Sharing and community curation of mass spectrometry data with GNPS
The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well suited to high throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http:/gnps.ucsd.edu) an openaccess knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community wide reference MS libraries will underpin improved annotations. Datadriven social networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data. Introduction Natural products (NPs) from marine and terrestrial environments, including their inhabiting microorganisms, plants, animals, and humans, are routinely analyzed using mass spectrometry. However a single mass spectrometry experiment can collect thousands of MS/MS spectra in minutes and individual projects can acquire millions of spectra. These datasets are too large for manual analysis. Further, comprehensive software and proper computational infrastructure are not readily available and only low throughput sharing of either raw or annotated spectra is feasible, even among members of the same lab. The potentially useful information in MS/MS datasets can thus remain buried in papers, laboratory notebooks, and private databases, hindering retrieval, mining, and sharing of data and knowledge. Although there are several NP databases Dictionary of Natural Products, AntiBase and MarinLit that assist in dereplication (identification of known compounds) these resources are not freely available and do not process mass spectrometry data. Conversely, mass spectrometry databases including Massbank, Metlin, mzCloud, and ReSpect host MS/MS spectra but limit data analyses to several individual spectra or a few LC MS files. While Metlin and mzCloud provide a spectrum search function, unfortunately, their libraries are not freely available. Global genomics and proteomics research has been facilitated by the development of integral resources such as the National Center for Biotechnology Information (NCBI) and UniProt KnowledgeBase (UniProtKB) which provide robust platforms for data sharing and knowledge dissemination. Recognizing the need for an analogous community platform to effectively share and analyze natural products MS data, we present the Global Natural Products Social Molecular Networking (GNPS, available at gnps.ucsd.edu) GNPS is a data driven platform for the storage, analysis, and knowledge dissemination of MS/MS spectra that enables community sharing of raw spectra, continuous annotation of deposited data, and collaborative curation of reference spectra (referred to as spectral libraries) and experimental data (organized as datasets) GNPS provides the ability to analyze a dataset and to compare it to all publically available data. By building on the computational infrastructure of the University of California San Diego (UCSD) Center for Computational Mass Spectrometry (CCMS) GNPS provides public dataset deposition/retrieval through the Mass Spectrometry Interactive Virtual Environment (MassIVE) data repository. The GNPS analysis infrastructure further enables online dereplication, automated molecular networking analysis, and crowdsourced MS/MS spectrum curation. Each dataset added to the GNPS repository is automatically reanalyzed in the next monthly cycle of continuous identification (see Living Data by Continuous Analysis below) Each of these tens of millions of spectra in GNPS datasets is matched to reference spectral libraries to annotate molecules and to discover putative analogs (Fig. 1a) From January 2014 to November 2015, GNPS has grown to serve 9,267 users from 100 countries (Fig. 1b) with 42,486 analysis sessions that have processed more than 93 million spectra as molecular networks from a quarter million LC MS runs. Searches against a combined catalog of over 221,000 MS/MS reference library spectra from 18,163 compounds (Supplementary Table 1) are possible, and GNPS has matched almost one hundred million MS/MS spectra in all public and private search jobs using an estimated 84,000 compute hours. GNPS Spectral Libraries GNPS spectral libraries enable dereplication, variable dereplication (approximate matches to spectra of related molecules) and identification of spectra in molecular networks. GNPS has collected available MS/MS spectral libraries relevant to NPs (which also include other metabolites and molecules) including MassBank, ReSpect and NIST (Table 1, Fig. 2a, and Supplementary Table 1) Altogether, these third party libraries total 212,230 MS/MS spectra representing 12,694 unique compounds (Fig. 2b) While this combined collection of reference spectra, provides a starting point for dereplication, only 1.01% of all spectra public GNPS datasets has been matched to this collection, indicating insufficient chemical space coverage. Although the NP community is working to populate this "missing" chemical space, there is no way to report discoveries of chemistries in an easily verifiable and reusable format. To begin to address this pressing need, GNPS houses both newly acquired reference spectra (GNPS Collections) as well as a crowd sourced library of community contributed reference spectra (GNPS Community) GNPS Collections includes NPs and pharmacologically active compounds totaling 6,629 MS/MS spectra of 4,243 compounds (Fig 2b, Supplementary Table 1, Supplementary Note 1,2, and Supplementary Table 2) The GNPS Community library has grown to include 2,224 MS/MS spectra of 1,325 compounds from 55 worldwide contributors. While the total number of MS/MS spectra in GNPS libraries is only 4% of the MS/MS spectra collected in third party libraries, GNPS libraries contribute matches of MS/MS spectra at a scale disproportionate to their size (Fig. 2c) The GNPS libraries account for 29% of unique compound matches and 59% of the MS/MS matches in public (88% of public+private) data. This indicates that the GNPS libraries contain compounds that are complementary to the chemical space represented in other libraries (Fig. 2c,d) Moreover, in contrast to third party libraries, spectra submitted to GNPS Community libraries are immediately searchable by the whole community, such that submissions seamlessly transfer knowledge between laboratories (Fig. 1a) in a process that is akin to the addition of genome annotations to GenBank. In order to create a robust library, it is important for submissions to be peer reviewed and, if necessary, annotations corrected or updated as appropriate. Reference spectra submitted to the GNPS Community library are categorized by the estimated reliability of the proposed submissions. Gold reference spectra must be derived from structurally characterized synthetic or purified compounds and can only be submitted by approved users. Approval is given to contributors who have undergone training. Training is initiated by contacting the corresponding authors or CCMS administrators. Silver reference spectra need to be supported by an associated publication, while Bronze reference spectra are all remaining putative annotations (Supplementary Table 3) This type of division of spectra is reminiscent of RefSeq/TPA/GenBank (genomics) and Swiss Prot/TrEMBL/UniProt (proteomics) allowing for varying tradeoffs between comprehensiveness and reliability of annotations defined as Gold, Silver, and Bronze (Fig. 2e) To enable refinements or corrections of annotations, GNPS allows for community driven, iterative re annotation of reference MS/MS spectra in a wiki like fashion, to progressively improve the library and converge towards consensus annotation of all MS/MS spectra of interest. This is a process similar to the iterative annotation of the human genome (e.g. see series of papers on NCBI GenBank) To date, 563 annotation revisions have been made in GNPS (Supplementary Table 4) most of which added metadata to library spectra or refined compound names. The history of each annotation is retained so that users can discuss the proper annotation and address disagreements via comment threads. Dereplication using GNPS High throughput dereplication of NP MS/MS data is implemented in GNPS by querying newly acquired MS/MS spectra against all the accumulated reference spectra in GNPS spectral libraries (Fig. 3a) To date, more than 93 million MS/MS spectra from various instruments (including Orbitrap, Ion Trap, qTof, and FT ICR) have been searched at GNPS, yielding putative dereplication matches of 7.7 million spectra to 15,477 compounds. In the second stage of dereplication, GNPS goes beyond re identification by utilizing variable dereplication, which is a modification tolerant spectral library search that is mediated by a spectral alignment algorithm. Variable dereplication enables the detection of significant matches to either putative analogs of known compounds (e.g. differing by one modification or substitution of a chemical group) or compounds belonging to the same general class of molecules (Fig. 3b).Variable dereplication is not available through any other computational platform. For example, GNPS variable dereplication has detected compounds with different levels of glycosylation on various substrates. As MS/MS fragmentation preferentially results in peaks from glycan fragments, it is possible to detect sets of compounds with related glycans even when the substrates to which the glycans are attached are themselves unrelated. To date, 3,891 putative analogs have been identified in publi