Open access repository-scale propagated nearest neighbor suspect spectral library for untargeted metabolomics.

Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of MS/MS spectra originating from published untargeted metabolomics experiments. Entries in this library, or "suspects," were derived from unannotated spectra that could be linked in a molecular network to an annotated spectrum. Annotations were propagated to unknowns based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer's brain phenotype. The nearest neighbor suspect spectral library is openly available for download or for data analysis through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data.

Host macrocyclic acylcarnitines mediate symbiotic interactions between frogs and their skin microbiome.

The host-microbiome associations occurring on the skin of vertebrates significantly influence hosts' health. However, the factors mediating their interactions remain largely unknown. Herein, we used integrated technical and ecological frameworks to investigate the skin metabolites sustaining a beneficial symbiosis between tree frogs and bacteria. We characterize macrocyclic acylcarnitines as the major metabolites secreted by the frogs' skin and trace their origin to an enzymatic unbalance of carnitine palmitoyltransferases. We found that these compounds colocalize with bacteria on the skin surface and are mostly represented by members of the Pseudomonas community. We showed that Pseudomonas sp. MPFS isolated from frogs' skin can exploit acylcarnitines as its sole carbon and nitrogen source, and this metabolic capability is widespread in Pseudomonas. We summarize frogs' multiple mechanisms to filter environmental bacteria and highlight that acylcarnitines likely evolved for another function but were co-opted to provide nutritional benefits to the symbionts.

Ex vivo study of molecular changes of stained teeth following hydrogen peroxide and peroxymonosulfate treatments.

White teeth can give confidence and tend to be associated with a healthier lifestyle in modern society. Therefore, tooth-bleaching strategies have been developed, including the use of hydrogen peroxide. Recently, peroxymonosulfate has been introduced as an alternative bleaching method to hydrogen peroxide. Although both chemicals are oxidizing agents, their effects on the molecular composition of the stained teeth are yet unknown. In this study, the molecular profiles of teeth bleached with hydrogen peroxide and peroxymonosulfate were compared using Liquid Chromatography-Tandem Mass Spectrometry. Statistical analyses were used to assess the samples. In addition, reference spectral libraries and in silico tools were used to perform metabolite annotation. Overall, principal component analysis showed a strong separation between control and hydrogen peroxide and peroxymonosulfate samples (p < 0.001). The analysis of molecular changes revealed amino acids and dipeptides in stained teeth samples after hydrogen peroxide and peroxymonosulfate treatments. Noteworthy, the two bleaching methods led to distinct molecular profiles. For example, diterpenoids were more prevalent after peroxymonosulfate treatment, while a greater abundance of alkaloids was detected after hydrogen peroxide treatment. Whereas non-bleached samples (controls) showed mainly lipids. Therefore, this study shows how two different tooth-whitening peroxides could affect the molecular profiles of human teeth.

Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

The molecular impact of life in an indoor environment.

The chemistry of indoor surfaces and the role of microbes in shaping and responding to that chemistry are largely unexplored. We found that, over 1 month, people's presence and activities profoundly reshaped the chemistry of a house. Molecules associated with eating/cooking, bathroom use, and personal care were found throughout the entire house, while molecules associated with medications, outdoor biocides, and microbially derived compounds were distributed in a location-dependent manner. The house and its microbial occupants, in turn, also introduced chemical transformations such as oxidation and transformations of foodborne molecules. The awareness of and the ability to observe the molecular changes introduced by people should influence future building designs.

Heterologous Expression in Anabaena of the Columbamide Pathway from the Cyanobacterium Moorena bouillonii and Production of New Analogs.

Columbamides are chlorinated acyl amide natural products, several of which exhibit cannabinomimetic activity. These compounds were originally discovered from a culture of the filamentous marine cyanobacterium Moorena bouillonii PNG5-198 collected from the coastal waters of Papua New Guinea. The columbamide biosynthetic gene cluster (BGC) had been identified using bioinformatics, but not confirmed by experimental evidence. Here, we report the heterologous expression in Anabaena (Nostoc) PCC 7120 of the 28.5 kb BGC that encodes for columbamide biosynthesis. The production of columbamides in Anabaena is investigated under several different culture conditions, and several new columbamide analogs are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition to previously characterized columbamides A, B, and C, new columbamides I-M are produced in these experiments, and the structure of the most abundant monochlorinated analog, columbamide K (11), is fully characterized. The other new columbamide analogs are produced in only small quantities, and structures are proposed based on high-resolution-MS, MS/MS, and 1H NMR data. Overexpression of the pathway's predicted halogenases resulted in increased productions of di- and trichlorinated compounds. The most significant change in production of columbamides in Anabaena is correlated with the concentration of NaCl in the medium.

Nerpa: A Tool for Discovering Biosynthetic Gene Clusters of Bacterial Nonribosomal Peptides.

Microbial natural products are a major source of bioactive compounds for drug discovery. Among these molecules, nonribosomal peptides (NRPs) represent a diverse class of natural products that include antibiotics, immunosuppressants, and anticancer agents. Recent breakthroughs in natural product discovery have revealed the chemical structure of several thousand NRPs. However, biosynthetic gene clusters (BGCs) encoding them are known only for a few hundred compounds. Here, we developed Nerpa, a computational method for the high-throughput discovery of novel BGCs responsible for producing known NRPs. After searching 13,399 representative bacterial genomes from the RefSeq repository against 8368 known NRPs, Nerpa linked 117 BGCs to their products. We further experimentally validated the predicted BGC of ngercheumicin from Photobacterium galatheae via mass spectrometry. Nerpa supports searching new genomes against thousands of known NRP structures, and novel molecular structures against tens of thousands of bacterial genomes. The availability of these tools can enhance our understanding of NRP synthesis and the function of their biosynthetic enzymes.

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.

Chemical Gradients of Plant Substrates in an Atta texana Fungus Garden.

Many ant species grow fungus gardens that predigest food as an essential step of the ants' nutrient uptake. These symbiotic fungus gardens have long been studied and feature a gradient of increasing substrate degradation from top to bottom. To further facilitate the study of fungus gardens and enable the understanding of the predigestion process in more detail than currently known, we applied recent mass spectrometry-based approaches and generated a three-dimensional (3D) molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments to compare with lab-maintained ecosystems. IMPORTANCE The study of complex ecosystems requires an understanding of the chemical processes involving molecules from several sources. Some of the molecules present in fungus-growing ants' symbiotic system originate from plants. To facilitate the study of fungus gardens from a chemical perspective, we provide a molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments.

Chemical interplay and complementary adaptative strategies toggle bacterial antagonism and co-existence.

Bacterial communities are in a continuous adaptive and evolutionary race for survival. In this work we expand our knowledge on the chemical interplay and specific mutations that modulate the transition from antagonism to co-existence between two plant-beneficial bacteria, Pseudomonas chlororaphis PCL1606 and Bacillus amyloliquefaciens FZB42. We reveal that the bacteriostatic activity of bacillaene produced by Bacillus relies on an interaction with the protein elongation factor FusA of P. chlororaphis and how mutations in this protein lead to tolerance to bacillaene and other protein translation inhibitors. Additionally, we describe how the unspecific tolerance of B. amyloliquefaciens to antimicrobials associated with mutations in the glycerol kinase GlpK is provoked by a decrease of Bacillus cell membrane permeability, among other pleiotropic responses. We conclude that nutrient specialization and mutations in basic biological functions are bacterial adaptive dynamics that lead to the coexistence of two primary competitive bacterial species rather than their mutual eradication.

Ion identity molecular networking for mass spectrometry-based metabolomics in the GNPS environment.

Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.

Integrating genomics and metabolomics for scalable non-ribosomal peptide discovery.

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.

Metabolites from Microbes Isolated from the Skin of the Panamanian Rocket Frog Colostethus panamansis (Anura: Dendrobatidae).

The Panamanian rocket frog Colostethus panamansis (family Dendrobatidae) has been affected by chytridiomycosis, a deadly disease caused by the fungus Batrachochytrium dendrobatidis (Bd). While there are still uninfected frogs, we set out to isolate microbes from anatomically distinct regions in an effort to create a cultivable resource within Panama for potential drug/agricultural/ecological applications that perhaps could also be used as part of a strategy to protect frogs from infections. To understand if there are specific anatomies that should be explored in future applications of this resource, we mapped skin-associated bacteria of C. panamansis and their metabolite production potential by mass spectrometry on a 3D model. Our results indicate that five bacterial families (Enterobacteriaceae, Comamonadaceae, Aeromonadaceae, Staphylococcaceae and Pseudomonadaceae) dominate the cultivable microbes from the skin of C. panamansis. The combination of microbial classification and molecular analysis in relation to the anti-Bd inhibitory databases reveals the resource has future potential for amphibian conservation.

Feature-based molecular networking in the GNPS analysis environment.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

ReDU: a framework to find and reanalyze public mass spectrometry data.

We present ReDU ( https://redu.ucsd.edu/ ), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.

Reproducible molecular networking of untargeted mass spectrometry data using GNPS.

Global Natural Product Social Molecular Networking (GNPS) is an interactive online small molecule-focused tandem mass spectrometry (MS2) data curation and analysis infrastructure. It is intended to provide as much chemical insight as possible into an untargeted MS2 dataset and to connect this chemical insight to the user's underlying biological questions. This can be performed within one liquid chromatography (LC)-MS2 experiment or at the repository scale. GNPS-MassIVE is a public data repository for untargeted MS2 data with sample information (metadata) and annotated MS2 spectra. These publicly accessible data can be annotated and updated with the GNPS infrastructure keeping a continuous record of all changes. This knowledge is disseminated across all public data; it is a living dataset. Molecular networking-one of the main analysis tools used within the GNPS platform-creates a structured data table that reflects the molecular diversity captured in tandem mass spectrometry experiments by computing the relationships of the MS2 spectra as spectral similarity. This protocol provides step-by-step instructions for creating reproducible, high-quality molecular networks. For training purposes, the reader is led through a 90- to 120-min procedure that starts by recalling an example public dataset and its sample information and proceeds to creating and interpreting a molecular network. Each data analysis job can be shared or cloned to disseminate the knowledge gained, thus propagating information that can lead to the discovery of molecules, metabolic pathways, and ecosystem/community interactions.

A Convolutional Neural Network-Based Approach for the Rapid Annotation of Molecularly Diverse Natural Products.

This report describes the first application of the novel NMR-based machine learning tool "Small Molecule Accurate Recognition Technology" (SMART 2.0) for mixture analysis and subsequent accelerated discovery and characterization of new natural products. The concept was applied to the extract of a filamentous marine cyanobacterium known to be a prolific producer of cytotoxic natural products. This environmental Symploca extract was roughly fractionated, and then prioritized and guided by cancer cell cytotoxicity, NMR-based SMART 2.0, and MS2-based molecular networking. This led to the isolation and rapid identification of a new chimeric swinholide-like macrolide, symplocolide A, as well as the annotation of swinholide A, samholides A-I, and several new derivatives. The planar structure of symplocolide A was confirmed to be a structural hybrid between swinholide A and luminaolide B by 1D/2D NMR and LC-MS2 analysis. A second example applies SMART 2.0 to the characterization of structurally novel cyclic peptides, and compares this approach to the recently appearing "atomic sort" method. This study exemplifies the revolutionary potential of combined traditional and deep learning-assisted analytical approaches to overcome longstanding challenges in natural products drug discovery.

Cryptic Species Account for the Seemingly Idiosyncratic Secondary Metabolism of Sarcophyton glaucum Specimens Collected in Palau.

Sarcophyton glaucum is one of the most abundant and chemically studied soft corals with over 100 natural products reported in the literature, primarily cembrane diterpenoids. Yet, wide variation in the chemistry observed from S. glaucum over the past 50 years has led to its reputation as a capricious producer of bioactive metabolites. Recent molecular phylogenetic analysis revealed that S. glaucum is not a single species but a complex of at least seven genetically distinct species not distinguishable using traditional taxonomic criteria. We hypothesized that perceived intraspecific chemical variation observed in S. glaucum was actually due to differences between cryptic species (interspecific variation). To test this hypothesis, we collected Sarcophyton samples in Palau, performed molecular phylogenetic analysis, and prepared chemical profiles of sample extracts using gas chromatography-flame ionization detection. Both unsupervised (principal component analysis) and supervised (linear discriminant analysis) statistical analyses of these profiles revealed a strong relationship between cryptic species membership and chemical profiles. Liquid chromatography with tandem mass spectrometry-based analysis using feature-based molecular networking permitted identification of the chemical drivers of this difference between clades, including cembranoid diterpenes (2R,11R,12R)-isosarcophytoxide (5), (2S,11R,12R)-isosarcophytoxide (6), and isosarcophine (7). Our results suggest that early chemical studies of Sarcophyton may have unknowingly conflated different cryptic species of S. glaucum, leading to apparently idiosyncratic chemical variation.

Protocol for community-created public MS/MS reference spectra within the Global Natural Products Social Molecular Networking infrastructure.

RATIONALE: A major hurdle in identifying chemicals in mass spectrometry experiments is the availability of tandem mass spectrometry (MS/MS) reference spectra in public databases. Currently, scientists purchase databases or use public databases such as Global Natural Products Social Molecular Networking (GNPS). The MSMS-Chooser workflow is an open-source protocol for the creation of MS/MS reference spectra directly in the GNPS infrastructure. METHODS: An MSMS-Chooser Sample Template is provided and completed manually. The MSMS-Chooser Submission File and Sequence Table for data acquisition were programmatically generated. Standards from the Mass Spectrometry Metabolite Library (MSMLS) suspended in a methanol-water (1:1) solution were analyzed. Flow injection on an LC/MS/MS system was used to generate negative and positive mode data using data-dependent acquisition. The MS/MS spectra and Submission File were uploaded to MSMS-Chooser workflow in GNPS for automatic selection of MS/MS spectra. RESULTS: Data acquisition and processing required ~2 h and ~2 min, respectively, per 96-well plate using MSMS-Chooser. Analysis of the MSMLS, over 600 small molecules, using MSMS-Chooser added 889 spectra (including multiple adducts) to the public library in GNPS. Manual validation of one plate indicated accurate selection of MS/MS scans (true positive rate of 0.96 and a true negative rate of 0.99). The MSMS-Chooser output includes a table formatted for inclusion in the GNPS library as well as the ability to directly launch searches via MASST. CONCLUSIONS: MSMS-Chooser enables rapid data acquisition, data analysis (selection of MS/MS spectra), and a formatted table for inspection and upload to GNPS. Open file-format data (.mzML or.mzXML) from most mass spectrometry platforms containing MS/MS spectra can be processed using MSMS-Chooser. MSMS-Chooser democratizes the creation of MS/MS reference spectra in GNPS which will improve annotation and strengthen the tools which use the annotation information.