Non-targeted Tandem Mass Spectrometry Research Articles

Urban and agricultural influences on the coastal dissolved organic matter pool in the Algoa Bay estuaries

While anthropogenic pollution is a major threat to aquatic ecosystem health, our knowledge of the presence of xenobiotics in coastal Dissolved Organic Matter (DOM) is still relatively poor. This is especially true for water bodies in the Global South with limited information gained mostly from targeted studies that rely on comparison with authentic standards. In recent years, non-targeted tandem mass spectrometry has emerged as a powerful tool to collectively detect and identify pollutants and biogenic DOM components in the environment, but this approach has yet to be widely utilized for monitoring ecologically important aquatic systems. In this study we compared the DOM composition of Algoa Bay, Eastern Cape, South Africa, and its two estuaries. The Swartkops Estuary is highly urbanized and severely impacted by anthropogenic pollution, while the Sundays Estuary is impacted by commercial agriculture in its catchment. We employed solid-phase extraction followed by liquid chromatography tandem mass spectrometry to annotate more than 200 pharmaceuticals, pesticides, urban xenobiotics, and natural products based on spectral matching. The identification with authentic standards confirmed the presence of methamphetamine, carbamazepine, sulfamethoxazole, N-acetylsulfamethoxazole, imazapyr, caffeine and hexa(methoxymethyl)melamine, and allowed semi-quantitative estimations for annotated xenobiotics. The Swartkops Estuary DOM composition was strongly impacted by features annotated as urban pollutants including pharmaceuticals such as melamines and antiretrovirals. By contrast, the Sundays Estuary exhibited significant enrichment of molecules annotated as agrochemicals widely used in the citrus farming industry, with predicted concentrations for some of them exceeding predicted no-effect concentrations. This study provides new insight into anthropogenic impact on the Algoa Bay system and demonstrates the utility of non-targeted tandem mass spectrometry as a sensitive tool for assessing the health of ecologically important coastal ecosystems and will serve as a valuable foundation for strategizing long-term monitoring efforts.

Bacterial and Chemical Evidence of Coastal Water Pollution from the Tijuana River in Sea Spray Aerosol.

Roughly half of the human population lives near the coast, and coastal water pollution (CWP) is widespread. Coastal waters along Tijuana, Mexico, and Imperial Beach (IB), USA, are frequently polluted by millions of gallons of untreated sewage and stormwater runoff. Entering coastal waters causes over 100 million global annual illnesses, but CWP has the potential to reach many more people on land via transfer in sea spray aerosol (SSA). Using 16S rRNA gene amplicon sequencing, we found sewage-associated bacteria in the polluted Tijuana River flowing into coastal waters and returning to land in marine aerosol. Tentative chemical identification from non-targeted tandem mass spectrometry identified anthropogenic compounds as chemical indicators of aerosolized CWP, but they were ubiquitous and present at highest concentrations in continental aerosol. Bacteria were better tracers of airborne CWP, and 40 tracer bacteria comprised up to 76% of the bacteria community in IB air. These findings confirm that CWP transfers in SSA and exposes many people along the coast. Climate change may exacerbate CWP with more extreme storms, and our findings call for minimizing CWP and investigating the health effects of airborne exposure.

Chemical Proportionality within Molecular Networks.

Molecular networking of non-targeted tandem mass spectrometry data connects structurally related molecules based on similar fragmentation spectra. Here, we report the Chemical Proportionality (ChemProp) contextualization of molecular networks. ChemProp scores the changes of abundance between two connected nodes over sequential data series (e.g., temporal or spatial relationships), which can be displayed as a direction within the network to prioritize potential biological and chemical transformations or proportional changes of (biosynthetically) related compounds. We tested the ChemProp workflow on a ground truth data set of a defined mixture and highlighted the utility of the tool to prioritize specific molecules within biological samples, including bacterial transformations of bile acids, human drug metabolism, and bacterial natural products biosynthesis. The ChemProp workflow is freely available through the Global Natural Products Social Molecular Networking (GNPS) environment.

Non-targeted tandem mass spectrometry enables the visualization of organic matter chemotype shifts in coastal seawater

Urbanization along coastlines alters marine ecosystems including contributing molecules of anthropogenic origin to the coastal dissolved organic matter (DOM) pool. A broad assessment of the nature and extent of anthropogenic impacts on coastal ecosystems is urgently needed to inform regulatory guidelines and ecosystem management. Recently, non-targeted tandem mass spectrometry approaches are gaining momentum for the analysis of global organic matter composition (chemotypes) including a wide array of natural and anthropogenic compounds. In line with these efforts, we developed a non-targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) workflow that utilizes advanced data analysis approaches such as feature-based molecular networking and repository-scale spectrum searches. This workflow allows the scalable comparison and mapping of seawater chemotypes from large-scale spatial surveys as well as molecular family level annotation of unknown compounds. As a case study, we visualized organic matter chemotype shifts in coastal environments in northern San Diego, USA, after notable rain fall in winter 2017/2018 and highlight potential anthropogenic impacts. The observed seawater chemotype, consisting of 4384 LC-MS/MS features, shifted significantly after a major rain event. Molecular drivers of this shift could be attributed to multiple anthropogenic compounds, including pesticides (Imazapyr and Isoxaben), cleaning products (Benzyl-tetradecyl-dimethylammonium) and chemical additives (Hexa (methoxymethyl)melamine) and potential degradation products. By expanding the search of identified xenobiotics to other public tandem mass spectrometry datasets, we further contextualized their possible origin and show their importance in other ecosystems. The mass spectrometry and data analysis pipelines applied here offer a scalable framework for future molecular mapping and monitoring of marine ecosystems, which will contribute to a deliberate assessment of how chemical pollution impacts our oceans.

Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra.

Metabolomics using nontargeted tandem mass spectrometry can detect thousands of molecules in a biological sample. However, structural molecule annotation is limited to structures present in libraries or databases, restricting analysis and interpretation of experimental data. Here we describe CANOPUS (class assignment and ontology prediction using mass spectrometry), a computational tool for systematic compound class annotation. CANOPUS uses a deep neural network to predict 2,497 compound classes from fragmentation spectra, including all biologically relevant classes. CANOPUS explicitly targets compounds for which neither spectral nor structural reference data are available and predicts classes lacking tandem mass spectrometry training data. In evaluation using reference data, CANOPUS reached very high prediction performance (average accuracy of 99.7% in cross-validation) and outperformed four baseline methods. We demonstrate the broad utility of CANOPUS by investigating the effect of microbial colonization in the mouse digestive system, through analysis of the chemodiversity of different Euphorbia plants and regarding the discovery of a marine natural product, revealing biological insights at the compound class level.

Total Synthesis and Biological Assessment of Novel Albicidins Discovered by Mass Spectrometric Networking.

Natural products represent an important source of potential novel antimicrobial drug leads. Low production by microorganisms in cell culture often delays the structural elucidation or even prevents a timely discovery. Starting from the anti-Gram-negative antibacterial compound albicidin produced by Xanthomonas albilineans, we describe a bioactivity-guided approach combined with non-targeted tandem mass spectrometry and spectral (molecular) networking for the discovery of novel antimicrobial compounds. We report eight new natural albicidin derivatives, four of which bear a β-methoxy cyanoalanine or β-methoxy asparagine as the central α-amino acid. We present the total synthesis of these albicidins, which facilitated the unambiguous determination of the (2 S,3 R)-stereoconfiguration which is complemented by the assessment of the stereochemistry on antibacterial activity.

Comparative Lipidomics of Caenorhabditis elegans Metabolic Disease Models by SWATH Non-Targeted Tandem Mass Spectrometry.

Tandem mass spectrometry (MS/MS) with Sequential Window Acquisition of all Theoretical (SWATH) mass spectra generates a comprehensive archive of lipid species within an extract for retrospective, quantitative MS/MS analysis. Here we apply this new technology in Caenorhabditis elegans (C. elegans) to identify potential lipid mediators and pathways. The DAF-1 type I TGF-β and DAF-2 insulin receptors transmit endocrine signals that couple metabolic status to fertility and lifespan. Mutations in daf-1 and daf-2 reduce prostaglandin-endoperoxide synthase (i.e., Cox)-independent prostaglandin synthesis, increase triacylglyceride storage, and alter transcription of numerous lipid metabolism genes. However, the extent to which DAF-1 and DAF-2 signaling modulate lipid metabolism and the underlying mechanisms are not well understood. MS/MSALL with SWATH analysis across the groups identified significant changes in numerous lipids, including specific triacylglycerols, diacylglycerols, and phosphatidylinositols. Examples are provided, using retrospective neutral loss and precursor ion scans as well as MS/MS spectra, to help identify annotated lipids and search libraries for lipids of interest. As proof of principle, we used comparative lipidomics to investigate the prostaglandin metabolism pathway. SWATH data support an unanticipated model: Cox-independent prostaglandin synthesis may involve lysophosphatidylcholine and other lyso glycerophospholipids. This study showcases the power of comprehensive, retrospectively searchable lipid archives as a systems approach for biological discovery in genetic animal models.