Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA240143

Local Sample ID:	#715_mito
Subject ID:	SU002492
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

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Subject:

Subject ID:	SU002492
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
#715_mito	SA240143	FL030268	red	group
#715_mito	SA240143	FL030268	mito	type

Collection:

Collection ID:	CO002485
Collection Summary:	Mitochondria were isolated by differential centrifugation. Freshly harvested renal cortex (50 mg) was finely minced and gently homogenized with glass Teflon tissue grinders in 2 ml ice-cold isolation medium, pH 7.2 (70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1mM EGTA). The homogenate was centrifuged at 800 g for 5 min at 4C and the resulting supernatant was centrifuged at 8,000 g for 10 min at 4C. After washing with 0.5 ml ice-cold isolation buffer, the mitochondrial pellet was resuspended in 200 l ice-cold isolation medium. Total protein was determined by the bicinchoninic acid method according to the manufacturer’s instructions (BCA Protein Assay Kit, Pierce-Thermo Fisher Scientific, Melbourne, Australia).
Sample Type:	Mitochondria

Treatment:

Treatment ID:	TR002504
Treatment Summary:	Male Sprague Dawley rats were housed in groups of three rats per cage in a temperature-controlled environment, with a 12 h light/dark cycle and ad libitum access to food and water. Experimental diabetes was induced in six week old male Sprague Dawley rats (200-250 g, n = 35) by i.v. injection of streptozotocin (55 mg/kg, sodium citrate buffer pH 4.5) following an overnight fast, as previously described (79). One group of rats received citrate buffer vehicle (0.42% in sterile saline, pH 4.5) as a non-diabetic control with normal blood glucose (NG) (n = 16). One week following STZ treatment, diabetic rats were further assigned to two groups: standard insulin therapy (n = 17 rats), resulting in severe hyperglycemia (SHG) and intensive insulin therapy (n = 19 rats), resulting in moderate hyperglycemia (MHG) using a single daily insulin injection (long-lasting Humulin NPH; Eli Lilly, Indianapolis, USA) to titrate blood glucose levels to >28 mM (1-2 units, s.c. per day) and ∼20 mM (6-7 units, s.c. per day) as required, respectively.

Treatment ID:

TR002504

Treatment Summary:

Male Sprague Dawley rats were housed in groups of three rats per cage in a temperature-controlled environment, with a 12 h light/dark cycle and ad libitum access to food and water. Experimental diabetes was induced in six week old male Sprague Dawley rats (200-250 g, n = 35) by i.v. injection of streptozotocin (55 mg/kg, sodium citrate buffer pH 4.5) following an overnight fast, as previously described (79). One group of rats received citrate buffer vehicle (0.42% in sterile saline, pH 4.5) as a non-diabetic control with normal blood glucose (NG) (n = 16). One week following STZ treatment, diabetic rats were further assigned to two groups: standard insulin therapy (n = 17 rats), resulting in severe hyperglycemia (SHG) and intensive insulin therapy (n = 19 rats), resulting in moderate hyperglycemia (MHG) using a single daily insulin injection (long-lasting Humulin NPH; Eli Lilly, Indianapolis, USA) to titrate blood glucose levels to >28 mM (1-2 units, s.c. per day) and ∼20 mM (6-7 units, s.c. per day) as required, respectively.

Sample Preparation:

Sampleprep ID:	SP002498
Sampleprep Summary:	Lipidomic analysis. Lipids were extracted from mitochondrial isolates using a single-phase chloroform/methanol extraction as described previously (87). Briefly, 20 volumes of chloroform:methanol (2:1) were added to the sample along with a series of internal standards. Samples were vortexed and centrifuged on a rotary mixer for 10 min. Following 30 min of sonication on a sonicator bath, samples were rested for 20 min before being centrifuged at 13,000 g for 10 min. Supernatants were transferred into a 96 well plate, dried down, and reconstituted in 50 µL H2O saturated butanol, before being sonicated for 10 min. Following the addition of 50 µL of methanol with 10 mM ammonium formate, samples were centrifuged at 4000 rpm on a plate centrifuge and transferred into glass vials with inserts for mass spectrometry analysis.

Sampleprep ID:

SP002498

Sampleprep Summary:

Lipidomic analysis. Lipids were extracted from mitochondrial isolates using a single-phase chloroform/methanol extraction as described previously (87). Briefly, 20 volumes of chloroform:methanol (2:1) were added to the sample along with a series of internal standards. Samples were vortexed and centrifuged on a rotary mixer for 10 min. Following 30 min of sonication on a sonicator bath, samples were rested for 20 min before being centrifuged at 13,000 g for 10 min. Supernatants were transferred into a 96 well plate, dried down, and reconstituted in 50 µL H2O saturated butanol, before being sonicated for 10 min. Following the addition of 50 µL of methanol with 10 mM ammonium formate, samples were centrifuged at 4000 rpm on a plate centrifuge and transferred into glass vials with inserts for mass spectrometry analysis.

Combined analysis:

Analysis ID	AN003917
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity II
Column	Agilent ZORBAX Eclipse Plus C18 (100 x 2.1mm,1.8um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6490 QQQ
Ion Mode	POSITIVE
Units	pmol per mg

Chromatography:

Chromatography ID:	CH002899
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent ZORBAX Eclipse Plus C18 (100 x 2.1mm,1.8um)
Column Temperature:	60
Flow Gradient:	Non-linear
Flow Rate:	0.4ml/minute
Solvent A:	50% water/30% acetonitrile/20% isopropanol; 10mM ammonium formate
Solvent B:	1% water/9% acetonitrile/90% isopropanol; 10mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003656
Analysis ID:	AN003917
Instrument Name:	Agilent 6490 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Lipid extraction. Muscle homogenates/mitochondrial isolates were extracted using a modified single-phase chloroform/methanol extraction as described previously [Weir et al. 2016]. In brief, 20 volumes of chloroform:methanol (2:1) was added to the sample along with a series of internal standards. Samples were vortexed and spun on a rotoary mixer for 10 minutes. After sonication on a sonicator bath for 30 minutes, samples were rested for a further 20 minutes prior to centrifugation at 13,000 x g for 10 minutes. Supernatants were transferred into a 96 well plated, dried down and reconstituted in 50L water saturated butanol and sonicated for 10 minutes. After the addition of 50l of methanol with 10mM ammonium formate, the samples were spun down again at 4000RPM on a plate centrifuge (Heraeus multifuge 1S-R, ThermoFisher) and transferred into glass vials with inserts for mass spectrometry analysis. Targeted lipidomics analysis. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed according to previously published methods, with slight modification for tissue samples [Huynh et al. 2019]. Sample extracts were analysed using either (i) a 4000 QTRAP mass spectrometer (Sciex) for cardiolipins as described preciously [Tan et al. 2020] or (ii) an Agilent 6490 QQQ mass spectrometer all other lipid species. Lipids run on the Agilent 6490 were measured using scheduled multiple reaction monitoring with the following conditions: Isolation widths for Q1 and Q3 were set to “unit” resolution (0.7 amu), gas temperature, 150°C, nebulizer 20psi, sheath gas temperature 200°C, gas flow rate 17L/min, capillary voltage 3500V and sheath gas flow 10L/min. The list of MRMs used and chromatographic conditions were extensively described previously [Huynh et al. 2019]
Ion Mode:	POSITIVE