Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174198

Local Sample ID:	P H-514
Subject ID:	SU001930
Subject Type:	Mammal
Subject Species:	Mesocricetus auratus
Taxonomy ID:	10036

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Subject:

Subject ID:	SU001930
Subject Type:	Mammal
Subject Species:	Mesocricetus auratus
Taxonomy ID:	10036

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
P H-514	SA174198	FL021366	SARS-CoV-2	Group
P H-514	SA174198	FL021366	4	Day post infection

Collection:

Collection ID:	CO001923
Collection Summary:	Outbred female LVG golden Syrian hamsters (6-8 weeks of age) were obtained from Charles River Laboratories (Kingston, NY). The hamsters were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine prior to intranasal inoculation with 0.1 mL of 1e5 plaque-forming units (PFU) of SARS-CoV-2 (WA-1) or H1N1 influenza A virus (A/California/04/2009). On day 2, 4, 6, and 14 after infection, 3-6 anesthetized hamsters per infection group were euthanized by exsanguination followed by intracardiac injection of veterinary euthanasia solution (SleepAway; Fort Dodge). Plasma samples were treated by exposure to germicidal UV-C light.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR001942
Treatment Summary:	The hamsters were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine prior to intranasal inoculation with 0.1 mL of 1e5 plaque-forming units (PFU) of SARS-CoV-2 (WA-1) or H1N1 influenza A virus (A/California/04/2009).

Sample Preparation:

Sampleprep ID:	SP001936
Sampleprep Summary:	Hamster plasma samples were diluted 1:4 with methanol (v/v), vortexed for 30 seconds, and incubated at -20 C for 2 hours. Samples were centrifuged for 10 minutes at 13,500 x g at 4°C and supernatant was transferred to a new centrifuge tube, concentrated, and stored at -80 C until reconstitution. Hamster plasma was thawed on ice. A 50 µL aliquot was transferred onto the solid-phase-extraction (SPE)-system CAPTIVA-EMR Lipid 96-wellplate (Agilent Technologies) before addition of 250 µL of acetonitrile containing 1% formic acid (v/v) and 10 µM internal standard (consisting of uniformly 13C and 15N labeled amino acids from Cambridge Isotope Laboratories, Inc). The samples were mixed for 1 min at 360 rpm on an orbital shaker at room temperature prior to a 10 min incubation period at 4 C. Afterwards, 200 µL 80% acetonitrile in water (v/v) were added to the samples. The samples were mixed on an orbital shaker (360 rpm) for an additional 10 min at room temperature. The samples were then eluted into a 96-deepwell collection plate by centrifugation (10 min, 57 x g, 4 C followed by 2 min, 1000 x g, 4 C). Polar eluates were stored at -80 C until the day of LC/MS analysis. The SPE-plates were then washed twice with 500 µL 80% acetonitrile in water (v/v). Lipids still bound to the SPE-material were then released into a second elution plate, in two elution steps applying 2x 500 µL 1:1 methyl tert-butyl ether:methanol (v/v) onto the SPE cartridge and centrifuging for 2 min at 1000 g and 4 C. The combined eluates were dried under a stream of nitrogen (Biotage SPE Dry Evaporation System) at room temperature and reconstituted with 100 µL 1:1 2-propanol:methanol (v/v) prior to LC/MS analysis.

Sampleprep ID:

SP001936

Sampleprep Summary:

Hamster plasma samples were diluted 1:4 with methanol (v/v), vortexed for 30 seconds, and incubated at -20 C for 2 hours. Samples were centrifuged for 10 minutes at 13,500 x g at 4°C and supernatant was transferred to a new centrifuge tube, concentrated, and stored at -80 C until reconstitution. Hamster plasma was thawed on ice. A 50 µL aliquot was transferred onto the solid-phase-extraction (SPE)-system CAPTIVA-EMR Lipid 96-wellplate (Agilent Technologies) before addition of 250 µL of acetonitrile containing 1% formic acid (v/v) and 10 µM internal standard (consisting of uniformly 13C and 15N labeled amino acids from Cambridge Isotope Laboratories, Inc). The samples were mixed for 1 min at 360 rpm on an orbital shaker at room temperature prior to a 10 min incubation period at 4 C. Afterwards, 200 µL 80% acetonitrile in water (v/v) were added to the samples. The samples were mixed on an orbital shaker (360 rpm) for an additional 10 min at room temperature. The samples were then eluted into a 96-deepwell collection plate by centrifugation (10 min, 57 x g, 4 C followed by 2 min, 1000 x g, 4 C). Polar eluates were stored at -80 C until the day of LC/MS analysis. The SPE-plates were then washed twice with 500 µL 80% acetonitrile in water (v/v). Lipids still bound to the SPE-material were then released into a second elution plate, in two elution steps applying 2x 500 µL 1:1 methyl tert-butyl ether:methanol (v/v) onto the SPE cartridge and centrifuging for 2 min at 1000 g and 4 C. The combined eluates were dried under a stream of nitrogen (Biotage SPE Dry Evaporation System) at room temperature and reconstituted with 100 µL 1:1 2-propanol:methanol (v/v) prior to LC/MS analysis.

Combined analysis:

Analysis ID	AN003001	AN003002	AN003003	AN003004
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	SeQuant ZIC-pHILIC (100 x 2.1mm,5um) including a ZIC-pHILIC guard (2.1 mm x 20 mm,5um)	SeQuant ZIC-pHILIC (100 x 2.1mm,5um) including a ZIC-pHILIC guard (2.1 mm x 20 mm,5um)	Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) including an Acquity UPLC® HSS T3 VanGuard Pre-Column (2.1 x 5mm,1.8 µm)	Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) including an Acquity UPLC® HSS T3 VanGuard Pre-Column (2.1 x 5mm,1.8 µm)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF	QTOF
MS instrument name	Agilent 6540 QTOF	Agilent 6540 QTOF	Agilent 6545 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Relative Intensity	Relative Intensity	Relative Intensity	Relative Intensity

Chromatography:

Chromatography ID:	CH002226
Chromatography Summary:	An aliquot of 2 µL of polar metabolite extract was subjected to LC/MS analysis by using an Agilent 1290 Infinity II liquid-chromatography (LC) system coupled to an Agilent 6540 Quadrupole-Time-of-Flight (Q-TOF) mass spectrometer with a dual Agilent Jet Stream electrospray ionization source. Polar metabolites were separated on a SeQuant® ZIC®-pHILIC column (100 x 2.1 mm, 5 µm, polymer, Merck-Millipore) including a ZIC®-pHILIC guard column (2.1 mm x 20 mm, 5 µm). The column compartment temperature was maintained at 40 C and the flow rate was set to 250 µLmin-1. The mobile phases consisted of A: 95% water, 5% acetonitrile, 20 mM ammonium bicarbonate, 0.1% ammonium hydroxide solution (25% ammonia in water), 2.5 µM medronic acid, and B: 95% acetonitrile, 5% water, 2.5 µM medronic acid. The following linear gradient was applied: 0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B followed by a re-equilibration phase of 4 min at 400 µLmin-1 and 2 min at 250 µLmin-1.
Instrument Name:	Agilent 1290 Infinity II
Column Name:	SeQuant ZIC-pHILIC (100 x 2.1mm,5um) including a ZIC-pHILIC guard (2.1 mm x 20 mm,5um)
Column Temperature:	40
Flow Gradient:	0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B followed by a re-equilibration phase of 4 min
Flow Rate:	250ul/min
Solvent A:	95% water/5% acetonitrile; 20 mM ammonium bicarbonate; 0.1% ammonium hydroxide; 2.5 µM medronic acid
Solvent B:	95% acetonitrile/5% water; 2.5 µM medronic acid
Chromatography Type:	HILIC

Chromatography ID:	CH002227
Chromatography Summary:	An aliquot of 2 µL of lipid extract was subjected to LC/MS analysis by using an Agilent 1290 Infinity II LC-system coupled to an Agilent 6545 Q-TOF mass spectrometer with a dual Agilent Jet Stream electrospray ionization source. Lipids were separated on an Acquity UPLC® HSS T3 column (2.1 x 150 mm, 1.8 µm) including an Acquity UPLC® HSS T3 VanGuard Pre-Column (2.1 x 5mm, 1.8 µm) at a temperature of 60 C and a flow rate of 250 µLmin-1. The mobile phases consisted of A: 60% acetonitrile, 40% water, 0.1% formic acid, 10 mM ammonium formate, 2.5 µM medronic acid, and B: 90% 2-propanol, 10% acetonitrile, 0.1% formic acid, 10 mM ammonium formate (dissolved in 1 mL water). The following linear gradient was used: 0-2 min, 30% B; 17 min, 75% B; 20 min, 85%; 23-26 min, 100% B; 26, 30% B followed by a re-equilibration phase of 5 min.
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) including an Acquity UPLC® HSS T3 VanGuard Pre-Column (2.1 x 5mm,1.8 µm)
Column Temperature:	60
Flow Gradient:	The following linear gradient was used: 0-2 min, 30% B; 17 min, 75% B; 20 min, 85%; 23-26 min, 100% B; 26, 30% B followed by a re-equilibration phase of 5 min.
Flow Rate:	0.25ml/min
Solvent A:	60% acetonitrile/40% water; 0.1% formic acid; 10mM ammonium formate; 2.5uM medronic acid
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002790
Analysis ID:	AN003001
Instrument Name:	Agilent 6540 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	gas temperature 200 C, drying gas flow 10 Lmin-1, nebulizer pressure 44 psi, sheath gas temperature 300 C, sheath gas flow 12 Lmin-1, VCap 3000 V, nozzle voltage 2000 V, Fragmentor 100 V, Skimmer 65 V, Oct 1 RF Vpp 750 V, and m/z range 50-1700. Data were acquired under continuous reference mass correction at m/z 121.0509 and 922.0890 for positive ion mode and m/z 119.0363 and 966.0007 for negative ion mode. Polar metabolite identifications were supported by matching the retention time, accurate mass, and MS/MS fragmentation data to our in-house retention time and MS/MS library created from authentic reference standards (Mass Spectrometry Metabolite Library supplied by IROA Technologies, Millipore Sigma, St. Louis, MO, USA) and online MS/MS libraries (Human Metabolome Database (HMDB, https://hmdb.ca), Mass Bank of North America (MoNA, https://mona.fiehnlab.ucdavis.edu/), and mzCloud (https://mzcloud.org). Lipid iterative MS/MS data were annotated with the Agilent Lipid Annotator software. All data files were then analyzed in Skyline (Version 20.1.0.155) to obtain peak areas.
Ion Mode:	POSITIVE

MS ID:	MS002791
Analysis ID:	AN003002
Instrument Name:	Agilent 6540 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	gas temperature 200 C, drying gas flow 10 Lmin-1, nebulizer pressure 44 psi, sheath gas temperature 300 C, sheath gas flow 12 Lmin-1, VCap 3000 V, nozzle voltage 2000 V, Fragmentor 100 V, Skimmer 65 V, Oct 1 RF Vpp 750 V, and m/z range 50-1700. Data were acquired under continuous reference mass correction at m/z 121.0509 and 922.0890 for positive ion mode and m/z 119.0363 and 966.0007 for negative ion mode. Polar metabolite identifications were supported by matching the retention time, accurate mass, and MS/MS fragmentation data to our in-house retention time and MS/MS library created from authentic reference standards (Mass Spectrometry Metabolite Library supplied by IROA Technologies, Millipore Sigma, St. Louis, MO, USA) and online MS/MS libraries (Human Metabolome Database (HMDB, https://hmdb.ca), Mass Bank of North America (MoNA, https://mona.fiehnlab.ucdavis.edu/), and mzCloud (https://mzcloud.org). Lipid iterative MS/MS data were annotated with the Agilent Lipid Annotator software. All data files were then analyzed in Skyline (Version 20.1.0.155) to obtain peak areas.
Ion Mode:	NEGATIVE

MS ID:	MS002792
Analysis ID:	AN003003
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	gas temperature 250 C, drying gas flow 11 Lmin-1, nebulizer pressure 35 psi, sheath gas temperature 300 C, sheath gas flow 12 Lmin-1, VCap 3000 V, nozzle voltage 500 V, Fragmentor 160 V, Skimmer 65 V, Oct 1 RF Vpp 750 V, and m/z range 50-1700. Data were acquired under continuous reference mass correction at m/z 121.0509 and 922.0890 in positive ion mode and m/z 119.0363 and 966.0007 in negative ion mode. Polar metabolite identifications were supported by matching the retention time, accurate mass, and MS/MS fragmentation data to our in-house retention time and MS/MS library created from authentic reference standards (Mass Spectrometry Metabolite Library supplied by IROA Technologies, Millipore Sigma, St. Louis, MO, USA) and online MS/MS libraries (Human Metabolome Database (HMDB, https://hmdb.ca), Mass Bank of North America (MoNA, https://mona.fiehnlab.ucdavis.edu/), and mzCloud (https://mzcloud.org). Lipid iterative MS/MS data were annotated with the Agilent Lipid Annotator software. All data files were then analyzed in Skyline (Version 20.1.0.155) to obtain peak areas.
Ion Mode:	POSITIVE

MS ID:	MS002793
Analysis ID:	AN003004
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	gas temperature 250 C, drying gas flow 11 Lmin-1, nebulizer pressure 35 psi, sheath gas temperature 300 C, sheath gas flow 12 Lmin-1, VCap 3000 V, nozzle voltage 500 V, Fragmentor 160 V, Skimmer 65 V, Oct 1 RF Vpp 750 V, and m/z range 50-1700. Data were acquired under continuous reference mass correction at m/z 121.0509 and 922.0890 in positive ion mode and m/z 119.0363 and 966.0007 in negative ion mode. Polar metabolite identifications were supported by matching the retention time, accurate mass, and MS/MS fragmentation data to our in-house retention time and MS/MS library created from authentic reference standards (Mass Spectrometry Metabolite Library supplied by IROA Technologies, Millipore Sigma, St. Louis, MO, USA) and online MS/MS libraries (Human Metabolome Database (HMDB, https://hmdb.ca), Mass Bank of North America (MoNA, https://mona.fiehnlab.ucdavis.edu/), and mzCloud (https://mzcloud.org). Lipid iterative MS/MS data were annotated with the Agilent Lipid Annotator software. All data files were then analyzed in Skyline (Version 20.1.0.155) to obtain peak areas.
Ion Mode:	NEGATIVE