Summary of Study ST002551

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001643. The data can be accessed directly via it's Project DOI: 10.21228/M8B41R This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002551
Study Title	Metabolomics dataset of CNTF induced axon regeneration in mice post optic nerve crush
Study Summary	Axons are processes or extensions of a neuron that help connect one neuron with the next. In the eye all retinal ganglion cells (RGCs) reside within the retina but their axons travel a very long distance traversing through the optic nerve they connect with other neurons in the lateral geniculate nucleus in the brain. Loss of axons results in blindness in glaucoma and traumatic optic neuropathies. Optic nerve crush (ONC) is mouse is an assay system that enable pharmacological induction of axon regeneration from existing RGCs. Lipids form the outer boundary of axons, their synthesis or alterations are associated with metabolite changes. Our motivation was to understand what metabolite changes occurred when ONC axons regenerated due to ciliary neurotrophic factor (CNTF) treatment. We found metabolite profile changes associated with regeneration after crush induced by CNTF. This metabolite dataset was collected from C57Bl/6 mice expressing either AAV2-CNTF to promote regeneration or AAV2-Green Lantern as a control. Animals were subjected to optic nerve crush injury and allowed to recover for either 7 days or 14 days. At the respective time points, animals were euthanized and optic nerves were collected. Nerves underwent two rounds of extraction using a Precellys 24 Touch Homogenizer and a two solvent system of 1:1 Methanol/Water and 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) using a Vanquish Horizon Binary HPLC coupled to a Q Exactive Orbitrap mass spectrometer. Metabolites were identified using Compound Discoverer 3.3 and quantified using isotopic internal metabolite standards.
Institute	University of Miami
Department	McKnight - Ophthalmology
Laboratory	Bhattacharya Lab
Last Name	Bhattacharya
First Name	Sanjoy
Address	1638 NW 10th Avenue, Room 706-A, Miami, FL 33136
Email	sbhattacharya@med.miami.edu
Phone	3054824103
Submit Date	2023-04-05
Num Groups	4
Total Subjects	23
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2023-04-21
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001643
Project DOI:	doi: 10.21228/M8B41R
Project Title:	Metabolomics dataset of CNTF induced axon regeneration in mice post optic nerve crush
Project Summary:	Untargeted metabolomics of AAV_CNTF mice at 7 days and 14 days post-crush, and AAV_Green Lantern mice at 7 days and 14 days post-crush. Both genders included. Metabolites were extracted in serial extraction method: 1) 1:1 Methanol/H2O; 2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were separated using hydrophilic interaction liquid chromatography (HILIC) and run on a Q Exactive mass spectrometer in both positive and negative modes.
Institute:	University of Miami
Department:	McKnight - Ophthalmology
Laboratory:	Bhattacharya Lab
Last Name:	Bhattacharya
First Name:	Sanjoy
Address:	1638 NW 10th Avenue, Room 706-A, Miami, FL 33136
Email:	sbhattacharya@med.miami.edu
Phone:	3054824103

Subject:

Subject ID:	SU002651
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male and female

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA256177	175516_2_GREENL_CTL_OD_7dpi_NEG	Control
SA256178	158618_2_CNTF_CTL_OD_14dpi_POS	Control
SA256179	180969_2_CNTF_CTL_OD_7dpi_NEG	Control
SA256180	159201_3_CNTF_CTL_OD_14dpi_POS	Control
SA256181	180969_2_CNTF_CTL_OD_7dpi_POS	Control
SA256182	159200_1_GREENL_CTL_OD_14dpi_POS	Control
SA256183	175516_2_GREENL_CTL_OD_14dpi_POS	Control
SA256184	158618_2_CNTF_CTL_OD_7dpi_NEG	Control
SA256185	180969_1_CNTF_CTL_OD_7dpi_NEG	Control
SA256186	159201_1_CNTF_CTL_OD_7dpi_NEG	Control
SA256187	159200_3_GREENL_CTL_OD_14dpi_POS	Control
SA256188	180969_3_CNTF_CTL_OD_7dpi_POS	Control
SA256189	180968_1_GREENL_CTL_OD_7dpi_NEG	Control
SA256190	180969_3_CNTF_CTL_OD_7dpi_NEG	Control
SA256191	159200_3_GREENL_CTL_OD_7dpi_NEG	Control
SA256192	159201_3_CNTF_CTL_OD_7dpi_NEG	Control
SA256193	159200_1_GREENL_CTL_OD_7dpi_NEG	Control
SA256194	180968_1_GREENL_CTL_OD_7dpi_POS	Control
SA256195	180968_2_GREENL_CTL_OD_7dpi_POS	Control
SA256196	159201_1_CNTF_CTL_OD_14dpi_POS	Control
SA256197	180968_2_GREENL_CTL_OD_7dpi_NEG	Control
SA256198	180968_3_GREENL_CTL_OD_7dpi_NEG	Control
SA256199	180969_1_CNTF_CTL_OD_7dpi_POS	Control
SA256200	180968_3_GREENL_CTL_OD_7dpi_POS	Control
SA256201	158618_1_CNTF_ONC_OS_7dpi_NEG	Crush
SA256202	175516_1_GREENL_ONC_OS_7dpi_NEG	Crush
SA256203	180968_2_GREENL_ONC_OS_7dpi_NEG	Crush
SA256204	180968_3_GREENL_ONC_OS_7dpi_NEG	Crush
SA256205	180968_1_GREENL_ONC_OS_7dpi_NEG	Crush
SA256206	159201_2_CNTF_ONC_OS_7dpi_NEG	Crush
SA256207	159200_2_GREENL_ONC_OS_7dpi_NEG	Crush
SA256208	159200_3_GREENL_ONC_OS_7dpi_NEG	Crush
SA256209	159200_2_GREENL_ONC_OS_14dpi_POS	Crush
SA256210	180968_2_GREENL_ONC_OS_7dpi_POS	Crush
SA256211	180968_3_GREENL_ONC_OS_7dpi_POS	Crush
SA256212	180968_1_GREENL_ONC_OS_7dpi_POS	Crush
SA256213	180969_1_CNTF_ONC_OS_7dpi_NEG	Crush
SA256214	180969_3_CNTF_ONC_OS_7dpi_NEG	Crush
SA256215	180969_2_CNTF_ONC_OS_7dpi_NEG	Crush
SA256216	180969_1_CNTF_ONC_OS_7dpi_POS	Crush
SA256217	180969_2_CNTF_ONC_OS_7dpi_POS	Crush
SA256218	159201_2_CNTF_ONC_OS_14dpi_POS	Crush
SA256219	159201_3_CNTF_ONC_OS_14dpi_POS	Crush
SA256220	158618_1_CNTF_ONC_OS_14dpi_POS	Crush
SA256221	159200_3_GREENL_ONC_OS_14dpi_POS	Crush
SA256222	180969_3_CNTF_ONC_OS_7dpi_POS	Crush
SA256223	175516_1_GREENL_ONC_OS_14dpi_POS	Crush
SA256224	159201_3_CNTF_ONC_OS_7dpi_NEG	Crush
SA256225	Pooled_QC_5_NEG	N/A
SA256226	Pooled_QC_MS2_4_NEG	N/A
SA256227	System_Suitability_Blank_2_NEG	N/A
SA256228	Pooled_QC_MS2_3_NEG	N/A
SA256229	Pooled_QC_MS2_2_NEG	N/A
SA256230	Extraction_Blank_2_NEG	N/A
SA256231	Pooled_QC_MS2_1_NEG	N/A
SA256232	Pooled_QC_4_NEG	N/A
SA256233	System_Suitability_Blank_NEG	N/A
SA256234	Pooled_QC_MS2_2_POS	N/A
SA256235	Pooled_QC3_POS	N/A
SA256236	Pooled_QC_4_POS	N/A
SA256237	Pooled_QC_MS2_1_POS	N/A
SA256238	Pooled_QC2_POS	N/A
SA256239	QC1_POS	N/A
SA256240	Extraction_Blank_1_POS	N/A
SA256241	Pooled_QC1_POS	N/A
SA256242	Pooled_QC_5_POS	N/A
SA256243	Extraction_Blank_2_POS	N/A
SA256244	Extraction_Blank_1_NEG	N/A
SA256245	Pooled_QC1_NEG	N/A
SA256246	Pooled_QC2_NEG	N/A
SA256247	QC1_NEG	N/A
SA256248	System_Suitability_Blank_POS	N/A
SA256249	Pooled_QC_MS2_3_POS	N/A
SA256250	Pooled_QC_MS2_4_POS	N/A
SA256251	System_Suitability_Blank_2_POS	N/A
SA256252	Pooled_QC3_NEG	N/A

Showing results 1 to 76 of 76

Showing results 1 to 76 of 76

Collection:

Collection ID:	CO002644
Collection Summary:	For tissue collection, animals were euthanized using CO2 and the optic nerve was removed by dissection from the optic nerve head to the optic chiasm.
Sample Type:	Eye tissue

Treatment:

Treatment ID:	TR002663
Treatment Summary:	The C57BL/6 mice were injected with AAV2-CNTF at titer of 1x10e13 vg/ml (viral genomes per mL) with 1 uL of AAV-CNTF injected in each eye. Mice labeled as AAV2-Green-Lantern were injected as a control. Prior to surgery, mice received a cocktail of ketamine and xylazine for anesthesia. The optic nerve crush was performed by exposing the optic nerve by blunt dissection. The nerve was crushed using forceps for 10 seconds 1 mm behind the globe. At 7 and 14 days post crush mice were euthanized and optic nerves were collected.

Treatment ID:

TR002663

Treatment Summary:

The C57BL/6 mice were injected with AAV2-CNTF at titer of 1x10e13 vg/ml (viral genomes per mL) with 1 uL of AAV-CNTF injected in each eye. Mice labeled as AAV2-Green-Lantern were injected as a control. Prior to surgery, mice received a cocktail of ketamine and xylazine for anesthesia. The optic nerve crush was performed by exposing the optic nerve by blunt dissection. The nerve was crushed using forceps for 10 seconds 1 mm behind the globe. At 7 and 14 days post crush mice were euthanized and optic nerves were collected.

Sample Preparation:

Sampleprep ID:	SP002657
Sampleprep Summary:	Metabolite extraction was carried out while keeping the optic nerve tissues on dry ice to prevent 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:

SP002657

Sampleprep Summary:

Metabolite extraction was carried out while keeping the optic nerve tissues on dry ice to prevent 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	AN004200	AN004201
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	µg/ml	µg/ml

Chromatography:

Chromatography ID:	CH003113
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
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:	95% acetonitrile/5% water; 10mM Ammonium Formate; 0.1% formic acid
Solvent B:	50% acetonitrile/50% water; 10mM Ammonium Formate; 0.1% formic acid
Chromatography Type:	HILIC

MS:

MS ID:	MS003947
Analysis ID:	AN004200
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:	MS003948
Analysis ID:	AN004201
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