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MB Sample ID: SA306495
| Local Sample ID: | PBS 1 |
| Subject ID: | SU002939 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002939 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| PBS 1 | SA306495 | FL036868 | PBS | Group |
Collection:
| Collection ID: | CO002932 |
| Collection Summary: | Retinas were collected at P10 following a single incision across the sclera and immediately snap frozen in liquid nitrogen and stored at -80 ºC until sample processing 2. Samples were processed and analyzed by LC-MS/MS by the NYU Metabolomics Core Resource Laboratory, New York, NY, USA, as described previously 3,4. Briefly, samples were homogenized using a bead blaster for 10 cycles with 30 seconds on and 30 seconds off. Metabolites were extracted using 80% methanol and dried down using a speedvac. Next, samples were reconstituted in 50 µL MS-grade water and sonicated for two minutes. Samples were then spun down in a centrifuge at 10 G for 4 min and finally transferred to MS vials for analysis. Internal standards were used for correction of retention time and identification of metabolites. Six retinas were pooled as one replicate to reduce biological variability for metabolomics analysis in each group, n=6 per group (HAR vs. control) |
| Sample Type: | Retina |
Treatment:
| Treatment ID: | TR002948 |
| Treatment Summary: | To study the metabolic alterations occurring in hyperglycemia-associated Phase I ROP, we applied quantitative metabolomics and proteomics on mouse retinas from HAR and normal control mice. C57BL/6J (Jackson Laboratory, Bar Harbor, ME) mice, of each sex, aged 10-12 weeks, were purchased, housed and bred in the institutional vivarium and maintained on a 12hour/12hour light/dark cycle with mouse chow provided ad libitum. Neonatal mice were randomly assigned to experimental groups. Induction of hyperglycemia was accomplished as previously described 1. Neonatal mice were intraperitoneally injected with 50mg/kg/day STZ consecutively from P1 to P9 using a 34-G needle (Hamilton syringe) (Fig. 1B). Vehicle control animals received equal volumes of vehicle phosphate-buffered saline (PBS, Gibco, Waltham, MA). Hyperglycemia is induced around P8 and delayed retinal vascularization is found at P10 1. Mice with weight range 4 to 5 grams were used for further metabolomics and proteomics analysis. Mouse litters were randomly assigned to HAR or control groups, both sexes were used. The cages were located at close spots to minimize the potential housing influences. All procedures were approved by our Institutional Animal Care and Use Committee and adhered to ARRIVE guidelines and the NIH Guide for the Care and Use of Laboratory Animals. With conditions tested with β=0.8 and α=0.05, at least n=6 per group will be needed for the analysis. Control was re-named as group 1 and HAR was re-named as group 2 for analysis. |
Sample Preparation:
| Sampleprep ID: | SP002945 |
| Sampleprep Summary: | Both retinas from each mouse were collected, pooled, and prepared for targeted MS-based proteomics as described previously 1. After sample preparation, LC-tandem MS analysis was performed using selected reaction monitoring (SRM) 5 on a Thermo Scientific TSQ Vantage mass spectrometer equipped with an Eksigent splitless nanoflow HPLC system. 7 µL aliquots of each sample were injected onto a 10 cm x 75 µm i.d. capillary column packed with Phenomenex Jupiter C18 reversed phase beads. The column was eluted at 150 nL/min with a 60 min linear gradient of acetonitrile in 0.1% formic acid. The SRM assays were developed and validated to monitor two peptides per protein. Each peptide was monitored in a 6-min window centered on the known elution time of the peptide 6-8. Approximately 30 protein assays are grouped into a panel of proteins that are measured in a single LC-tandem MS run. |
| Sampleprep Protocol ID: | SP002600 |
Combined analysis:
| Analysis ID | AN004623 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Ultimate 3000 |
| Column | 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 | abundance/intensity |
Chromatography:
| Chromatography ID: | CH003479 |
| Instrument Name: | Thermo Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm, 5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min) |
| Flow Rate: | 0.1mL/min |
| Solvent A: | 100% water; 10 mM ammonium carbonate, pH 9.0 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004369 |
| Analysis ID: | AN004623 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Xcalibur, chromeleon unspecified because both negative and positive ion mode were collected |
| Ion Mode: | UNSPECIFIED |
