-containing compounds to dissolved organic matter. A combination of targeted and untargeted metabolomics and genomic tools was used to characterize the intracellular and extracellular metabolites of S. elongatus. Aromatic compounds, such as 4-hydroxybenzoic acid and phenylalanine, as well as nucleosides (e.g. thymidine, 5′-methylthioadenosine, xanthosine), the organosulfur compound 3-mercaptopropionate, and the plant auxin indole 3-acetic acid, were released by S. elongatus at multiple time points during its growth. Further, the amino acid kynurenine was found to accumulate in the media even though it was not present in the predicted metabolome of S. elongatus. This indicates that some metabolites, including those not predicted by an organism's genome, are likely excreted into the environment as waste; however, these molecules may have broader ecological relevance if they are labile to nearby microbes. The compounds described herein provide excellent targets for quantitative analysis in field settings to assess the source and lability of dissolved organic matter in situ.
Dissolved Organic Matter
Molecular characterization of dissolved organic matter associated with the Greenland ice sheet (Bhatia, M. P., S. B. Das, K. Longnecker, Matthew A. Charette, E. B. Kujawinski. 2010. Geochimica et Cosmochimica Acta, 74: 3768-3784)
Instrument: FT-ICR MS
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Summary: Subsurface microbial oxidation of overridden soils and vegetation beneath glaciers and ice sheets may affect global carbon budgets on glacial–interglacial timescales. The likelihood and magnitude of this process depends on the chemical nature and reactivity of the subglacial organic carbon stores. We examined the composition of carbon pools associated with different regions of the Greenland ice sheet (subglacial, supraglacial, proglacial) in order to elucidate the type of dissolved organic matter (DOM) present in the subglacial discharge over a melt season. Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry coupled to multivariate statistics permitted unprecedented molecular level characterization of this material and revealed that carbon pools associated with discrete glacial regions are comprised of different compound classes. Specifically, a larger proportion of protein-like compounds were observed in the supraglacial samples and in the early melt season (spring) subglacial discharge. In contrast, the late melt season (summer) subglacial discharge contained a greater fraction of lignin-like and other material presumably derived from underlying vegetation and soil. These results suggest (1) that the majority of supraglacial DOM originates from autochthonous microbial processes on the ice sheet surface, (2) that the subglacial DOM contains allochthonous carbon derived from overridden soils and vegetation as well as autochthonous carbon derived from in situ microbial metabolism, and (3) that the relative contribution of allochthonous and autochthonous material in subglacial discharge varies during the melt season. These conclusions are consistent with the hypothesis that, given sufficient time (e.g., overwinter storage), resident subglacial microbial communities may oxidize terrestrial material beneath the Greenland ice sheet.
Atmospheric Organic Matter
The chemical composition of organic nitrogen in marine rainwater and aerosols (Altieri, K. E., Hastings, M. G., Peters, A., Sigman, D. M., 2010 Fall Meeting, American Geophysical Union)
Instrument: FT-ICR MS
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Summary: The current state of knowledge on organic nitrogen in the atmosphere is very limited. Atmospheric water soluble organic nitrogen (WSON) is a subset of the complex water soluble organic matter measured in atmospheric aerosols and rainwater; as such, it impacts cloud condensation processes and aerosol chemical and optical properties. In marine and continental atmospheric deposition, the organic N fraction can be 20-80% of total N potentially influencing receiving ecosystems. Therefore, atmospheric WSON plays an important role in both atmospheric chemistry and the global biogeochemical N cycle. However, the sources (i.e., anthropogenic vs. terrestrial vs. marine), composition (e.g., reduced or oxidized N), potential connections to inorganic N (NO3- and NH4+), and spatio-temporal variability of atmospheric WSON are largely unknown. Samples were collected on or near the island of Bermuda (32.27°N, 64.87°W), which is located in the western North Atlantic and experiences seasonal changes in transport that allow for study of both anthropogenically and primarily marine influenced air masses. Rainwater samples (n=7) and aqueous extracted aerosol samples (n=4) were analyzed by positive ion ultra-high resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to characterize the chemical composition of the water soluble organic N on a per compound level. We found ~ 800 N containing compounds in 8 compound classes. The CHON+ compound class contained the largest number of N compounds (~ 460). Compared to continental rainwater [Altieri et al., ES&T, 2009], the CHON+ compounds in the marine samples are as dominant in number, yet have less regular patterns and lower O:C ratios for comparable N:C ratios. In fact, average O:C ratios of all N containing compound classes were lower in the marine samples than in continental rainwater samples. No organosulfates or nitrooxy-organosulfates were detected in the marine samples, both of which are known secondary anthropogenic compounds detected in continental rainwater. This suggests that the lifetime of these compounds is insufficient for transport to the remote marine atmosphere. Possible explanations for the differences between continental and marine samples include 1) anthropogenic emissions might lead to more secondary formation closer to the source regions, 2) there are more oxidants in air masses dominated by anthropogenic emissions leading to higher O:C ratios, or 3) higher NOx concentrations produce more water soluble carbonyls and thus more secondary formation via aqueous reactions in the anthropogenic environment. The marine rainwater and aerosols also had a large number of mixed functional compounds (i.e., sulfur and phosphorous present with nitrogen) suggesting a potentially increased importance of organic S and organic P in remote marine environments. In addition to the detailed compositional information above, we will also discuss the influence of air mass origin on the sources of WSON using air mass back trajectory data, and the potential inter-relationships of inorganic N and WSON will be investigated using the nitrogen and oxygen isotopic ratios of nitrate.
Ultrahigh-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Identification of Water-Soluble Atmospheric Organic Matter in Polluted Fog Waters (Mazzoleni, L.R., Ehrmann, B. M., Shen, X., Marshall, A. G., Collett, J. L. 2010. Environmental Science & Technology, 44: 3690-3697)
Instrument: FT-ICR MS
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Summary: The detailed molecular composition of water-soluble atmospheric organic matter (AOM) contained in fog water was studied by use of electrospray ionization ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry. We identified hundreds of individual molecular masses in the range of 100-400 u as negative quasi-molecular ions. In our fog water samples, we found a high degree of complexity across the mass range of 100 to 400 u and in some cases our mass range extended up to 1000 u. The detected negative organic ions were multifunctional compounds which included C, H, N, O, and S elements. We observed organic nitrogen (CHNO), organic sulfur (CHOS), and organic nitrooxy-sulfate compounds (CHNOS) as well as many masses with only CHO elemental composition. Analysis of the double bond equivalents (DBE), the number of rings plus the number of double bonds to carbon, suggests that these compound structures range from highly aliphatic to aromatic with DBE values of 1-11. The compounds ranged in their extent of oxidation with oxygen to carbon ratios from 0.2 to 2 with an average value of 0.43. Several CH2 and C3H4O2 series of compounds were identified in this AOM. The high extent of CH2 homologous series of compounds likely originates from primary components that have become oxidized. Over 400 C3H4O2 series (sometimes referred to as oligomers) were also found. Overall, approximately 80% of the CHO and CHNO compounds can be linked through C3H4O2 series. The series appear to represent atmospheric processing of primary and secondary compounds. However, they may also result coincidentally by atomic valence of these elements and the very high number of ions detected in these AOM samples. In general, the isolated water-soluble components identified here are amphiphilic, thus they contain both hydrophilic oxygenated functional groups and hydrophobic aliphatic and aromatic structural moieties. Results and implications from our analysis of several samples of polluted fog water will be presented.
Environmental Contaminants
Fate of Dispersants Associated with the Deepwater Horizon Oil Spill (Kujawinski, E. B., M. C. Kido Soule, D. L. Valentine, A. K. Boysen, K. Longnecker, M. C. Redmond. 2011. Environmental Science and Technology, 45: 1298-1306)
Instrument: LTQ-MS
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Summary: Response actions to the Deepwater Horizon oil spill included the injection of ~ 771,000 gallons (2,900,000 L) of chemical dispersant into the flow of oil near the seafloor. Prior to this incident, no deepwater applications of dispersant had been conducted, and thus no data exist on the environmental fate of dispersants in deepwater. We used ultrahigh resolution mass spectrometry and liquid chromatography with tandem mass spectrometry (LC/MS/MS) to identify and quantify one key ingredient of the dispersant, the anionic surfactant DOSS (dioctyl sodium sulfosuccinate), in the Gulf of Mexico deepwater during active flow and again after flow had ceased. Here we show that DOSS was sequestered in deepwater hydrocarbon plumes at 1000−1200 m water depth and did not intermingle with surface dispersant applications. Further, its concentration distribution was consistent with conservative transport and dilution at depth and it persisted up to 300 km from the well, 64 days after deepwater dispersant applications ceased. We conclude that DOSS was selectively associated with the oil and gas phases in the deepwater plume, yet underwent negligible, or slow, rates of biodegradation in the affected waters. These results provide important constraints on accurate modeling of the deepwater plume and critical geochemical contexts for future toxicological studies.
Ultrahigh Resolving Power and Mass Accuracy
Viral Glycosphingolipids Induce Lytic Infection and Cell Death in Marine Phytoplankton (Vardi A., Van Mooy B. A. S., Fredricks H. F., Popendorf K. J., Ossolinski J. E., Haramaty L., and Bidle K. A. Science 6 November 2009 326: 861-865)
Instrument: FT-ICR MS
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Summary: Marine viruses that infect phytoplankton are recognized as a major ecological and evolutionary driving force, shaping community structure and nutrient cycling in the marine environment. Little is known about the signal transduction pathways mediating viral infection. We show that viral glycosphingolipids regulate infection of Emiliania huxleyi, a cosmopolitan coccolithophore that plays a major role in the global carbon cycle. These sphingolipids derive from an unprecedented cluster of biosynthetic genes in Coccolithovirus genomes, are synthesized de novo during lytic infection, and are enriched in virion membranes. Purified glycosphingolipids induced biochemical hallmarks of programmed cell death in an uninfected host. These lipids were detected in coccolithophore populations in the North Atlantic, which highlights their potential as biomarkers for viral infection in the oceans.
E. huxleyi cells were collected by filtration on precombusted GF/F filters, which were snap frozen in liquid nitrogen. Subsequently, lipids were extracted using a modified Bligh–Dyer method, as described (S4). Cellular polar membrane lipids were analyzed by HPLC/ESI-MS as described (S5) using an Agilent 1100 HPLC and Thermo Finnigan LCQ Deca XP ion-trap mass spectrometer. Authentic glycosphingolipid standards (Avanti Polar Lipids, brain cerebrosides) were used for initial identification of retention times and to identify characteristic MS2 and MS3 fragmentation. These authentic standards were also used to construct standard curves for quantification. A subset of the samples were analyzed using identical HPLC and ESI conditions on a Thermo FTQ high-resolution Fourier-transform ion cyclotron resonance mass spectrometer (FT-MS) for confirmation of elemental formulas in glycosphingolipid molecular ions and MS2 fragment ions.
Prefluorescent Nitroxide Probe for the Highly Sensitive Determination of Peroxyl and Other Radical Oxidants (Jia, M., Y. Tang, Y. Lam, S.A. Green, N.V. Blough. 2009. Analytical Chemistry, 81: 8033-8040)
Instrument: FT-ICR MS
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Summary: Fluorescamine derivatized 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (I) is shown to undergo an irreversible reaction with peroxyl radicals and other radical oxidants to generate a more highly fluorescent diamagnetic product (II) and thus can be used as a highly sensitive and versatile probe to determine oxidant production optically, either by monitoring the changes in fluorescence intensity, by HPLC analysis with fluorescence detection, or by a combination of both approaches. By changing the [O2]/[I] ratio, we show that peroxyl radicals can be detected and quantified preferentially in the presence of other radical oxidants. Detection of photochemically produced peroxyl radicals is achieved by employing 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) alone, followed by derivatization with fluorescamine. With employment of HPLC analysis, the detection limit of II at a S/N of 2 is 
3 nM for a 125 μL injection. Preliminary applications include the detection of peroxyl radicals generated thermally in soybean phosphatidylcholine liposomes and produced photochemically in tap water.
Marine Lipids
Archaeal lipid distributions in oxygen minimum zones of the eastern South Pacific, eastern tropical North Pacific and the Gulf of California. (Sara A. Lincoln, L. Bird, E. F. DeLong, R. E. Summons. The 24th International Meeting on Organic Geochemistry, Bremen, Germany. September 2009.)
Instrument: LTQ-MS
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Summary: Oxygen minimum zones (OMZs; dissolved oxygen < 22 μM) are sites of globally important biogeochemical processes. Although they represent less than one percent of ocean volume, they account for as much as one half of marine denitrification and are significant sources of the greenhouse gases carbon dioxide and nitrous oxide. Microbes mediate these processes, but microbial ecology and community structure in OMZs are not well understood. In particular, information about the roles of marine archaea in these environments is lacking.
Here, we approach this question by determining correlations between abundances of archaeal membrane lipids in OMZ particulate organic matter [using HPLC-MS] and factors including depth, salinity, in situ temperatures, and concentrations of oxygen, nitrate, nitrite, ammonia and phosphate. The strong redox gradients of OMZs give rise to sharp biogeographic boundaries, making these sites valuable for assessing the influence of population composition on the pool of archaeal lipids.
We report abundances of glycerol dialkyl glycerol tetraether (GDGT) lipids from depth profiles of particulate organic matter from OMZs in the eastern South Pacific (20S, 70W; sampled from 0-800 m), the Gulf of California (27N, 111W; sampled from 3-400 m) and the eastern tropical North Pacific (10N, 104W; sampled from 3-1000 m). The oxygen minima in these sites differ; the shallow eastern South Pacific OMZ (~75-300 m) is driven by intense upwelling-fueled biological productivity, while the deeper (~350-650 m) eastern tropical North Pacific OMZ is viewed as representative of oxygen minima in open-ocean sites with productive waters underlain by poorly ventilated intermediate waters. A more complex interplay of physical oceanographic phenomena is thought to be responsible for maintaining the OMZ in the Gulf of California (~400-700 m when sampled). Water column GDGTs may comprise both a core or fossil component and a living, intact polar lipid (IPL) component in which labile polar head groups remain intact. Previous OMZ studies have focused on the core lipid component, and upwelling has been hypothesized to carry core GDGTs from dept. We have analyzed both IPLs and core lipids from a subset of samples to determine whether the two components of the archaeal lipid pool contain different GDGTs and whether differences are more pronounced in sites of strong upwelling.
The distribution of bacteriohopanepolyols in marine environments: probing the biogeochemical significance of hopanoids in the geologic record (James P. Sáenz, Roger E. Summons, Timothy I. Eglinton, Ann Pearson, John B. Waterbury, The 24th International Meeting on Organic Geochemistry, Bremen, Germany. September 2009.)
Instrument: LTQ-MS
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Summary: Bacteriohopanepolyols (BHPs) are a class of isoprenoids synthesized exclusively by bacteria as membrane lipids. The detection of BHPs and their degradation products in ancient sediments can informus about bacterial ecology and evolution through Earth’s history. However, our limited understanding of the function, source, and environmental distribution of hopanoids has prevented organic geochemists from taking full advantage of the biological information associated with hopanoids preserved in ancient sediments. Indeed, knowledge of the environmental distribution of hopanoids will help to inform us of their functions and bacterial sources. We have, therefore, set out to develop a picture of the natural distribution of hopanoids in marine environments through analysis of intact BHPs in environmental samples by HPLC-MS.
We have investigated the structural diversity and abundance of BHPs in a diverse suite of samples from marine sediments and the upper water column from both coastal and open ocean environments. In general, we observe the highest structural diversity in benthic bacterial mats, biofilms, and ooids in the intertidal zone, and the lowest diversity in particulate organic carbon filtered from the water column. For instance, in ooids collected from the Bahamas we identified 11 BHPs including a novel putative 2-methylbacteriohopaneribonolactone, whereas in the upper 200 meters of water column in the North Atlantic near Cape Verde we could only identify six compounds, with bacteriohopanepentol and 35-aminobacteriohopane-32,33,34-triol only occurring below 100m depth. We detected 2- methylbacteriohopanepolyols, putative biomarkers for cyanobacteria, in many shallow benthic bacterial communities, including samples from Shark Bay, Australia, San Salvador, Bahamas, and Sao Vicente, Cape Verde. We detected 2-methylbacteriohopanetetrol in marine sediments off the coast of N.W. Africa, but not in particulate organic carbon samples from the overlying water column. This could suggest a seasonal influence in the production of 2-methylBHPs in the water column, redistribution of sediments, or possibly a sedimentary source. However, good agreement in BHP structural diversity and relative abundances of individual BHPs between the water column and core top sediments goes some way to argue against a significant sedimentary source of hopanoids. We are investigating additional sites including water column and sediment samples from the coast of N.W. Africa, spanning climatic and geochemical gradients in order to understand how such patterns of BHP abundance and diversity might be linked to environmental and ecological conditions. We are also screening cyanobacteria that we have isolated and cultured directly from several benthic communities in which 2-methylhopanoids were detected, in order to determine which BHPs are being produced by cyanobacteria in these environments.