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MB Sample ID: SA183963

Local Sample ID:	4A_T1_I3_G2_L1
Subject ID:	SU002028
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Genotype Strain:	Sprague-Dawley
Age Or Age Range:	8 weeks
Weight Or Weight Range:	250-450 g
Gender:	Male and female
Animal Animal Supplier:	Charles River
Animal Light Cycle:	12 h reverse light-dark cycles
Animal Feed:	ad libitum
Animal Water:	ad libitum

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU002028
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Genotype Strain:	Sprague-Dawley
Age Or Age Range:	8 weeks
Weight Or Weight Range:	250-450 g
Gender:	Male and female
Animal Animal Supplier:	Charles River
Animal Light Cycle:	12 h reverse light-dark cycles
Animal Feed:	ad libitum
Animal Water:	ad libitum

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
4A_T1_I3_G2_L1	SA183963	FL022632	Repeat-Impact	Injury Severity
4A_T1_I3_G2_L1	SA183963	FL022632	M	Sex
4A_T1_I3_G2_L1	SA183963	FL022632	Baseline	Blood Collection

Collection:

Collection ID:	CO002021
Collection Summary:	Approximately 200 µL of whole blood was collected from a tail vein punctured by 20-gauge Precision Glide needles and stored on ice. Whole blood samples were allowed to coagulate at room temperature for 45 minutes. Samples were then centrifuged at 4 °C for 15 min at 2500 x g, and serum was collected in 50 μL aliquots and stored at -80 °C.
Collection Protocol ID:	A100188
Collection Protocol Comments:	All procedures involving Sprague-Dawley rat models were performed in accordance with guidelines set forth in the Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services, Pub no. 85-23, 1985) and were approved by the Georgia Institute of Technology Institutional Animal Care and Use Committee
Sample Type:	Blood (serum)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002040
Treatment Summary:	A CCI device (Pittsburgh Precision Instruments, Pittsburgh, PA) was used to induce single and repetitive closed-head injuries to the cortex. Prior to injuries, all rat groups were anesthetized with isoflurane (induction: 5% isoflurane; maintenance: 2-3% isoflurane) and a toe pinch was administered to evaluate loss of consciousness and ensure minimal pain during injury. A pneumatic piston on the CCI device with a 5 mm tip diameter was positioned 15 degrees below the vertical axis of the coronal plane to induce injury to the closed skull, 30 s after removal of the isoflurane supply. All injury groups received impacts from the pneumatic piston at a velocity of 5 m/s. The single impact injury group received one injury with a 5 mm head displacement. The repeat injury group received a total of 3 injuries at 2 min intervals, with head displacements of 5 mm, 2 mm, and 2 mm, respectively. Sham-operated animals received a treatment identical to injured animals but excluding the injury procedure. Following final injury, time-to-right was recorded, and animals were monitored to survey the presence of neurological deficits. Animals were returned to home cages and singly housed with soft bedding during recovery.

Treatment ID:

TR002040

Treatment Summary:

A CCI device (Pittsburgh Precision Instruments, Pittsburgh, PA) was used to induce single and repetitive closed-head injuries to the cortex. Prior to injuries, all rat groups were anesthetized with isoflurane (induction: 5% isoflurane; maintenance: 2-3% isoflurane) and a toe pinch was administered to evaluate loss of consciousness and ensure minimal pain during injury. A pneumatic piston on the CCI device with a 5 mm tip diameter was positioned 15 degrees below the vertical axis of the coronal plane to induce injury to the closed skull, 30 s after removal of the isoflurane supply. All injury groups received impacts from the pneumatic piston at a velocity of 5 m/s. The single impact injury group received one injury with a 5 mm head displacement. The repeat injury group received a total of 3 injuries at 2 min intervals, with head displacements of 5 mm, 2 mm, and 2 mm, respectively. Sham-operated animals received a treatment identical to injured animals but excluding the injury procedure. Following final injury, time-to-right was recorded, and animals were monitored to survey the presence of neurological deficits. Animals were returned to home cages and singly housed with soft bedding during recovery.

Sample Preparation:

Sampleprep ID:	SP002034
Sampleprep Summary:	A standard spiked IPA solution was prepared with 250 µL of SPLASH II Lipidomix and 14.750 mL of IPA. Serum samples were thawed on ice for one hour prior to the addition of the IPA solution in a 1:3 v/v ratio to separate lipids and small non-polar metabolites from proteins. Mixtures of serum and IPA solution were vortexed for 10 s and centrifuged at 16000g for 7 min. The supernatant was then collected for LC-MS analysis. Sample blanks were prepared with 50 µL of LC-MS grade water, and pooled QC samples were prepared from 5 µL aliquots of all study subject serum samples. Serum reference samples from uninjured Sprague-Dawley rat serum were processed in the same manner as study subject serum samples.

Sampleprep ID:

SP002034

Sampleprep Summary:

A standard spiked IPA solution was prepared with 250 µL of SPLASH II Lipidomix and 14.750 mL of IPA. Serum samples were thawed on ice for one hour prior to the addition of the IPA solution in a 1:3 v/v ratio to separate lipids and small non-polar metabolites from proteins. Mixtures of serum and IPA solution were vortexed for 10 s and centrifuged at 16000g for 7 min. The supernatant was then collected for LC-MS analysis. Sample blanks were prepared with 50 µL of LC-MS grade water, and pooled QC samples were prepared from 5 µL aliquots of all study subject serum samples. Serum reference samples from uninjured Sprague-Dawley rat serum were processed in the same manner as study subject serum samples.

Combined analysis:

Analysis ID	AN003174	AN003175
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Thermo Accucore C30 (50 x 2.1mm,2.1um)	Thermo Accucore C30 (50 x 2.1mm,2.1um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo ID-X Orbitrap Tribrid	Thermo ID-X Orbitrap Tribrid
Ion Mode	POSITIVE	NEGATIVE
Units	Normalized Peak Area	Normalized Peak Area

Chromatography:

Chromatography ID:	CH002347
Chromatography Summary:	All samples were run in a randomized order over two and a half days of consecutive instrument time. QC samples were interleaved every 24 runs to evaluate LC-MS system stability and to account for time-dependent batch effects.
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Accucore C30 (50 x 2.1mm,2.1um)
Column Temperature:	60 ℃
Flow Gradient:	(Time: A/B) 0 min 80/20, 1 min 40/60, 5 min 30/70, 5.5 min 15/85, 8 min 10/90, 8.2 min 0/100, 10.7 min 80/20, 12 min 80/20
Flow Rate:	0.3 mL/min
Solvent A:	40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002952
Analysis ID:	AN003174
Instrument Name:	Thermo ID-X Orbitrap Tribrid
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS experiments were preformed over a scan range of 150-2000 m/z, maximum ion injection time was set to 200 ms, and orbitrap resolution was 24000. Raw spectral data from LC-MS experiments were pre-processed using Compound Discoverer v3.0.0 software (Thermo Fischer Scientific, Inc., Waltham, MA) and the XCMS web-based application (xcmsonline.scripps.edu). Initial steps involved retention time alignment between samples, peak area integration, peak picking, and QC area normalization. Features eluting with the solvent front or having retention times below 0.75 min were removed to account for potential ion suppression effects in that retention time region. ChemSpider and in-house mzVault database searches were used to obtain a list of tentative IDs based on accurate mass, isotope pattern, and MS/MS data whenever possible. Each lipid feature was identified according to the following confidence levels: (1) compounds matched to existing in house database standards by accurate mass (<2 ppm), isotopic abundance, fragmentation spectrum, and retention time; (2) compounds annotated according to accurate mass, isotopic abundance, and fragmentation consistent with Lipid Maps and Human Metabolome Database (HMDB) entries; (3) accurate mass match matched to Lipid Maps and HMDB entries and fragmentation showing a few matching characteristic fragment ions.
Ion Mode:	POSITIVE
Ion Source Temperature:	275 ℃
Ion Spray Voltage:	3500 V
Source Temperature:	320 ℃

MS ID:	MS002953
Analysis ID:	AN003175
Instrument Name:	Thermo ID-X Orbitrap Tribrid
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS experiments were preformed over a scan range of 150-2000 m/z, maximum ion injection time was set to 200 ms, and orbitrap resolution was 24000. Raw spectral data from LC-MS experiments were pre-processed using Compound Discoverer v3.0.0 software (Thermo Fischer Scientific, Inc., Waltham, MA) and the XCMS web-based application (xcmsonline.scripps.edu). Initial steps involved retention time alignment between samples, peak area integration, peak picking, and QC area normalization. Features eluting with the solvent front or having retention times below 0.75 min were removed to account for potential ion suppression effects in that retention time region. ChemSpider and in-house mzVault database searches were used to obtain a list of tentative IDs based on accurate mass, isotope pattern, and MS/MS data whenever possible. Each lipid feature was identified according to the following confidence levels: (1) compounds matched to existing in house database standards by accurate mass (<2 ppm), isotopic abundance, fragmentation spectrum, and retention time; (2) compounds annotated according to accurate mass, isotopic abundance, and fragmentation consistent with Lipid Maps and Human Metabolome Database (HMDB) entries; (3) accurate mass match matched to Lipid Maps and HMDB entries and fragmentation showing a few matching characteristic fragment ions.
Ion Mode:	NEGATIVE
Ion Source Temperature:	275 ℃
Ion Spray Voltage:	-2500 V
Source Temperature:	320 ℃