Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA151366

Local Sample ID:	13_B2_D2_4S-
Subject ID:	SU001721
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001721
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
13_B2_D2_4S-	SA151366	FL017704	4S	Treatment
13_B2_D2_4S-	SA151366	FL017704	2	Day

Collection:

Collection ID:	CO001714
Collection Summary:	Whole blood was collected by Canadian Blood Services from donors providing signed informed consent. Buffy coat platelet concentrates suspended in plasma were produced by the Canadian Blood Services Blood4Research Facility in Vancouver, Canada. For each biological replicate, three buffy coat platelet units were pooled and split into three identical platelet units to minimize the effect of donor variability. One unit (RP) was stored at 22°C under constant agitation (Helmer Scientific, PC1200-Pro Platelet Incubator, Noblesville, IN). One unit (CPA) was stored in a walk-in fridge (4°C) in a platelet agitator (Forma platelet agitator, Thermo Electron Corporation. Waltham, MA). The last unit (CP) was stored in walk-in fridge (4°C) without agitation. Platelet units were sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) using a sterile syringe in a biosafety cabinet. Platelet units were sampled on days 1, 2, 4, 7, 9, 11, and 14 of storage. This study was approved by both the University of British Columbia Ethics Committee (H17-02708) and the Canadian Blood Services Ethics Board (2018-003).
Sample Type:	Platelets

Treatment:

Treatment ID:	TR001734
Treatment Summary:	Whole blood was collected by Canadian Blood Services from donors providing signed informed consent. Buffy coat platelet concentrates suspended in plasma were produced by the Canadian Blood Services Blood4Research Facility in Vancouver, Canada. For each biological replicate, three buffy coat platelet units were pooled and split into three identical platelet units to minimize the effect of donor variability. One unit (RP) was stored at 22°C under constant agitation (Helmer Scientific, PC1200-Pro Platelet Incubator, Noblesville, IN). One unit (CPA) was stored in a walk-in fridge (4°C) in a platelet agitator (Forma platelet agitator, Thermo Electron Corporation. Waltham, MA). The last unit (CP) was stored in walk-in fridge (4°C) without agitation. Platelet units were sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) using a sterile syringe in a biosafety cabinet. Platelet units were sampled on days 1, 2, 4, 7, 9, 11, and 14 of storage. This study was approved by both the University of British Columbia Ethics Committee (H17-02708) and the Canadian Blood Services Ethics Board (2018-003).

Treatment ID:

TR001734

Treatment Summary:

Whole blood was collected by Canadian Blood Services from donors providing signed informed consent. Buffy coat platelet concentrates suspended in plasma were produced by the Canadian Blood Services Blood4Research Facility in Vancouver, Canada. For each biological replicate, three buffy coat platelet units were pooled and split into three identical platelet units to minimize the effect of donor variability. One unit (RP) was stored at 22°C under constant agitation (Helmer Scientific, PC1200-Pro Platelet Incubator, Noblesville, IN). One unit (CPA) was stored in a walk-in fridge (4°C) in a platelet agitator (Forma platelet agitator, Thermo Electron Corporation. Waltham, MA). The last unit (CP) was stored in walk-in fridge (4°C) without agitation. Platelet units were sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) using a sterile syringe in a biosafety cabinet. Platelet units were sampled on days 1, 2, 4, 7, 9, 11, and 14 of storage. This study was approved by both the University of British Columbia Ethics Committee (H17-02708) and the Canadian Blood Services Ethics Board (2018-003).

Sample Preparation:

Sampleprep ID:	SP001727
Sampleprep Summary:	A volume of 50μl of platelet concentrates were extracted at a 1:10 dilution in methanol:acetonitrile:water (5:3:2, v/v/v). After vortexing at 4°C for 30 min, extracts were separated from the protein pellet by centrifugation for 10 min at 10,000g at 4°C and stored at −80°C until analysis. For polar metabolites, half of the extracts were dried down via SpeedVac prior to resuspension in ddH2O + 0.1% formic acid, to facilitate ionization and chromatographic resolution of certain polar metabolites (e.g., betaine, kyurenine, etc.).

Sampleprep ID:

SP001727

Sampleprep Summary:

A volume of 50μl of platelet concentrates were extracted at a 1:10 dilution in methanol:acetonitrile:water (5:3:2, v/v/v). After vortexing at 4°C for 30 min, extracts were separated from the protein pellet by centrifugation for 10 min at 10,000g at 4°C and stored at −80°C until analysis. For polar metabolites, half of the extracts were dried down via SpeedVac prior to resuspension in ddH2O + 0.1% formic acid, to facilitate ionization and chromatographic resolution of certain polar metabolites (e.g., betaine, kyurenine, etc.).

Combined analysis:

Analysis ID	AN002689	AN002690
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	NEGATIVE	POSITIVE
Units	Relative Abundance	Relative Abundance

Chromatography:

Chromatography ID:	CH001983
Chromatography Summary:	UHPLC-MS metabolomics analyses were performed as described,(1, 3) using a Vanquish UHPLC system coupled online to a high-resolution Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7 µm; Phenomenex, Torrance, CA, USA) at 45°C. A volume of 10 μl of sample extracts was injected into the UHPLC-MS. Each sample was injected and run with two different chromatographic and MS conditions using a 5 minute gradient at 450 µL/minute from 5-95% B (A: 5% acetonitrile, 95%water/1 mM ammonium acetate; B:95%acetonitrile/5% water, 1 mM ammonium acetate) and the MS was operated in negative ion mode.
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:	45
Flow Gradient:	5 minute gradient from 5-95% B
Flow Rate:	450 µL/min
Solvent A:	5% acetonitrile/95% water; 1 mM ammonium acetate
Solvent B:	95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:	Reversed phase

Chromatography ID:	CH001984
Chromatography Summary:	UHPLC-MS metabolomics analyses were performed as described,(1, 3) using a Vanquish UHPLC system coupled online to a high-resolution Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7 µm; Phenomenex, Torrance, CA, USA) at 45°C. A volume of 10 μl of sample extracts was injected into the UHPLC-MS. Each sample was injected and run with two different chromatographic and MS conditions, using a 5 minute gradient at 450 µL/minute from 5-95% B (A: water/0.1% formic acid; B:acetonitrile/0.1% formic acid) and the MS was operated in positive mode.
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:	45
Flow Gradient:	5 minute gradient from 5-95% B
Flow Rate:	450 µL/min
Solvent A:	5% acetonitrile/95% water; 1 mM ammonium acetate
Solvent B:	95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002488
Analysis ID:	AN002689
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC system was coupled online with a Q Exactive (Thermo, San Jose, CA, USA) scanning in Full MS mode at 70,000 resolution in the 60-900 m/z range, 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in negative or positive ion mode (separate runs).
Ion Mode:	NEGATIVE

MS ID:	MS002489
Analysis ID:	AN002690
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC system was coupled online with a Q Exactive (Thermo, San Jose, CA, USA) scanning in Full MS mode at 70,000 resolution in the 60-900 m/z range, 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in negative or positive ion mode (separate runs).
Ion Mode:	POSITIVE