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MB Sample ID: SA354934
Local Sample ID: | CX_12dpi_Tect2_NEG2 |
Subject ID: | SU003399 |
Subject Type: | Amphibian |
Subject Species: | Xenopus laevis |
Taxonomy ID: | 8355 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003399 |
Subject Type: | Amphibian |
Subject Species: | Xenopus laevis |
Taxonomy ID: | 8355 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
CX_12dpi_Tect2_NEG2 | SA354934 | FL041434 | Tectum | Sample source |
CX_12dpi_Tect2_NEG2 | SA354934 | FL041434 | Crush | Treatment |
Collection:
Collection ID: | CO003392 |
Collection Summary: | The tissue was removed via dissection from the optic nerve head to the optic chiasm. The tecta were collected at 12- and 27-days post crush and separated into biological samples. Due to the small tissue and metabolomics resolution constraints, tissues were pooled to generate higher signal intensities. A total of 3 tecta were pooled into one tube. |
Sample Type: | Eye tissue |
Treatment:
Treatment ID: | TR003408 |
Treatment Summary: | Optic nerves from each transgenic Tg(Islet2b:EGFP-RPL10a) Xenopus laevis frogs, 3.5 - 5.0 cm in length, underwent monocular surgery of either a left optic crush injury (crush) or sham surgery (sham). The matching controls of uninjured right optic nerves were also collected (control). Operated individuals were anesthetized with 0.05% ethyl 3-aminobenzoate methanesulfonate (Sigma, USA). |
Sample Preparation:
Sampleprep ID: | SP003406 |
Sampleprep Summary: | Tecta remained on dry ice to prevent metabolite degradation while the metabolite extraction was conducted. Tissues were transferred to 0.5mL Soft Tissue Lysing Kit Precellys tubes containing beads. Then, 84 µL of chilled 1:1 MeOH/H2O were added to Precellys tube. Pre-extraction internal standards were added to the tubes: 5µl of 1mg/ml Caffeine 13C6, 5µl of 1mg/ml D-Glucose 13C6, 5µl of 1mg/ml Oleic Acid 13C5, and 1µl of 5mg/mL Isoleucine 13C6 to each sample. Tissues were homogenized using Precellys 24 Touch. Cycle parameters: 2 cycles: 30 seconds homogenization at 4500 rpm, 10 seconds rest. Homogenate was transferred to a microcentrifuge tube and centrifuged at 18000xrcf for 20 min at 4°C. Then, collect supernatant and transfer pellet to Precellys Lysing Kit tube. Add 84uL of 8:1:1 Acetonitrile/Methanol/Acetone to pellet and add the rest of the pre-extraction internal standards: 5µl of 1mg/ml Caffeine 13C6, 5µl of 1mg/ml D-Glucose 13C6, 5µl of 1mg/ml Oleic Acid 13C5, 1µl of 5mg/mL Isoleucine 13C6. Final pre-extraction internal standards concentrations are 50μg/mL. Homogenization cycles were repeating using Precellys 24 Touch. Centrifuge as before and add second supernatant to first round of collected supernatant. Centrifuge at 1800xrcf for 20 min once more to remove any remaining tissue debris. Collect supernatant and dry supernatant in Speedvac. Two extraction blanks were prepared in the same manner as the biological samples. Dried samples were reconstituted immediately in 0.1% formic acid in 44.75µL of HPLC-MS grade water. Post-extraction internal standards were added: 25 µl of 5mg/ml Phenylalanine 13C6, 2.5 µl of .5mg/ml Uracil 13C 15N2, 1.25 µl of 1mg/ml Arginine 13C6, 1.25 µl of 1mg/ml Serine 13C3 to each sample. |
Combined analysis:
Analysis ID | AN005371 | AN005372 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Thermo Vanquish | Thermo Vanquish |
Column | Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) | Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | Peak area | Peak area |
Chromatography:
Chromatography ID: | CH004070 |
Chromatography Summary: | Positive ion mode |
Instrument Name: | Thermo Vanquish |
Column Name: | Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) |
Column Temperature: | 35 C |
Flow Gradient: | The gradient began at 1.0% B for 1 min, then shifted to 95.0% B for 9 minutes, then stayed at 95.0% B for 1 min before ramping down quickly to 1.0% B for 5 minutes. |
Flow Rate: | 0.5 ml/min |
Solvent A: | 95% acetonitrile/5% water/10mM Ammonium Formate/0.1% formic acid; 95% acetonitrile/5% water/10mM Ammonium Acetate/0.1% acetic acid |
Solvent B: | 50% acetonitrile/50% water/10mM Ammonium Formate/0.1% formic acid |
Chromatography Type: | HILIC |
Chromatography ID: | CH004071 |
Chromatography Summary: | Negative ion mode |
Instrument Name: | Thermo Vanquish |
Column Name: | Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) |
Column Temperature: | 35 C |
Flow Gradient: | The gradient began at 1.0% B for 1 min, then shifted to 95.0% B for 9 minutes, then stayed at 95.0% B for 1 min before ramping down quickly to 1.0% B for 5 minutes. |
Flow Rate: | 0.5 ml/min |
Solvent A: | 95% acetonitrile/5% water; 10mM Ammonium Acetate; 0.1% acetic acid |
Solvent B: | 50% acetonitrile/50% water; 10mM Ammonium Acetate; 0.1% acetic acid |
Chromatography Type: | HILIC |
MS:
MS ID: | MS005100 |
Analysis ID: | AN005371 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The samples were run using a Q ExactiveTM mass spectrometer coupled to a heated electrospray ionization (HESI) source. The spray voltage was set to 3.50 kV, capillary temperature to 350°C, sheath gas to 55, aux gas to 14, sweep gas to 4, and S-Lens RF Level to 30.0. The mass range was set to 67 – 1000 m/z, resolution 140,000 for full scan and 35,000 for ddMS2. AGC target was set to 1e6 for full scan and 2e5 for ddMS2. The max injection time (IT) was 100 seconds for full scan mode and 50 seconds for ddMS2. The number of microscans was 2, and normalized collision energy (NCE) was set to 20, 35, and 50. Samples were run in both positive and negative ion mode separately. The parameters for negative mode were the same except the spray voltage, which was set to 2.50 kV and capillary temperature to 380°C. Metabolites were identified from their Thermo.RAW scans using Compound DiscovererTM 3.3 software. Extraction blanks were used to determine and correct for reagent effects, allow for the creation of exclusions lists, mark background components, and filters the background components from the results table in Compound DiscovererTM 3.3. Pooled QCs were used for initial compound normalization and identification. All non-identified compounds were removed. |
Ion Mode: | POSITIVE |
MS ID: | MS005101 |
Analysis ID: | AN005372 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The samples were run using a Q ExactiveTM mass spectrometer coupled to a heated electrospray ionization (HESI) source. The spray voltage was set to 3.50 kV, capillary temperature to 350°C, sheath gas to 55, aux gas to 14, sweep gas to 4, and S-Lens RF Level to 30.0. The mass range was set to 67 – 1000 m/z, resolution 140,000 for full scan and 35,000 for ddMS2. AGC target was set to 1e6 for full scan and 2e5 for ddMS2. The max injection time (IT) was 100 seconds for full scan mode and 50 seconds for ddMS2. The number of microscans was 2, and normalized collision energy (NCE) was set to 20, 35, and 50. Samples were run in both positive and negative ion mode separately. The parameters for negative mode were the same except the spray voltage, which was set to 2.50 kV and capillary temperature to 380°C. Metabolites were identified from their Thermo.RAW scans using Compound DiscovererTM 3.3 software. Extraction blanks were used to determine and correct for reagent effects, allow for the creation of exclusions lists, mark background components, and filters the background components from the results table in Compound DiscovererTM 3.3. Pooled QCs were used for initial compound normalization and identification. All non-identified compounds were removed. |
Ion Mode: | NEGATIVE |