Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA242074

Local Sample ID:	Ther-1707-02-01_35_1_1485
Subject ID:	SU002505
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU002505
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Ther-1707-02-01_35_1_1485	SA242074	FL030339	Therapeutic1707	Group

Collection:

Collection ID:	CO002498
Collection Summary:	The experiments on the mice were approved by the University of Sharjah animal care and use committee (ACUC) (ACUC approval number: ACUC-16-003-EAE and ACUC-21-09-06-01). The mice were housed at the University of Sharjah animal facility, in plastic cages under a controlled environment of 24 ± 2 °C, 50% ± 5% humidity, 12 h/12 h light-dark cycle with unlimited food and water access.
Sample Type:	Brain

Treatment:

Treatment ID:	TR002517
Treatment Summary:	From Normal, EAE vehicle-treated, and therapeutic SIMR1707 treated mice, the cerebellum part of the brain was cut, and immediately snap frozen using liquid nitrogen. The samples then were weighed, made in powder form and homogenized using pistol method under liquid nitrogen. The cerebellum sample was lysed using 400 uL RIBA lysis buffer and let to set for 5-10 minutes to allow lysis. 50 ul of the homogenized sample was taken for RNA extraction using norgen biotek RNA/DNA purification kit (cat#:47700) according to the manufacturer’s instructions. The remaining 350 uL was processed for metabolites and proteins extraction. First, it was centrifuged for 5 minutes at 4 C, 13000 rpm. The supernatant was collected in a new 1.5 mL eppendorf tube. The supernatant was centrifuged again for 1 minute at 4 C, at maximum speed (>13000 rpm), and the supernatant was collected again in a new 1.5 mL tube and kept on ice as it was used in the following steps, while the pellet was discarded.

Treatment ID:

TR002517

Treatment Summary:

From Normal, EAE vehicle-treated, and therapeutic SIMR1707 treated mice, the cerebellum part of the brain was cut, and immediately snap frozen using liquid nitrogen. The samples then were weighed, made in powder form and homogenized using pistol method under liquid nitrogen. The cerebellum sample was lysed using 400 uL RIBA lysis buffer and let to set for 5-10 minutes to allow lysis. 50 ul of the homogenized sample was taken for RNA extraction using norgen biotek RNA/DNA purification kit (cat#:47700) according to the manufacturer’s instructions. The remaining 350 uL was processed for metabolites and proteins extraction. First, it was centrifuged for 5 minutes at 4 C, 13000 rpm. The supernatant was collected in a new 1.5 mL eppendorf tube. The supernatant was centrifuged again for 1 minute at 4 C, at maximum speed (>13000 rpm), and the supernatant was collected again in a new 1.5 mL tube and kept on ice as it was used in the following steps, while the pellet was discarded.

Sample Preparation:

Sampleprep ID:	SP002511
Sampleprep Summary:	400 uL of methanol, and 300 uL of chloroform were added to the protein sample, and centrifuged for 5 minutes, at 4 C, 13000 rpm. Three phases appeared: the upper phase is the metabolites, the middle is the protein, and the lower phase is other metabolites. The metabolites' upper phase was kept in a new eppendorf tube and kept at -20 C. 300 uL methanol was added to the middle and the lower phases then they were mixed and centrifuged for 1 minute at 13000 rpm. The supernatant was added to the metabolite tube and then it was kept at - 80C for longer storage.

Sampleprep ID:

SP002511

Sampleprep Summary:

400 uL of methanol, and 300 uL of chloroform were added to the protein sample, and centrifuged for 5 minutes, at 4 C, 13000 rpm. Three phases appeared: the upper phase is the metabolites, the middle is the protein, and the lower phase is other metabolites. The metabolites' upper phase was kept in a new eppendorf tube and kept at -20 C. 300 uL methanol was added to the middle and the lower phases then they were mixed and centrifuged for 1 minute at 13000 rpm. The supernatant was added to the metabolite tube and then it was kept at - 80C for longer storage.

Combined analysis:

Analysis ID	AN003937
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker Elute
Column	Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH002916
Instrument Name:	Bruker Elute
Column Name:	Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um)
Column Temperature:	35
Flow Gradient:	1%B to 99%B in 15 min
Flow Rate:	250 uL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003675
Analysis ID:	AN003937
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Metabolomics Data Processing. For metabolomic analysis, MetaboScape® 4.0 program (Bruker Daltonics, Billerica, MA, USA) was employed for data processing, feature extrac-tion and metabolite identification. The T-ReX 2D/3D workflow was used to identify the molecular features with the following settings: The minimum peak length was set to 7 spectra and the minimum intensity threshold was 1000 counts for peaks detection. The peak area was employed for quantification and the injected external calibrant in the in-terval of 0–0.3 min was used to recalibrate the mass spectra. The selected mass to charge ratio (m/z) and retention time for scanning were in the ranges of 20–1300 m/z and 0.3–30 min, respectively. MS/MS spectra for features were averaged on import and features found at least in 12 of the 40 injections were taken into further consideration. Metabolites were identified by matching to the human metabolome database (HMDB) by combined MS/MS, precursor m/z values, and isotopic pattern scores. Where multiple features matched a given database entry, the annotation quality score (AQ score) was used to select only the best matching feature.
Ion Mode:	POSITIVE