Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli.
Daniel C SévinTobias FuhrerNicola ZamboniUwe SauerPublished in: Nature methods (2016)
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.
Keyphrases
- high throughput
- mass spectrometry
- escherichia coli
- liquid chromatography
- single cell
- high resolution mass spectrometry
- rna seq
- gas chromatography
- high resolution
- capillary electrophoresis
- high performance liquid chromatography
- healthcare
- tandem mass spectrometry
- ms ms
- cell proliferation
- hydrogen peroxide
- emergency department
- small molecule
- cell therapy
- bone marrow
- staphylococcus aureus
- climate change
- mesenchymal stem cells
- multidrug resistant
- drug induced