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MB Sample ID: SA272308
| Local Sample ID: | RF33 |
| Subject ID: | SU002813 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002813 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| RF33 | SA272308 | FL035305 | AM LV_ChP (FB extraction) | Diurnal_Cycle |
Collection:
| Collection ID: | CO002806 |
| 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). Mice (CD-1 males) kept in circadian cabinet housing with 12-hour light cycle (7 a.m. on/ 7 p.m. off) aged 8 weeks (N =16 at each time—9 a.m. and 9 p.m.), were decapitated and brain tissue was immediately dissected and frozen on dry ice. ChP were extracted by brief sonication in either 200µl 80% LC/MS-grade methanol extraction solvent or 200µl of ”FB” extraction solvent (80% LC/MS-grade methanol, 20% 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC/MS water and supplemented with isotopically labeled internal standards (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 cleared supernatant was 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. |
| Sample Type: | Choroid Plexus |
Treatment:
| Treatment ID: | TR002822 |
| Treatment Summary: | Mice (CD-1 males) kept in circadian cabinet housing with 12-hour light cycle (7 a.m. on/ 7 p.m. off). Two tissues were examined (left ventricle choroid plexus and 4th ventricle choroid plexus). Two separate extractions were performed to capture a wider set of metabolites. |
Sample Preparation:
| Sampleprep ID: | SP002819 |
| Sampleprep Summary: | ChP were extracted by brief sonication in either 200µl 80% LC/MS-grade methanol extraction solvent or 200µl of ”FB” extraction solvent (80% LC/MS-grade methanol, 20% 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC/MS water and supplemented with isotopically labeled internal standards (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 cleared supernatant was 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. |
Combined analysis:
| Analysis ID | AN004390 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Vanquish™ Flex UHPLC |
| Column | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | a.u. |
Chromatography:
| Chromatography ID: | CH003293 |
| Chromatography Summary: | ZIC-pHILIC 150 × 2.1 mm (5 µm particle size) column (EMD Millipore) operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA). |
| Instrument Name: | Vanquish™ Flex UHPLC |
| Column Name: | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B |
| Flow Rate: | 0.150µl/min |
| Solvent A: | 100% acetonitrile |
| Solvent B: | 100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004139 |
| Analysis ID: | AN004390 |
| Instrument Name: | Thermo Q Exactive 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 in positive and negative ionization mode in a range of m/z = 70–1000, with the resolution set at 70,000, the AGC target at 1x106, and the maximum injection time (Max IT) at 20 msec. A narrower scan in positive mode at m/z = 600-800 was used 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. For polar metabolites HESI conditions were: Sheath gas frow rate: 35; Aug gas flow rate: 8; Sweet gas flow rate: 1; Spray voltage: 3.5kV (pos), 2.8kV (neg); Capillary temperature: 320ºC; S-lens RF: 50; Aux gas heater temperature: 350 ºC. |
| Ion Mode: | UNSPECIFIED |
