Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA090772

Local Sample ID:	SC_20180803_RPLCp_FMS_Sup_S10_J1
Subject ID:	SU001312
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562
Gender:	Not applicable

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Subject:

Subject ID:	SU001312
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562
Gender:	Not applicable

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SC_20180803_RPLCp_FMS_Sup_S10_J1	SA090772	FL013074	Cystitis_1	Group

Collection:

Collection ID:	CO001306
Collection Summary:	Urine-associated E. coli isolates were collected in accordance with an approved IRB
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR001327
Treatment Summary:	For each isolate, a single colony from an agar dish was inoculated in 5 mL LB and shaken overnight at 37°C under ambient atmospheric conditions. Cultures were then diluted 1:1000 in the combined human urine and grown for 6 hours to mid-log phase (37°C, shaking), under 4% oxygen to emulate the bladder environment. After 6 hours, OD600 of each isolate was measured – and cultures were normalized by volume to yield equal number of organisms from each strain – prior to pooling into groups of 8 isolates each. CFUs were enumerated for each pool to confirm bacterial denisty (~109 total E. coli per pool). Each pool was then centrifuged to separate the cellular (pellet) and supernatant fraction. Pellets and supernatants were flash frozen and stored at -80°C until for metabolomic analysis.

Treatment ID:

TR001327

Treatment Summary:

For each isolate, a single colony from an agar dish was inoculated in 5 mL LB and shaken overnight at 37°C under ambient atmospheric conditions. Cultures were then diluted 1:1000 in the combined human urine and grown for 6 hours to mid-log phase (37°C, shaking), under 4% oxygen to emulate the bladder environment. After 6 hours, OD600 of each isolate was measured – and cultures were normalized by volume to yield equal number of organisms from each strain – prior to pooling into groups of 8 isolates each. CFUs were enumerated for each pool to confirm bacterial denisty (~109 total E. coli per pool). Each pool was then centrifuged to separate the cellular (pellet) and supernatant fraction. Pellets and supernatants were flash frozen and stored at -80°C until for metabolomic analysis.

Sample Preparation:

Sampleprep ID:	SP001320
Sampleprep Summary:	Global untargeted metabolomic analyses were performed on the supernatant-fraction of ASB and cystitis pools. Aliquots of each pool (200µL) were added to individual Eppendorf tubes containing 200µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0)) (LC-MS grade). Labeled creatinine-D3 and lysine-D4 were added to each sample to assess the metabolite extraction (sample preparation) step. Samples were first subjected to protein precipitation by addition of 800µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samples were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing supernatant was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis. The pellet-fraction of each sample pool prepared as described above was lysed using 400µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0) (LC-MS grade), followed by sonication in an ice bath for 10 min. Sample volume for each pool was adjusted such that all samples have the same cell number in each vial. Samples were first subjected to protein precipitation by addition of 1000µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samplwere were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing extract was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis.

Sampleprep ID:

SP001320

Sampleprep Summary:

Global untargeted metabolomic analyses were performed on the supernatant-fraction of ASB and cystitis pools. Aliquots of each pool (200µL) were added to individual Eppendorf tubes containing 200µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0)) (LC-MS grade). Labeled creatinine-D3 and lysine-D4 were added to each sample to assess the metabolite extraction (sample preparation) step. Samples were first subjected to protein precipitation by addition of 800µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samples were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing supernatant was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis. The pellet-fraction of each sample pool prepared as described above was lysed using 400µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0) (LC-MS grade), followed by sonication in an ice bath for 10 min. Sample volume for each pool was adjusted such that all samples have the same cell number in each vial. Samples were first subjected to protein precipitation by addition of 1000µL of ice cold methanol (4x by volume), then incubated at -80C overnight. Following incubation, samplwere were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing extract was transferred to a clean Eppendorf tube, dried in vacuo and stored at -80C until further LC-MS analysis.

Combined analysis:

Analysis ID	AN002067
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Thermo Hypersil Gold
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE
Units	abundance

Chromatography:

Chromatography ID:	CH001505
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Hypersil Gold
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001918
Analysis ID:	AN002067
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Progenesis QI
Ion Mode:	POSITIVE
Analysis Protocol File:	ARutledge_MS_Protocol.docx