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MB Sample ID: SA163344
Local Sample ID: | NYSM-00474 |
Subject ID: | SU001822 |
Subject Type: | Mammal |
Subject Species: | Rattus norvegicus |
Taxonomy ID: | 10116 |
Genotype Strain: | Long Evans |
Age Or Age Range: | From post-natal day 1 (PN1) to PN80 |
Weight Or Weight Range: | 5g-250g |
Gender: | Male and female |
Animal Animal Supplier: | Envigo |
Animal Housing: | Animal facility at NYU |
Animal Light Cycle: | 7am-7pm |
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Subject:
Subject ID: | SU001822 |
Subject Type: | Mammal |
Subject Species: | Rattus norvegicus |
Taxonomy ID: | 10116 |
Genotype Strain: | Long Evans |
Age Or Age Range: | From post-natal day 1 (PN1) to PN80 |
Weight Or Weight Range: | 5g-250g |
Gender: | Male and female |
Animal Animal Supplier: | Envigo |
Animal Housing: | Animal facility at NYU |
Animal Light Cycle: | 7am-7pm |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
NYSM-00474 | SA163344 | FL019447 | trained | Treatment |
Collection:
Collection ID: | CO001815 |
Collection Summary: | Rats trained in inhibitory avoidance at PN17, PN24, and PN80, and the untrained (naive) control rats were euthanized by decapitation (1 hour after IA training). Their brains were quickly removed and placed in ice-cold phosphate-buffered saline (1X). Whole hippocampal samples were dissected and immediately snap-frozen in isopentane on dry ice. All samples were stored at -80°C until processing. Frozen samples were shipped in dry ice to Metabolon Inc. (Morrisville, NC, USA) for metabolomic analysis using a proprietary methodology. Extraction of samples was performed using a MicroLab STAR automated liquid handling robot (Hamilton Robotics, Inc., Reno, NV, USA); 450 μL of methanol was added to 100 μo of sample to precipitate proteins. Four recovery standards (DL-2-fluorophenylglycine, tridecanoic acid, cholesterol-d6 and 4-chlorophenylalanine) were added to each sample to determine extraction efficiency. To remove proteins, dissociate small molecules bound to protein, and recover metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation (1300 g for 10 min at room temperature). The resultant supernatants were placed in a TurboVap (Zymark) to remove the organic solvent. Each sample extract was then divided into five equal aliquots, dried under nitrogen, and stored in vacuo prior to metabolomic profiling. |
Sample Type: | Brain |
Collection Method: | Brain dissection |
Collection Location: | New York University |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR001835 |
Treatment Summary: | Metabolomic profiling was carried out on hippocampal extracts (five to seven individual replicates) obtained from untrained rats at four early, prepuberal developmental ages [postnatal day 1 (PN1), PN7, PN17 and PN24] and compared to the profile obtained from young adults (PN80). Additionally, the metabolomic profiles of the hippocampus 1 hour after IA training at PN17, PN24, and PN80 were compared with those of age-matched untrained (naive) control rats. |
Sample Preparation:
Sampleprep ID: | SP001828 |
Sampleprep Summary: | Extraction of samples was performed using a MicroLab STAR automated liquid handling robot (Hamilton Robotics, Inc., Reno, NV, USA); 450 μL of methanol was added to 100 μL of sample to precipitate proteins. Four recovery standards (DL-2-fluorophenylglycine, tridecanoic acid, cholesterol-d6 and 4-chlorophenylalanine) were added to each sample to determine extraction efficiency. To remove proteins, dissociate small molecules bound to protein, and recover metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation (1300 g for 10 min at room temperature). The resultant supernatants were placed in a TurboVap (Zymark) to remove the organic solvent. Each sample extract was then divided into five equal aliquots, dried under nitrogen, and stored in vacuo prior to metabolomic profiling. |
Combined analysis:
Analysis ID | AN002838 | AN002839 | AN002840 | AN002841 |
---|---|---|---|---|
Analysis type | MS | MS | MS | MS |
Chromatography type | Reversed phase | Reversed phase | Reversed phase | HILIC |
Chromatography system | Waters Acquity | Waters Acquity | Waters Acquity | Waters Acquity |
Column | Waters Acquity BEH C18 (100 x 2mm,1.7um) | Waters Acquity BEH C18 (100 x 2mm,1.7um) | Waters Acquity BEH C18 (100 x 2mm,1.7um) | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
MS Type | ESI | ESI | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
Ion Mode | POSITIVE | POSITIVE | NEGATIVE | NEGATIVE |
Units | pmoles/l | pmoles/l | pmoles/l | pmoles/l |
Chromatography:
Chromatography ID: | CH002101 |
Chromatography Summary: | The first two aliquots were analyzed by two separate reverse-phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI): one aliquot was analyzed using acidic positive-ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient-eluted from a C18 column (2 mm × 100 mm Waters UPLC BEH C18 1.7 µm column held at 40°C) using 5% water and 95% methanol and containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA) at pH 3.5. The second aliquot was also analyzed using acidic positive-ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient-eluted from the aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA at pH 3.5. A third aliquot was analyzed by RP/UPLC-MS/MS using basic negative-ion mode ESI in a separate dedicated C18 column. The basic extracts were gradient-eluted from the column using methanol (95%) and water (5%) with 6.5 mM ammonium bicarbonate at pH 8. |
Instrument Name: | Waters Acquity |
Column Name: | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
Column Temperature: | 40 |
Solvent A: | 5% water/95% methanol; 6.5 mM ammonium bicarbonate, pH 8 |
Chromatography Type: | Reversed phase |
Chromatography ID: | CH002102 |
Chromatography Summary: | The fourth aliquot was analyzed via negative ionization following elution from a Hydrophilic-Interaction Chromatography (HILIC) column (2.1 mm x 150 mm Waters UPLC BEH Amide, 1.7 µm column held at 40°C) using a mobile phase consisting of 10 mM ammonium formate in 15% water, 5% methanol, 80% acetonitrile (pH 10.8). |
Instrument Name: | Waters Acquity |
Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
Solvent A: | 15% water/5% methanol/80% acetonitrile; 10 mM ammonium formate, pH 10.8 |
Chromatography Type: | HILIC |
MS:
MS ID: | MS002631 |
Analysis ID: | AN002838 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | One aliquot was analyzed using acidic positive-ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient-eluted from a C18 column (2.1 mm × 100 mm Waters UPLC BEH C18 1.7 µm column held at 40°C) using 5% water and 95% methanol and containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA) at pH 3.5. |
Ion Mode: | POSITIVE |
MS ID: | MS002632 |
Analysis ID: | AN002839 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The second aliquot was also analyzed using acidic positive-ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient-eluted from the aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA at pH 3.5. |
Ion Mode: | POSITIVE |
MS ID: | MS002633 |
Analysis ID: | AN002840 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | A third aliquot was analyzed by RP/UPLC-MS/MS using basic negative-ion mode ESI in a separate dedicated C18 column. The basic extracts were gradient-eluted from the column using methanol (95%) and water (5%) with 6.5 mM ammonium bicarbonate at pH 8. |
Ion Mode: | NEGATIVE |
MS ID: | MS002634 |
Analysis ID: | AN002841 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The fourth aliquot was analyzed via negative ionization following elution from a Hydrophilic-Interaction Chromatography (HILIC) column (2.1 mm x 150 mm Waters UPLC BEH Amide, 1.7 µm column held at 40°C) using a mobile phase consisting of 10 mM ammonium formate in 15% water, 5% methanol, 80% acetonitrile (pH 10.8). |
Ion Mode: | NEGATIVE |