A Fast and Efficient Molecular Networking Approach for Reactivity of Natural Products Exploration in Plant Extracts: Application to Diterpene Esters from Euphorbia dendroides.

Natural products represent a rich source of bioactive compounds, covering a large chemical space. Even if challenging, this diversity can be extended by applying chemical modifications. However, these studies generally require multigram amounts of isolated natural products and face frequent testing failures. To overcome this limitation, we propose a rapid and efficient approach that uses molecular networking (MN) to visualize the new chemical diversity generated by simple chemical modifications of natural extracts. Moreover, the strategy deployed enables the most appropriate reagents to be defined quickly upstream of a reaction on a pure compound, in order to maximize chemical diversity. This methodology was applied to the latex extract of Euphorbia dendroides to follow the reactivity toward a series of Brønsted and Lewis acids of three class of diterpene esters identified in this species: jatrophane, terracinolide, and phorbol. Through the molecular networking interpretation, with the aim to illustrate our approach, BF3·OEt2 was selected for chemical modification on isolated jatrophane esters. Three rearranged compounds (3-5) were obtained, showing that the most appropriate reagents can be selected by MN interpretation.

Standardized multi-omics of Earth's microbiomes reveals microbial and metabolite diversity.

Despite advances in sequencing, lack of standardization makes comparisons across studies challenging and hampers insights into the structure and function of microbial communities across multiple habitats on a planetary scale. Here we present a multi-omics analysis of a diverse set of 880 microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry). We used standardized protocols and analytical methods to characterize microbial communities, focusing on relationships and co-occurrences of microbially related metabolites and microbial taxa across environments, thus allowing us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of an evolving community resource. We demonstrate the utility of this database by testing the hypothesis that every microbe and metabolite is everywhere but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment, whereas the relative abundances of microbially related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner. We additionally show the power of certain chemistry, in particular terpenoids, in distinguishing Earth's environments (for example, terrestrial plant surfaces and soils, freshwater and marine animal stool), as well as that of certain microbes including Conexibacter woesei (terrestrial soils), Haloquadratum walsbyi (marine deposits) and Pantoea dispersa (terrestrial plant detritus). This Resource provides insight into the taxa and metabolites within microbial communities from diverse habitats across Earth, informing both microbial and chemical ecology, and provides a foundation and methods for multi-omics microbiome studies of hosts and the environment.

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.

Chemically informed analyses of metabolomics mass spectrometry data with Qemistree.

Untargeted mass spectrometry is employed to detect small molecules in complex biospecimens, generating data that are difficult to interpret. We developed Qemistree, a data exploration strategy based on the hierarchical organization of molecular fingerprints predicted from fragmentation spectra. Qemistree allows mass spectrometry data to be represented in the context of sample metadata and chemical ontologies. By expressing molecular relationships as a tree, we can apply ecological tools that are designed to analyze and visualize the relatedness of DNA sequences to metabolomics data. Here we demonstrate the use of tree-guided data exploration tools to compare metabolomics samples across different experimental conditions such as chromatographic shifts. Additionally, we leverage a tree representation to visualize chemical diversity in a heterogeneous collection of samples. The Qemistree software pipeline is freely available to the microbiome and metabolomics communities in the form of a QIIME2 plugin, and a global natural products social molecular networking workflow.

Auto-deconvolution and molecular networking of gas chromatography-mass spectrometry data.

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.

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.

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.

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.

Investigation of Premyrsinane and Myrsinane Esters in Euphorbia cupanii and Euphobia pithyusa with MS2LDA and Combinatorial Molecular Network Annotation Propagation.

The species Euphorbia pithyusa and Euphorbia cupanii are two closely related Mediterranean spurges for which their taxonomic relationships are still being debated. Herein, the diterpene ester content of E. cupanii was investigated using liquid chromatography coupled to tandem mass spectrometry. The use of molecular networking coupled to unsupervised substructure annotation ( MS2LDA) indicated the presence of new premyrsinane/myrsinane diterpene esters in the E. cupanii fractions. A structure-guided isolation procedure yielded 16 myrsinane (11a-h, 12, and 13) and premyrsinane esters (14a-c and 15a-c), along with four 4ß-phorbol esters (16a-c and 17) that showed inhibitory activity against chikungunya virus replication. The structures of the 16 new compounds (11a-c, 11h, 12, 13, 14a-c, 15a-c, 16a-c, and 17) were characterized by NMR spectroscopy and X-ray crystallography. To further uncover the diterpene ester content of these two species, the concept of combinatorial network annotation propagation (C-NAP) was developed. By leveraging the fact that the diterpene esters of Euphorbia species are made up of limited building blocks, a combinatorial database of theoretical structures was created and used for C-NAP that made possible the annotation of 123 premyrsinane or myrsinane esters, from which 74% are not found in any compound database.

Bioactivity-Based Molecular Networking for the Discovery of Drug Leads in Natural Product Bioassay-Guided Fractionation.

It is a common problem in natural product therapeutic lead discovery programs that despite good bioassay results in the initial extract, the active compound(s) may not be isolated during subsequent bioassay-guided purification. Herein, we present the concept of bioactive molecular networking to find candidate active molecules directly from fractionated bioactive extracts. By employing tandem mass spectrometry, it is possible to accelerate the dereplication of molecules using molecular networking prior to subsequent isolation of the compounds, and it is also possible to expose potentially bioactive molecules using bioactivity score prediction. Indeed, bioactivity score prediction can be calculated with the relative abundance of a molecule in fractions and the bioactivity level of each fraction. For that reason, we have developed a bioinformatic workflow able to map bioactivity score in molecular networks and applied it for discovery of antiviral compounds from a previously investigated extract of Euphorbia dendroides where the bioactive candidate molecules were not discovered following a classical bioassay-guided fractionation procedure. It can be expected that this approach will be implemented as a systematic strategy, not only in current and future bioactive lead discovery from natural extract collections but also for the reinvestigation of the untapped reservoir of bioactive analogues in previous bioassay-guided fractionation efforts.