Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA244596

Local Sample ID:	Left_Optic_Nerve_2_2_NEG
Subject ID:	SU002533
Subject Type:	Fish
Subject Species:	Danio rerio
Taxonomy ID:	7955
Gender:	Male and female

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Subject:

Subject ID:	SU002533
Subject Type:	Fish
Subject Species:	Danio rerio
Taxonomy ID:	7955
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Left_Optic_Nerve_2_2_NEG	SA244596	FL030671	Male	Gender
Left_Optic_Nerve_2_2_NEG	SA244596	FL030671	Uninjured	Treatment

Collection:

Collection ID:	CO002526
Collection Summary:	For tissue collection, animals were euthanized by overdose of MS-222 and optic nerve removed by dissection from the optic nerve head to the optic chiasm. Female and male Zebrafish optic nerves were collected separated and are separate biological samples. Due to the small tissue and metabolomics resolution constraints, optic nerves were pooled to generate higher signal intensities. 31 and 36 crushed optic nerves were pooled for female and male zebrafish samples, respectively. The contralateral uncrushed optic nerves were pooled in the same way.
Sample Type:	Eye tissue

Treatment:

Treatment ID:	TR002545
Treatment Summary:	For optic nerve crush, animals were deeply anesthetized in 0.033% tricaine methane-sulfonate (MS-222). The right optic nerve was exposed by gently removing the connective tissue on the dorsal half of the eye and rotating the eye ventrally out of the orbit with a number 5 forceps. A nerve crush was then performed using number 5 forceps to crush the nerve ~0.5–1 mm from the optic nerve head for 5 s. Success was observed by the generation of a translucent stripe in the nerve that completely separated two areas of white myelination with no bleeding. Fish were then revived in fresh aquatic system water in individual tanks. After 1 h the tanks were returned to the fish system and animals were maintained normally with daily feeding until 3 days post injury.

Treatment ID:

TR002545

Treatment Summary:

For optic nerve crush, animals were deeply anesthetized in 0.033% tricaine methane-sulfonate (MS-222). The right optic nerve was exposed by gently removing the connective tissue on the dorsal half of the eye and rotating the eye ventrally out of the orbit with a number 5 forceps. A nerve crush was then performed using number 5 forceps to crush the nerve ~0.5–1 mm from the optic nerve head for 5 s. Success was observed by the generation of a translucent stripe in the nerve that completely separated two areas of white myelination with no bleeding. Fish were then revived in fresh aquatic system water in individual tanks. After 1 h the tanks were returned to the fish system and animals were maintained normally with daily feeding until 3 days post injury.

Sample Preparation:

Sampleprep ID:	SP002539
Sampleprep Summary:	Metabolite extraction from samples was carried out quickly while keeping optic nerve tissues on dry ice to prevent metabolite degradation. Tissues were transferred to 0.5mL Soft Tissue Lysing Kit Precellys tubes containing beads. Add 84 µL of chilled 1:1 MeOH/H2O to Precellys tube. Pre-extraction internal standards were added to the tubes. Add pre-extraction internal standards: 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. Transfer homogenate to microcentrifuge tube and centrifuge at 18000xrcf for 20 min at 4°C. 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.

Sampleprep ID:

SP002539

Sampleprep Summary:

Metabolite extraction from samples was carried out quickly while keeping optic nerve tissues on dry ice to prevent metabolite degradation. Tissues were transferred to 0.5mL Soft Tissue Lysing Kit Precellys tubes containing beads. Add 84 µL of chilled 1:1 MeOH/H2O to Precellys tube. Pre-extraction internal standards were added to the tubes. Add pre-extraction internal standards: 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. Transfer homogenate to microcentrifuge tube and centrifuge at 18000xrcf for 20 min at 4°C. 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	AN003981	AN003982
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	normalized peak areas	normalized peak areas

Chromatography:

Chromatography ID:	CH002944
Chromatography Summary:	Mobile Phases: NEG A: 10mM Ammonium Acetate in 95% ACN w/ .1% acetic acid NEG B: 10mM Ammonium Acetate in 50% ACN w/ .1% acetic acid POS A: 10mM Ammonium Formate in 95% ACN w/ .1% formic acid POS B: 10mM Ammonium Formate in 50% ACN w/ .1% formic acid
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um)
Column Temperature:	35 C
Flow Gradient:	PumpModule.Pump.Pump_Pressure.AcqOn 0.000 [min] Run PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 1.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 9.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 95.0 [%] PumpModule.Pump.Curve: 5 10.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 95.0 [%] PumpModule.Pump.Curve: 5 10.500 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 15.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 15.000 [min] Stop Run
Flow Rate:	0.5 ml/min
Solvent A:	10mM Ammonium Formate in 95% ACN w/ .1% formic acid
Solvent B:	10mM Ammonium Formate in 50% ACN w/ .1% formic acid
Chromatography Type:	HILIC

MS:

MS ID:	MS003715
Analysis ID:	AN003981
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:	MS003716
Analysis ID:	AN003982
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