How Large Is the Metabolome? A Critical Analysis of Data Exchange Practices in Chemistry Tobias Kind, Martin Scholz, Oliver Fiehn* University of California Davis, Genome Center – Metabolomics, Davis, California, United States of America Abstract Background: Calculating the metabolome size of species by genome-guided reconstruction of metabolic pathways misses all products from orphan genes and from enzymes lacking annotated genes. Hence, metabolomes need to be determined experimentally. Annotations by mass spectrometry would greatly benefit if peer-reviewed public databases could be queried to compile target lists of structures that already have been reported for a given species. We detail current obstacles to compile such a knowledge base of metabolites. Results: As an example, results are presented for rice. Two rice (oryza sativa) subspecies have been fully sequenced, oryza japonica and oryza indica. Several major small molecule databases were compared for listing known rice metabolites comprising PubChem, Chemical Abstracts, Beilstein, Patent databases, Dictionary of Natural Products, SetupX/BinBase, KNApSAcK DB, and finally those databases which were obtained by computational approaches, i.e. RiceCyc, KEGG, and Reactome. More than 5,000 small molecules were retrieved when searching these databases. Unfortunately, most often, genuine rice metabolites were retrieved together with non-metabolite database entries such as pesticides. Overlaps from database compound lists were very difficult to compare because structures were either not encoded in machine-readable format or because compound identifiers were not cross-referenced between databases. Conclusions: We conclude that present databases are not capable of comprehensively retrieving all known metabolites. Metabolome lists are yet mostly restricted to genome-reconstructed pathways. We suggest that providers of (bio)chemical databases enrich their database identifiers to PubChem IDs and InChIKeys to enable cross-database queries. In addition, peer-reviewed journal repositories need to mandate submission of structures and spectra in machine readable format to allow automated semantic annotation of articles containing chemical structures. Such changes in publication standards and database architectures will enable researchers to compile current knowledge about the metabolome of species, which may extend to derived information such as spectral libraries, organ-specific metabolites, and cross-study comparisons. Citation: Kind T, Scholz M, Fiehn O (2009) How Large Is the Metabolome? A Critical Analysis of Data Exchange Practices in Chemistry. PLoS ONE 4(5): e5440. doi:10.1371/journal.pone.0005440 Editor: Michael Polymenis, Texas A&M University, United States of America Received December 16, 2008; Accepted April 15, 2009; Published May 5, 2009 Copyright: 2009 Kind et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NIEHS R01 13932: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ofiehn@ucdavis.edu Introduction Unlike animals, plants produce all metabolites by their cellular biochemical machinery from carbon dioxide and inorganic nutrients. Hence, well-studied plant species should present a best-case scenario for retrieving a comprehensive overview about all metabolites that have previously been reported and identified, or that can be deduced to be present by pathway reconstruction from genomic information. We have thus chosen rice (oryza sativa) with its two subspecies oryza japonica and oryza indica as test case. Due to the importance of rice as one of the world’s staple foods [1], the question arises: what is naturally found in rice (see Figure 1)? How many primary and secondary metabolites can be found in oryza sativa species that are established in the literature and databases? What kind of structural and spectral information for rice compounds has already been collected? Are de novo metabolite identifications needed for every species that is subjected to metabolome studies? For genome sequenced species, information about taxonomy specific metabolic pathways and metabolites can be compiled in pathway databases. Figure 2 shows that there are three general approaches: information retrieved from literature, from genome annotations or from experimental data. The first method obtains pathways and chemical structures from the peer-reviewed scientific literature by manual or automatic curation. A second way is to use in-silico methods and methods similar to gene ortholog mapping approaches and deducing metabolites using computational methods. The third approach is to obtain metabolite data directly from experimental result databases together with a complete study design and metadata. For this experimental approach, it is critical to unambiguously identify the chemical structures of the detected metabolites as these com- pounds will then be used to assign enzyme E.C. numbers and subsequently be used to annotate gene functions. It is therefore crucial to include target lists of known compounds in order to avoid redundant rediscovery. Preferentially, molecule target lists would be further constrained by information of taxonomy and organs. Structure elucidation of unknown small molecules is a tedious and complex process requiring sophisticated chromato- graphic separations [2] with multiple structural assignment tools PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5440