Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA272221

Local Sample ID:	RF150
Subject ID:	SU002812
Subject Type:	Mammal
Subject Species:	Mus musculus

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Subject:

Subject ID:	SU002812
Subject Type:	Mammal
Subject Species:	Mus musculus

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
RF150	SA272221	FL035271	AM_CSF (TopPhase)	Diurnal_Cycle

Collection:

Collection ID:	CO002805
Collection Summary:	All animal studies were performed under the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Boston Children’s Hospital (BCH). All mice were housed in a 12h light-dark cycle with ad libitum access to food and water. CSF, eCSF, was collected from the cisterna magna of the pup embryos and mother, and centrifuged at 5,000g for 10 min. at 4 °C to remove any tissue debris. CSF samples were kept on ice and analyzed the same day as collection.
Sample Type:	CSF

Treatment:

Treatment ID:	TR002821
Treatment Summary:	Mice (CD-1 males) kept in circadian cabinet housing with 12-hour light cycle (7 a.m. on/ 7 p.m. off).

Sample Preparation:

Sampleprep ID:	SP002818
Sampleprep Summary:	CSF was collected from adult (3 months old) wild-type CD1 mice. Samples were placed on wet ice, then spun 1000xg for 10 minutes at 4C. The supernatant was collected. Per condition, 5-10µL of fresh, cleared CSF or 5µl serum was extracted in 4:6:3 chlorophorm:methanol:water mixture supplemented with isotopically labeled T3 and T4 (Cambridge Isotope Laboratories, CLM-7185-C and CLM-8931-PK ) as well as isotopically labelled 17 amino acids and isotopically labelled reduced glutathione (Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). After centrifugation for 10 min at maximum speed on a benchtop centrifuge (Eppendorf) the top, hydrophilic layer (top phase) was transferred to a new tube, dried using a nitrogen dryer and reconstituted in 20 µl water (supplemented with QReSS, Cambridge Isotope Laboratories, MSK-QRESS-KIT) by brief vortexing. Extracted metabolites were spun again and cleared supernatant was transferred to LC-MS micro vials. A small amount of each sample was pooled and serially diluted 3- and 10-fold to be used as quality controls throughout the run of each batch. I calibration curve of unlabeled T3 and T4 standards was run per experiment for concentration calculations.

Sampleprep ID:

SP002818

Sampleprep Summary:

CSF was collected from adult (3 months old) wild-type CD1 mice. Samples were placed on wet ice, then spun 1000xg for 10 minutes at 4C. The supernatant was collected. Per condition, 5-10µL of fresh, cleared CSF or 5µl serum was extracted in 4:6:3 chlorophorm:methanol:water mixture supplemented with isotopically labeled T3 and T4 (Cambridge Isotope Laboratories, CLM-7185-C and CLM-8931-PK ) as well as isotopically labelled 17 amino acids and isotopically labelled reduced glutathione (Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). After centrifugation for 10 min at maximum speed on a benchtop centrifuge (Eppendorf) the top, hydrophilic layer (top phase) was transferred to a new tube, dried using a nitrogen dryer and reconstituted in 20 µl water (supplemented with QReSS, Cambridge Isotope Laboratories, MSK-QRESS-KIT) by brief vortexing. Extracted metabolites were spun again and cleared supernatant was transferred to LC-MS micro vials. A small amount of each sample was pooled and serially diluted 3- and 10-fold to be used as quality controls throughout the run of each batch. I calibration curve of unlabeled T3 and T4 standards was run per experiment for concentration calculations.

Combined analysis:

Analysis ID	AN004389
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Ascentis Express C18 (150 x 2.1mm,2.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap
Ion Mode	POSITIVE
Units	nM

Chromatography:

Chromatography ID:	CH003292
Chromatography Summary:	Ascentis Express C18 HPLC column (2.7 μm × 15 cm × 2.1 mm; Sigma Aldrich).
Instrument Name:	Thermo Vanquish
Column Name:	Ascentis Express C18 (150 x 2.1mm,2.7um)
Column Temperature:	30
Flow Gradient:	0–5 min: gradient was held at 5% B; 2–12.1 min: linear gradient of 5% to 95% B; 12.1–17.0 min: 95% B; 17.1–21.0 min: gradient was returned to 5% B
Flow Rate:	0.250 ml/min
Solvent A:	water, 0.1% formic acid
Solvent B:	acetonitrile, 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004138
Analysis ID:	AN004389
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS data acquisition was performed using a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe (Thermo Fisher Scientific, San Jose, CA) and was performed at a narrow scan in positive mode (m/z = 600-800) for more specific detection of thyroxine hormones, the resolution was set at 70,000, the AGC target was 5x105, and the max IT was 100 msec. HESI conditions were: Sheath gas flow rate: 40; Aug gas flow rate: 10; Sweet gas flow rate: 0; Spray voltage: 3.5kV (pos), 2.8kV (neg); Capillary temperature: 380ºC; S-lens RF: 60; Aux gas heater temperature: 420 ºC.
Ion Mode:	POSITIVE