High-Resolution Liquid Chromatography Tandem Mass Spectrometry Enables Large Scale Molecular Characterization of Dissolved Organic Matter

Abstract

Dissolved organic matter (DOM) is arguably one of the most complex exometabolomes on earth, and is comprised of thousands of compounds, that together contribute more than 600 × 10 15 g carbon. This reservoir is primarily the product of interactions between the upper ocean's microbial food web, yet abiotic processes that occur over millennia have also modified many of its molecules. The compounds within this reservoir play important roles in determining the rate and extent of element exchange between inorganic reservoirs and the marine biosphere, while also mediating microbe-microbe interactions. As such, there has been a widespread effort to characterize DOM using high-resolution analytical methods including nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). To date, molecular information in DOM has been primarily obtained through calculated molecular formulas from exact mass. This approach has the advantage of being non-targeted, accessing the inherent complexity of DOM. Molecular structures are however still elusive and the most commonly used instruments are costly. More recently, tandem mass spectrometry has been employed to more precisely identify DOM components through comparison to library mass spectra. Here we describe a data acquisition and analysis workflow that expands the repertoire of high-resolution analytical approaches available to access the complexity of DOM molecules that are amenable to electrospray ionization (ESI) MS. We couple liquid chromatographic separation with tandem MS (LC-MS/MS) and a data analysis pipeline, that integrates peak extraction from extracted ion chromatograms (XIC), molecular formula calculation and molecular networking. This provides more precise structural characterization. Although only around 1% of detectable DOM compounds can be annotated through publicly available spectral libraries, community-wide participation in populating and annotating DOM datasets could rapidly increase the annotation rate and should be broadly encouraged. Our analysis also identifies shortcomings of the current Petras et al. LC-MS/MS Analysis of DOMdata analysis workflow that need to be addressed by the community in the future. This work will lay the foundation for an integrative, non-targeted molecular analysis of DOM which, together with next generation sequencing, meta-proteomics and physical data, will pave the way to a more comprehensive understanding of the role of DOM in structuring marine ecosystems.

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

“…2B). The numbers of spectral clusters and the range of formula stoichiometries observed in DOM produced from this single cyanobacterial isolate are roughly equivalent to the numbers of spectral network nodes and formulas observed in natural marine DOM extracts by Petras et al (2017). The viral and mechanical lysates contained more high-MW compounds than did the exudate DOM ( Fig.…”

“…While candidate elemental formulas could be calculated for each cluster's intact mass (Supporting Information Table S1), none of the fragmentation patterns produced a clear MetFrag match that would indicate a particular molecular structure. Similarly low identification rates using spectral library matching were observed and some potential causes discussed by Petras et al (2017). In the absence of specific structural identification, we used elemental formula assignments (allowing for multiple assignments within parent mass uncertainty) in order to suggest at least the broad compound classes that these clusters might represent ( Fig.…”

Fluorescent organic exudates of corals and algae in tropical reefs are compositionally distinct and increase with nutrient enrichment

“…Methods of querying the composition of DOM vary widely (Repeta ), from relatively coarse bulk characterization of elemental content (C, N, P, etc.) to more fine‐scale multivariate methods that analyze a subset of extracted compounds, ranging from chromatographic analysis of sugars (Goldberg et al ; Nelson et al ) or amino acids (Yamashita and Tanoue ), to high resolution mass spectrometry (Kido Soule et al ; Petras et al ). A subset of light‐absorbing DOM is fluorescent (fDOM), allowing multivariate spectral characterization of putative humic‐like, fulvic‐like and aromatic proteinaceous compounds (Coble ).…”

“…Coral reefs are highly productive ecosystems that thrive in oligotrophic tropical waters, in part by intense recycling of limited nutrients through a highly diverse and active microbial community (Raina et al 2009;Cardini et al 2015). Benthic primary producer communities in tropical reef ecosystems are dominated by hermatypic corals, macroalgae, sandassociated microphytobenthos, and a variety of encrusting organisms, primarily crustose coralline algae (CCA) and mixed turf algal assemblages (Hatcher 1988).…”

Native Electrospray-based Metabolomics Enables the Detection of Metal-binding Compounds

“…Although metal-binding small molecules have a variety of biological functions and many potential biomedical applications, it is currently challenging to find metal-binding compounds present in complex mixtures such as microbial culture extracts 15 , dissolved organic matter 16 , or fecal extracts 17 , and it is additionally challenging to assess metal-binding preferences. Predictions of small molecule structures using genome mining strategies have improved tremendously in recent years 18,19 ; however, it is still not possible to predict the selectivity and affinity of metal coordination sites, and it is even difficult to predict whether a small molecule contains a metal-binding site 20,21 .…”