Summary of Study ST003659

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002269. The data can be accessed directly via it's Project DOI: 10.21228/M8BC15 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST003659
Study Title	The native small molecule interaction landscape of transcription factors and essential enzymes in E. coli
Study Type	untargeted metabolite-protein interactions
Study Summary	Knowledge of protein–metabolite interactions can enhance mechanistic understanding and chemical probing of biochemical processes, but the discovery of endogenous ligands remains challenging. Here, we combined rapid affinity-purification with precision mass spectrometry and highresolution molecular docking to precisely map the physical associations of 296 chemically diverse small molecule metabolite ligands with 69 distinct essential enzymes and 45 transcription factors in the Gram-negative bacterium Escherichia coli. We then conducted systematic metabolic pathway integration, pan-microbial evolutionary projections, and independent in-depth biophysical characterization experiments to define the functional significance of novel ligand interfaces. This effort revealed principles governing functional crosstalk on a network-level, divergent patterns of binding pocket conservation, and scaffolds for designing selective chemical probes. This structurally resolved ligand interactome mapping pipeline can be scaled to illuminate the native small molecule networks of complete cells and potentially entire microbial communities.
Institute	University of Toronto
Department	Chemistry
Last Name	peng
First Name	hui
Address	80 st george street, toronto, ON, M5S3H6, Canada
Email	hui.peng@utoronto.ca
Phone	6476730580
Submit Date	2024-06-11
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	GC-MS/LC-MS
Release Date	2025-01-26
Release Version	1

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Project:

Project ID:	PR002269
Project DOI:	doi: 10.21228/M8BC15
Project Title:	Protein-metabolite interactions (PMI) in E. coli
Project Type:	Untargeted metabolomics
Project Summary:	This project employed protein affinity selection in combination with untargeted metabolomics to systematically profile the interactions between endogenous metabolites and ~600 genes in E. Coli.
Institute:	University of Toronto
Department:	Chemistry
Last Name:	peng
First Name:	hui
Address:	80 st george street, toronto, ON, M5S3H6, Canada
Email:	hui.peng@utoronto.ca
Phone:	6476730580

Subject:

Subject ID:	SU003789
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

Factors:

Subject type: Bacteria; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Pulldowngroup	Batch
SA398957	metK_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398958	fldA_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398959	folA_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398960	folA_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398961	gapA_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398962	gapA_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398963	kdsB_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398964	kdsB_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398965	LpXB_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398966	LpXB_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398967	lpxH_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398968	lpxH_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398969	murB_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398970	DXS_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398971	murB_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398972	prmC_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398973	prmC_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398974	thrS_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398975	thrS_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398976	tmk_10	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398977	tmk_10R	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398978	trpS_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398979	trpS_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398980	tryS_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398981	tryS_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398982	fldA_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398983	metK_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398984	DXS_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398985	birA_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398986	adk_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398987	adk_TB_1	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398988	birA_TB_2	E. coli cultured in terrific broth (TB) medium	TB medium	batch2
SA398989	nanR_pos_2	E. coli	32 selected proteins	batch3
SA398990	murB_neg_1	E. coli	32 selected proteins	batch3
SA398991	murB_neg_2	E. coli	32 selected proteins	batch3
SA398992	murB_neg_3	E. coli	32 selected proteins	batch3
SA398993	murB_pos_1	E. coli	32 selected proteins	batch3
SA398994	murB_pos_2	E. coli	32 selected proteins	batch3
SA398995	murB_pos_3	E. coli	32 selected proteins	batch3
SA398996	nanR_neg_1	E. coli	32 selected proteins	batch3
SA398997	nanR_neg_2	E. coli	32 selected proteins	batch3
SA398998	nanR_neg_3	E. coli	32 selected proteins	batch3
SA398999	nanR_pos_1	E. coli	32 selected proteins	batch3
SA399000	nrdB_neg_1	E. coli	32 selected proteins	batch3
SA399001	nanR_pos_3	E. coli	32 selected proteins	batch3
SA399002	murA_pos_2	E. coli	32 selected proteins	batch3
SA399003	nrdB_neg_2	E. coli	32 selected proteins	batch3
SA399004	nrdB_neg_3	E. coli	32 selected proteins	batch3
SA399005	nrdB_pos_1	E. coli	32 selected proteins	batch3
SA399006	nrdB_pos_2	E. coli	32 selected proteins	batch3
SA399007	nrdB_pos_3	E. coli	32 selected proteins	batch3
SA399008	prmC_neg_1	E. coli	32 selected proteins	batch3
SA399009	prmC_neg_2	E. coli	32 selected proteins	batch3
SA399010	prmC_neg_3	E. coli	32 selected proteins	batch3
SA399011	prmC_pos_1	E. coli	32 selected proteins	batch3
SA399012	prmC_pos_2	E. coli	32 selected proteins	batch3
SA399013	murA_pos_3	E. coli	32 selected proteins	batch3
SA399014	murA_neg_2	E. coli	32 selected proteins	batch3
SA399015	murA_pos_1	E. coli	32 selected proteins	batch3
SA399016	kdsB_pos_1	E. coli	32 selected proteins	batch3
SA399017	murA_neg_1	E. coli	32 selected proteins	batch3
SA399018	metK_pos_3	E. coli	32 selected proteins	batch3
SA399019	metK_pos_2	E. coli	32 selected proteins	batch3
SA399020	metK_pos_1	E. coli	32 selected proteins	batch3
SA399021	metK_neg_3	E. coli	32 selected proteins	batch3
SA399022	metK_neg_2	E. coli	32 selected proteins	batch3
SA399023	metK_neg_1	E. coli	32 selected proteins	batch3
SA399024	kdsB_pos_3	E. coli	32 selected proteins	batch3
SA399025	kdsB_pos_2	E. coli	32 selected proteins	batch3
SA399026	pyrG_neg_2	E. coli	32 selected proteins	batch3
SA399027	kdsB_neg_3	E. coli	32 selected proteins	batch3
SA399028	murA_neg_3	E. coli	32 selected proteins	batch3
SA399029	kdsB_neg_2	E. coli	32 selected proteins	batch3
SA399030	kdsB_neg_1	E. coli	32 selected proteins	batch3
SA399031	ispF_pos_3	E. coli	32 selected proteins	batch3
SA399032	ispF_pos_2	E. coli	32 selected proteins	batch3
SA399033	ispF_pos_1	E. coli	32 selected proteins	batch3
SA399034	ispF_neg_3	E. coli	32 selected proteins	batch3
SA399035	ispF_neg_2	E. coli	32 selected proteins	batch3
SA399036	ispF_neg_1	E. coli	32 selected proteins	batch3
SA399037	ispD_pos_3	E. coli	32 selected proteins	batch3
SA399038	ispD_pos_2	E. coli	32 selected proteins	batch3
SA399039	ispD_pos_1	E. coli	32 selected proteins	batch3
SA399040	pyrG_neg_1	E. coli	32 selected proteins	batch3
SA399041	tmK_neg_1	E. coli	32 selected proteins	batch3
SA399042	pyrG_neg_3	E. coli	32 selected proteins	batch3
SA399043	TyrS_pos_3	E. coli	32 selected proteins	batch3
SA399044	TrpS_neg_2	E. coli	32 selected proteins	batch3
SA399045	TrpS_neg_3	E. coli	32 selected proteins	batch3
SA399046	TrpS_pos_1	E. coli	32 selected proteins	batch3
SA399047	TrpS_pos_2	E. coli	32 selected proteins	batch3
SA399048	TrpS_pos_3	E. coli	32 selected proteins	batch3
SA399049	TyrS_neg_1	E. coli	32 selected proteins	batch3
SA399050	TyrS_neg_2	E. coli	32 selected proteins	batch3
SA399051	TyrS_neg_3	E. coli	32 selected proteins	batch3
SA399052	TyrS_pos_1	E. coli	32 selected proteins	batch3
SA399053	TyrS_pos_2	E. coli	32 selected proteins	batch3
SA399054	UbiD_neg_1	E. coli	32 selected proteins	batch3
SA399055	trmD_pos_3	E. coli	32 selected proteins	batch3
SA399056	UbiD_neg_2	E. coli	32 selected proteins	batch3

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Collection:

Collection ID:	CO003782
Collection Summary:	The set of Escherichia coli K-12 strains overexpressing His6-tagged bacterial proteins was generated in a previous study (Kitagawa et al., DNA research, 2005, 12, 291-299) E. coli strain stocks were stored at -80°C prior to culture in Luria-Bertani (or Terrific Broth) medium containing 34 μg/mL chloramphenicol in 50 mL polypropylene tubes. After growth for ~5 hr at 37°C until the OD600 reached ~0.8, 1 mM isopropyl-β-dthiogalactoside (IPTG) was added to induce protein expression. The cells were then cultured overnight at 18°C and harvested by centrifugation at 6000 ×g for 5 min.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR003798
Treatment Summary:	Frozen E. coli cell pellets were suspended in 0.5 mL of lysis buffer (10 mM Tris-HCl, 500 mM NaCl, 0.1% NP40, 1× Protease Inhibitor Cocktail).and lysed by performing 3 freeze–thaw cycles alternating between -80 oC and a 37°C water baths, followed by sonication for 1 min. The lysate was centrifuged at 20,000 ×g for 30 min, and 0.4 mL of supernatant was transferred along with 7 μL of magnetic Ni-NTA beads (HIS-select nickel magnetic agarose beads (H9914, Sigma-Aldrich)) to a 96-well polypropylene microplate. After incubation at 4°C for 60 min, the beads were washed three times with 50 mM PBS, 500 mM NaCl, and 5 mM imidazole. N-terminal polyhistidine-tagged bait proteins and their bound metabolites were eluted with 50 μL of buffer containing 250 mM imidazole. To eliminate nonspecific components, the eluates were transferred to a 96-well size-exclusion microplate; native protein–ligand complexes were then recovered by brief centrifugation at 1,000 ×g for 2 min. After partial drying via SpeedVac, the ligands were eluted by reconstitution in 50 μL of 80% methanol in water.

Treatment ID:

TR003798

Treatment Summary:

Frozen E. coli cell pellets were suspended in 0.5 mL of lysis buffer (10 mM Tris-HCl, 500 mM NaCl, 0.1% NP40, 1× Protease Inhibitor Cocktail).and lysed by performing 3 freeze–thaw cycles alternating between -80 oC and a 37°C water baths, followed by sonication for 1 min. The lysate was centrifuged at 20,000 ×g for 30 min, and 0.4 mL of supernatant was transferred along with 7 μL of magnetic Ni-NTA beads (HIS-select nickel magnetic agarose beads (H9914, Sigma-Aldrich)) to a 96-well polypropylene microplate. After incubation at 4°C for 60 min, the beads were washed three times with 50 mM PBS, 500 mM NaCl, and 5 mM imidazole. N-terminal polyhistidine-tagged bait proteins and their bound metabolites were eluted with 50 μL of buffer containing 250 mM imidazole. To eliminate nonspecific components, the eluates were transferred to a 96-well size-exclusion microplate; native protein–ligand complexes were then recovered by brief centrifugation at 1,000 ×g for 2 min. After partial drying via SpeedVac, the ligands were eluted by reconstitution in 50 μL of 80% methanol in water.

Sample Preparation:

Sampleprep ID:	SP003796
Sampleprep Summary:	The pull-down assay samples from affinity purifications with incubated with methanol to denature proteins and release ligands. The solvent extracts were directly subjected to LC-MS analysis.

Chromatography:

Chromatography ID:	CH004566
Chromatography Summary:	LC was used with a C18 reversed-phase column
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Thermo Hypersil GOLD aQ C18 (100 x 2.1mm,1.9um)
Column Temperature:	30
Flow Gradient:	Acetonitrile (2% initially) was increased to 30% over 3 min, then to 100% at 10 min followed by a 3 min static hold, and then returned to initial conditions for 2 min to allow for equilibration.
Flow Rate:	0.2 mL/min
Solvent A:	100% water
Solvent B:	100% acetonitrile
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN006009
Laboratory Name:	Hui Peng
Analysis Type:	MS
Operator Name:	Jianxian Sun
Chromatography ID:	CH004566
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003659_AN006009_Results.txt
Units:	Peak intensity

Analysis ID:	AN006010
Laboratory Name:	Hui Peng
Analysis Type:	MS
Operator Name:	Jianxian Sun
Chromatography ID:	CH004566
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003659_AN006010_Results.txt
Units:	Peak intensity