ONTOLOGY SOURCE REFERENCE
Term Source Name	"NCIT"	"NCBITAXON"	"EDAM"	"EFO"	"BAO"	"OBI"	"MS"
Term Source File	"http://data.bioontology.org/ontologies/NCIT"	"http://data.bioontology.org/ontologies/NCBITAXON"	"http://data.bioontology.org/ontologies/EDAM"	"http://data.bioontology.org/ontologies/EFO"	"http://data.bioontology.org/ontologies/BAO"	"http://data.bioontology.org/ontologies/OBI"	"http://data.bioontology.org/ontologies/MS"
Term Source Version	"28"	"2"	"19"	"113"	"19"	"22"	"86"
Term Source Description	"National Cancer Institute Thesaurus"	"National Center for Biotechnology Information (NCBI) Organismal Classification"	"EDAM bioinformatics operations, data types, formats, identifiers and topics"	"Experimental Factor Ontology"	"BioAssay Ontology"	"Ontology for Biomedical Investigations"	"Mass Spectrometry Ontology"
INVESTIGATION
Investigation Identifier	"MTBLS373"
Investigation Title	"Investigation"
Investigation Description	""
Investigation Submission Date	"2016-08-24"
Investigation Public Release Date	"2016-01-11"
Comment[Created With Configuration]	""
Comment[Last Opened With Configuration]	""
INVESTIGATION PUBLICATIONS
Investigation PubMed ID	""
Investigation Publication DOI	"10.1038/nmeth.4103"
Investigation Publication Author List	""
Investigation Publication Title	""
Investigation Publication Status	""
Investigation Publication Status Term Accession Number	""
Investigation Publication Status Term Source REF	""
INVESTIGATION CONTACTS
Investigation Person Last Name	""
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Investigation Person Email	""
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Investigation Person Roles Term Accession Number	""
Investigation Person Roles Term Source REF	""
STUDY
Study Identifier	"MTBLS373"
Study Title	"Non-targeted in vitro metabolomics for proteome-scale identification of novel enzymes in Escherichia coli"
Study Description	"Many yet undiscovered enzymes still limit our understanding of metabolism. Here, we develop a high-throughput mass spectrometry approach to comprehensively profile proteome-scale protein collections 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 or depleting metabolite ions are determined by nontargeted mass spectrometry. Combining chemometrics and database approaches, we established an automated pipeline for unbiased annotation of novel enzyme functions that was demonstrated to achieve 75% predictive accuracy. In a screen of all 1,275 functionally uncharacterized Escherichia coli proteins, this low false-positive rate discovery method identified 241 potential novel enzymes. Twelve of these were experimentally validated to catalyze 29 diverse metabolic reactions, including the long-elusive multispecific cytidine monophosphate nucleosidase PpnN (YgdH) and the two fumarases FumD (YdhZ) and FumE (YggD). Thus, we establish high-throughput nontargeted in vitro metabolomics and the corresponding hypothesis generation pipeline as an approach for proteome-scale enzyme discovery and assign enzymatic functions to about 20% of the so far uncharacterized E. coli proteins."
Study Submission Date	"2016-08-24"
Study Public Release Date	"2016-01-11"
Study File Name	"s_MTBLS373.txt"
STUDY DESIGN DESCRIPTORS
Study Design Type	"Functional genomics"	"non-targeted metabolites"	"enzyme discovery"	"flow injection analysis"
Study Design Type Term Accession Number	"http://edamontology.org/topic_0085"	""	""	"http://purl.obolibrary.org/obo/MS_1000058"
Study Design Type Term Source REF	"EDAM"	""	""	"MS"
STUDY PUBLICATIONS
Study PubMed ID	""
Study Publication DOI	"10.1038/nmeth.4103"
Study Publication Author List	"Daniel C. Sévin, Tobias Fuhrer, Nicola Zamboni and Uwe Sauer"
Study Publication Title	"Nontargeted in vitro metabolomics for proteome-scale identification of novel enzymes in Escherichia coli"
Study Publication Status	"Submitted"
Study Publication Status Term Accession Number	""
Study Publication Status Term Source REF	""
STUDY FACTORS
Study Factor Name	"extract"	"gene"	"replicate"
Study Factor Type	"Group"	"gene"	"technical replicate"
Study Factor Type Term Accession Number	"http://purl.obolibrary.org/obo/NCIT_C43359"	"http://purl.obolibrary.org/obo/SO_0000704"	"http://www.ebi.ac.uk/efo/EFO_0002090"
Study Factor Type Term Source REF	"NCIT"	"EFO"	"EFO"
STUDY ASSAYS
Study Assay File Name	"a_MTBLS373_sevinaskascreen_metabolite_profiling_mass_spectrometry.txt"
Study Assay Measurement Type	"metabolite profiling"
Study Assay Measurement Type Term Accession Number	"http://purl.obolibrary.org/obo/OBI_0000366"
Study Assay Measurement Type Term Source REF	"OBI"
Study Assay Technology Type	"mass spectrometry"
Study Assay Technology Type Term Accession Number	"http://purl.obolibrary.org/obo/OBI_0000470"
Study Assay Technology Type Term Source REF	"OBI"
Study Assay Technology Platform	"6520 TOF-MS (Agilent)"
STUDY PROTOCOLS
Study Protocol Name	"Sample collection"	"Extraction"	"Chromatography"	"Mass spectrometry"	"Data transformation"	"Metabolite identification"
Study Protocol Type	"Sample collection"	"Extraction"	"Chromatography"	"Mass spectrometry"	"Data transformation"	"Metabolite identification"
Study Protocol Type Term Accession Number	""	""	""	""	""	""
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Study Protocol Description	"Media and bacterial cell cultivation:</br> All E. coli strains used in this study were obtained from the ASKA collection[1]. These strains were derived from E. coli K-12 AG1 and harbored plasmid pCA24N conferring chloramphenicol resistance and carrying a His6-tagged open reading frame under the control of an isopropyl-β-D-thiogalactoside (IPTG)-inducible T5-lac promoter. Cultures were inoculated from -80 °C glycerol stocks, and cultivation was performed in LB medium (10 g/l Bacto tryptone, 5 g/l Bacto yeast extract, 5 g/l NaCl, pH 7.4) in presence of 100 μM IPTG and 100 μM chloramphenicol for 16 h at 37 °C shaking at 300 rpm. For the screen, 96-well deepwell blocks with 1.5 ml medium/well were used, whereas for the validation experiments 500 ml baffled shake flasks with 50 ml medium were used. </p> Ref:</br> [1] Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 12, 291–299 (2005). doi:10.1093/dnares/dsi012. PMID:16769691</br>"	"Protein purification (enzyme screen):</br> Cells were pelleted by centrifugation (10 min at 4,000 x g) and resuspended in 400 μl of lysis buffer (20 mM sodium phosphate, 1 mM MgCl2, 20 mM imidazole, 500 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 2 mg/ml lysozyme (Fluka), 0.2 mg/ml DNAse I (Roche), pH 7.4). After incubation at 30 °C for 30 min, lysis was completed by three repeated cycles of freezing (-80 °C) and thawing (30 °C). 50 μl aliquots of the cell lysates were stored at -80 °C for the cell lysate activity assays. Remaining cell lysates were applied to 96-well format Ni2+-charged nitrilotriacetic acid (Ni-NTA) affinity columns (GE Healthcare) and washed twice with 20 column volumes of washing buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.5). Proteins were eluted from the column with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5) which was subsequently replaced by storage buffer (2 mM TrisHCl, 1 mM MgCl2, pH 7.4) using 96-well ultrafiltration plates with 10 kDa molecular weight cutoff, and proteins were stored at 4 °C at most 1 day before performing activity assays. We recorded for each purified protein the growth of its expression strain, the concentration of purified protein and whether the solution had a yellow color (see Supplementary Table 3). Four 96-well plates per workday can be easily handled in parallel, thus enabling a theoretical throughput of 384 purified proteins using this setup. </p> Non-targeted enzyme activity assays:</br> Each assay mixture contained 39 μl buffer (2 mM Tris-HCl, 2 mM MgCl2, pH 7.5), 5 μl 10-fold concentrated cofactor mix (1 mM of each NADH, NAD+, NADPH, NADP+, FAD, ATP, GTP, ITP, S-adenosyl-L-methionine, acetyl-CoA, CoA), 2.5 μl of E. coli metabolite extract cultivated on glucose minimal medium and 2.5 μl of E. coli metabolite extract cultivated on glycerol complex medium per well in 96-well plates. Assays were started by adding 1 μl of either purified protein or 1 μl of overexpression strain lysate to each well, allowed to proceed for 1 h at 22 °C and quenched by adding -80 °C-cold methanol to 75%-v/v. Each uncharacterized protein was analyzed in pentuplicates for both assay types."	"Not applicable - Flow Injection Analysis."	"Flow injection time-of-flight mass spectrometry:</br> The analysis of quenched assays was performed on a platform consisting of a Hitachi L-7100 liquid chromatography pump coupled to a Gerstel MPS2 autosampler and an Agilent 6520 QTOF mass spectrometer (Agilent, Santa Clara, CA) operated with published settings[1]. The isocratic flow rate was 150 μl/min of mobile phase consisting of isopropanol:water (60:40, v/v) buffered with 5 mM ammonium fluoride at pH 9 for negative ionization mode. For online mass axis correction, 2-propanol (in the mobile phase), taurocholic acid and Hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine were used. Mass spectra were recorded in profile mode from 50 to 1,000 m/z with a frequency of 1.4 spectra/sec using the highest available resolving power (4 GHz HiRes). Source temperature was set to 325 °C, with 5 l/min drying gas and a nebulizer pressure of 30 psig. Fragmentor, skimmer and octupole voltages were set to 175 V, 65 V and 750 V, respectively. </p> Ref:</br> [1] Fuhrer, T., Heer, D., Begemann, B. and Zamboni, N. High-throughput, accurate mass metabolome profiling of cellular extracts by flow injection-time-of-flight mass spectrometry. Anal. Chem. 83, 7074–80 (2011). doi:10.1021/ac201267k. PMID:21830798</br>"	"Spectral data processing and ion annotation. All steps of mass spectrometry data processing and analysis were performed with Matlab (The Mathworks, Natick) using functions embedded in the Bioinformatics, Statistics, Database, and Parallel Computing toolboxes as described previously[1]. Briefly, peak detection was done for each sample once on the total profile spectrum obtained by summing all single scans recorded over time and using wavelet decomposition as provided by the Bioinformatics toolbox. In this procedure, we applied a cutoff to filter peaks of less than 500 ion counts (in the summed spectrum) to avoid the detection of artifacts. Centroid lists from samples were then merged into a single matrix by binning the accurate centroid masses within the tolerance given by the instrument resolution (about 0.002 amu at m/z 300). The resulting matrix lists the intensity of each mass peak in each analyzed sample. An accurate common m/z was recalculated with a weighted average of the values obtained from independent centroiding. Because mass axis calibration was applied online during acquisition, no m/z correction was applied during processing to correct for potential drifts. After merging, negatively charged ions were tentatively annotated based on accurate mass using 0.001 Da tolerance (see Supplementary Table 1). </p> Ref:</br> [1] Fuhrer, T., Heer, D., Begemann, B. and Zamboni, N. High-throughput, accurate mass metabolome profiling of cellular extracts by flow injection-time-of-flight mass spectrometry. Anal. Chem. 83, 7074–80 (2011). doi:10.1021/ac201267k. PMID:21830798</br>"	"Annotation was based on assumption that [M-H]- and [M+F]- are the possible and dominant ionization options for negative mode. The following isotopes were considered: 12C->13C. The following neutral electrospray adducts were considered: exchange of H to K (H->K), H to Na (H->Na) and addition of sodium chloride (+NaCl). Ions and adducts were chosen based on occurrence frequency determined previously during method development[1] and are tentative in nature. We then correlated the abundance of non-annotated ions with the abundance of annotated ions across all data sets and, in case of a positive correlation (Pearson’s R > 0.8), transferred this annotation. Correlation-based annotation is highlighted by [CORR] in Supplementary Table 2. </p> Ref:</br> [1] Fuhrer, T., Heer, D., Begemann, B. and Zamboni, N. High-throughput, accurate mass metabolome profiling of cellular extracts by flow injection-time-of-flight mass spectrometry. Anal. Chem. 83, 7074–80 (2011). doi:10.1021/ac201267k. PMID:21830798</br>"
Study Protocol URI	""	""	""	""	""	""
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Study Protocol Parameters Name	""	"Post Extraction;Derivatization"	"Chromatography Instrument;Autosampler model;Column type;Column model"	"Scan polarity;Scan m/z range;Instrument;Mass analyzer;Ion source"	""	""
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STUDY CONTACTS
Study Person Last Name	"Sévin"	"Fuhrer"	"Zamboni"	"Sauer"
Study Person First Name	"Daniel"	"Tobias"	"Nicola"	"Uwe"
Study Person Mid Initials	""	""	""	""
Study Person Email	"daniel.x.sevin@gsk.com"	"fuhrer@imsb.biol.ethz.ch"	"zamboni@imsb.biol.ethz.ch"	"sauer@imsb.biol.ethz.ch"
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