title: Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli.
abstract: Our understanding of metabolism is limited by a lack of knowledge about the functions of many enzymes. Here, we develop a high-throughput mass spectrometry approach to comprehensively profile proteins for in vitro enzymatic activity. Overexpressed or purified proteins are incubated in a supplemented metabolome extract containing hundreds of biologically relevant candidate substrates, and accumulating and depleting metabolites are determined by nontargeted mass spectrometry. By combining chemometrics and database approaches, we established an automated pipeline for unbiased annotation of the functions of novel enzymes. In screening all 1,275 functionally uncharacterized Escherichia coli proteins, we discovered 241 potential novel enzymes, 12 of which we experimentally validated. Our high-throughput in vitro metabolomics method is generally applicable to any purified protein or crude cell lysate of its overexpression host and enables performing up to 1,200 nontargeted enzyme assays per working day.
authors.affiliation: Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.|PhD Program on Systems Biology, Life Science Zürich, Zürich, Switzerland.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Sévin
authors.affiliation: Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.
authors.forename: Tobias
authors.identifier: 0000-0001-5006-6874
authors.initials: T
authors.lastname: Fuhrer
authors.affiliation: Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.
authors.forename: Nicola
authors.identifier: 0000-0003-1271-1021
authors.initials: N
authors.lastname: Zamboni
authors.affiliation: Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.
authors.forename: Uwe
authors.initials: U
authors.lastname: Sauer
mesh_terms: D061249:Batch Cell Culture Techniques; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D055432:Metabolomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D004798:Enzymes; D029968:Escherichia coli Proteins