Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA289303

Local Sample ID:	VIAH002_BK_1_Pos_QE1_Lipids_006
Subject ID:	SU002854
Subject Type:	Cultured cells
Subject Species:	Rickettsia parkeri; Homo sapiens
Taxonomy ID:	35792; 9606

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU002854
Subject Type:	Cultured cells
Subject Species:	Rickettsia parkeri; Homo sapiens
Taxonomy ID:	35792; 9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
VIAH002_BK_1_Pos_QE1_Lipids_006	SA289303	FL035668	blank	Genotype
VIAH002_BK_1_Pos_QE1_Lipids_006	SA289303	FL035668	blank	Treatment

Collection:

Collection ID:	CO002847
Collection Summary:	A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).
Sample Type:	A549 cells

Treatment:

Treatment ID:	TR002863
Treatment Summary:	A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).

Treatment ID:

TR002863

Treatment Summary:

A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).

Sample Preparation:

Sampleprep ID:	SP002860
Sampleprep Summary:	A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).

Sampleprep ID:

SP002860

Sampleprep Summary:

Combined analysis:

Analysis ID	AN004454	AN004455	AN004456	AN004457
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish
Column	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	counts, height	counts, height	counts, height	counts, height

Chromatography:

Chromatography ID:	CH003345
Chromatography Summary:	HILIC
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	45C
Flow Gradient:	Gradient elution was performed from 100% (B) at 0–2 min to 70% (B) at 7.7 min, 40% (B) at 9.5 min, 30% (B) at 10.25 min, 100% (B) at 12.75 min, isocratic until 16.75 min with a column flow of
Flow Rate:	0.4 mL/min.
Solvent A:	100% water; 10mM ammonium formate; 0.125% formic acid
Solvent B:	95% acetonitrile; 10mM ammonium formate; 0.125% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH003346
Chromatography Summary:	Lipids (ammonium acetate replaces ammonium formate in neg mode analysis
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	65C
Flow Gradient:	Gradient elution was performed from 15% (B) at 0 min to 30% (B) at 2 min, 48% (B) at 2.5 min, 82% (B) at 11 min, 99% (B) at 11.5 min, isocratic until 12 and resetting to initial conditions at 12.1 min through the end of the run at 15 min.
Flow Rate:	is 0.6 mL/min.
Solvent A:	60% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Solvent B:	10% acetonitrile/90% isopropanol; 10 mM ammonium formate; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004201
Analysis ID:	AN004454
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from the in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 50, 100 eV.
Ion Mode:	POSITIVE

MS ID:	MS004202
Analysis ID:	AN004455
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from the in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 50, 100 eV.
Ion Mode:	NEGATIVE

MS ID:	MS004203
Analysis ID:	AN004456
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected. Mass range was 220-1600 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 40, 60 eV.
Ion Mode:	POSITIVE

MS ID:	MS004204
Analysis ID:	AN004457
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected. Mass range was 220-1600 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 40, 60 eV.
Ion Mode:	NEGATIVE