Summary of Study ST003280

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 PR002032. The data can be accessed directly via it's Project DOI: 10.21228/M8ZC0C This work is supported by NIH grant, U2C- DK119886.

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Study IDST003280
Study TitleMetabolomic analysis of Axon Regeneration in Xenopus laevis Optic Nerve
Study SummaryWe profile the metabolite changes in the optic nerve of a transgenic line of 1 year old Xenopus laevis Tg(islet2b:gfp) frogs that either had a 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). Tg(islet2b:gfp) frogs were allowed to recover for 12 and 27 days post optic nerve crush. Following euthanasia, the tissues were collected for metabolomic analysis. Samples were pooled for each category (crush, sham, and control) at n =3 to obtain sufficient metabolite concentrations for analysis. Metabolites were extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. Metabolites were identified and quantified using Compound Discoverer 3.3 and isotopic internal metabolites standards.
Institute
University of Miami
DepartmentMcKnight - Ophthalmology
LaboratoryBhattacharya Lab
Last NameBhattacharya
First NameSanjoy
Address1638 NW 10th Avenue, Room 706-A, Miami, FL 33136
Emailsbhattacharya@med.miami.edu
Phone3054824103
Submit Date2024-06-11
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-07-15
Release Version1
Sanjoy Bhattacharya Sanjoy Bhattacharya
https://dx.doi.org/10.21228/M8ZC0C
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002032
Project DOI:doi: 10.21228/M8ZC0C
Project Title:Metabolomic Analysis of Axon Regeneration in Xenopus laevis
Project Summary:CNS injuries of the anuran amphibian, Xenopus laevis, are uniquely befitted for studying the molecular compositions of neuronal regeneration of retinal ganglion cells (RGC) due to a functional recovery of optic axons disparate to adult mammalian analogues. RGCs and their optic nerve axons undergo irreversible neurodegeneration in glaucoma and associated optic neuropathies, resulting in blindness in mammals. Conversely, Xenopus demonstrates RGC lifetime-spanning regenerative capabilities after optic nerve crush, inciting opportunities to compare de novo regeneration and develop efficient pharmaceutical approaches for vision restoration. Studies revealing metabolome alterations during optic nerve regeneration are sparse and could serve as a solid foundation for these underlying molecular changes. We profile the metabolite changes in the optic tissues of a transgenic line of 1 year old Xenopus laevis Tg(islet2b:gfp) frogs that either had a 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). Tg(islet2b:gfp) frogs were allowed to recover for 12 and 27 days post optic nerve crush. Following euthanasia, the tissues were collected for metabolomic analysis. Samples were pooled for each category (crush, sham, and control) at n =3 to obtain sufficient metabolite concentrations for analysis. Metabolites were extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. Metabolites were identified and quantified using Compound Discoverer 3.3 and isotopic internal metabolites 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

Subject:

Subject ID:SU003400
Subject Type:Amphibian
Subject Species:Xenopus laevis
Taxonomy ID:8355
Gender:Not applicable

Factors:

Subject type: Amphibian; Subject species: Xenopus laevis (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA354985Blank_NEG3NA Extraction Blank
SA354986Blank_NEG2NA Extraction Blank
SA354987Blank_NEG1NA Extraction Blank
SA354988Blank_POS3NA Extraction Blank
SA354989Blank_POS2NA Extraction Blank
SA354990Blank_POS1NA Extraction Blank
SA354991CTL_27dpi_ON4_NEG2Optic Nerve Control
SA354992CTL_27dpi_ON3_NEG2Optic Nerve Control
SA354993CTL_27dpi_ON3_NEG3Optic Nerve Control
SA354994CTL_27dpi_ON4_POS1Optic Nerve Control
SA354995CTL_27dpi_ON4_POS2Optic Nerve Control
SA354996CTL_27dpi_ON4_POS3Optic Nerve Control
SA354997CTL_27dpi_ON4_NEG1Optic Nerve Control
SA354998CTL_27dpi_ON5_POS3Optic Nerve Control
SA354999CTL_27dpi_ON4_NEG3Optic Nerve Control
SA355000CTL_27dpi_ON5_POS1Optic Nerve Control
SA355001CTL_27dpi_ON5_POS2Optic Nerve Control
SA355002CTL_27dpi_ON3_POS3Optic Nerve Control
SA355003CTL_27dpi_ON5_NEG1Optic Nerve Control
SA355004CTL_27dpi_ON5_NEG2Optic Nerve Control
SA355005CTL_27dpi_ON5_NEG3Optic Nerve Control
SA355006CTL_12dpi_ON1_POS2Optic Nerve Control
SA355007CTL_27dpi_ON3_NEG1Optic Nerve Control
SA355008CTL_12dpi_ON1_POS1Optic Nerve Control
SA355009CTL_27dpi_ON3_POS2Optic Nerve Control
SA355010CTL_27dpi_ON1_POS1Optic Nerve Control
SA355011CTL_27dpi_ON3_POS1Optic Nerve Control
SA355012CTL_12dpi_ON2_POS3Optic Nerve Control
SA355013CTL_12dpi_ON2_POS2Optic Nerve Control
SA355014CTL_12dpi_ON2_POS1Optic Nerve Control
SA355015CTL_12dpi_ON1_NEG3Optic Nerve Control
SA355016CTL_12dpi_ON1_NEG2Optic Nerve Control
SA355017CTL_12dpi_ON1_NEG1Optic Nerve Control
SA355018CTL_12dpi_ON2_NEG1Optic Nerve Control
SA355019CTL_12dpi_ON2_NEG2Optic Nerve Control
SA355020CTL_12dpi_ON2_NEG3Optic Nerve Control
SA355021CTL_12dpi_ON1_POS3Optic Nerve Control
SA355022CTL_27dpi_ON1_POS2Optic Nerve Control
SA355023CTL_27dpi_ON2_NEG2Optic Nerve Control
SA355024CTL_27dpi_ON1_NEG1Optic Nerve Control
SA355025CTL_27dpi_ON1_NEG2Optic Nerve Control
SA355026CTL_27dpi_ON1_NEG3Optic Nerve Control
SA355027CTL_27dpi_ON2_POS1Optic Nerve Control
SA355028CTL_27dpi_ON2_POS2Optic Nerve Control
SA355029CTL_27dpi_ON2_POS3Optic Nerve Control
SA355030CTL_27dpi_ON2_NEG1Optic Nerve Control
SA355031CTL_27dpi_ON1_POS3Optic Nerve Control
SA355032CTL_27dpi_ON2_NEG3Optic Nerve Control
SA355033CX_27dpi_ON4_POS3Optic Nerve Crush
SA355034CX_27dpi_ON4_POS2Optic Nerve Crush
SA355035CX_27dpi_ON4_POS1Optic Nerve Crush
SA355036CX_27dpi_ON3_NEG3Optic Nerve Crush
SA355037CX_27dpi_ON3_POS1Optic Nerve Crush
SA355038CX_27dpi_ON3_NEG1Optic Nerve Crush
SA355039CX_27dpi_ON3_POS3Optic Nerve Crush
SA355040CX_27dpi_ON3_POS2Optic Nerve Crush
SA355041CX_27dpi_ON2_NEG3Optic Nerve Crush
SA355042CX_27dpi_ON4_NEG2Optic Nerve Crush
SA355043CX_27dpi_ON4_NEG1Optic Nerve Crush
SA355044CX_27dpi_ON6_NEG2Optic Nerve Crush
SA355045CX_27dpi_ON4_NEG3Optic Nerve Crush
SA355046CX_27dpi_ON5_POS1Optic Nerve Crush
SA355047CX_27dpi_ON5_POS2Optic Nerve Crush
SA355048CX_27dpi_ON5_POS3Optic Nerve Crush
SA355049CX_27dpi_ON5_NEG1Optic Nerve Crush
SA355050CX_27dpi_ON5_NEG2Optic Nerve Crush
SA355051CX_27dpi_ON6_POS1Optic Nerve Crush
SA355052CX_27dpi_ON6_POS2Optic Nerve Crush
SA355053CX_27dpi_ON6_POS3Optic Nerve Crush
SA355054CX_27dpi_ON6_NEG1Optic Nerve Crush
SA355055CX_27dpi_ON2_NEG1Optic Nerve Crush
SA355056CX_27dpi_ON6_NEG3Optic Nerve Crush
SA355057CX_27dpi_ON2_NEG2Optic Nerve Crush
SA355058CX_27dpi_ON5_NEG3Optic Nerve Crush
SA355059CX_27dpi_ON2_POS3Optic Nerve Crush
SA355060CX_12dpi_ON2_POS1Optic Nerve Crush
SA355061CX_12dpi_ON3_POS1Optic Nerve Crush
SA355062CX_12dpi_ON2_NEG3Optic Nerve Crush
SA355063CX_12dpi_ON2_NEG2Optic Nerve Crush
SA355064CX_12dpi_ON2_NEG1Optic Nerve Crush
SA355065CX_12dpi_ON2_POS3Optic Nerve Crush
SA355066CX_12dpi_ON2_POS2Optic Nerve Crush
SA355067CX_27dpi_ON2_POS2Optic Nerve Crush
SA355068CX_12dpi_ON3_POS3Optic Nerve Crush
SA355069CX_12dpi_ON1_NEG3Optic Nerve Crush
SA355070CX_12dpi_ON1_NEG2Optic Nerve Crush
SA355071CX_12dpi_ON1_NEG1Optic Nerve Crush
SA355072CX_12dpi_ON1_POS3Optic Nerve Crush
SA355073CX_12dpi_ON1_POS2Optic Nerve Crush
SA355074CX_12dpi_ON1_POS1Optic Nerve Crush
SA355075CX_12dpi_ON3_POS2Optic Nerve Crush
SA355076CX_27dpi_ON3_NEG2Optic Nerve Crush
SA355077CX_12dpi_ON3_NEG1Optic Nerve Crush
SA355078CX_27dpi_ON2_POS1Optic Nerve Crush
SA355079CX_12dpi_ON3_NEG2Optic Nerve Crush
SA355080CX_27dpi_ON1_POS2Optic Nerve Crush
SA355081CX_27dpi_ON1_POS3Optic Nerve Crush
SA355082CX_27dpi_ON1_NEG1Optic Nerve Crush
SA355083CX_27dpi_ON1_POS1Optic Nerve Crush
SA355084CX_27dpi_ON1_NEG3Optic Nerve Crush
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Collection:

Collection ID:CO003393
Collection Summary:The tissue was removed via dissection from the optic nerve head to the optic chiasm. The optic nerves 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 optic nerves were pooled into one tube.
Sample Type:Eye tissue

Treatment:

Treatment ID:TR003409
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:SP003407
Sampleprep Summary:Optic nerves 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 AN005373 AN005374
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:CH004072
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
Solvent B:50% acetonitrile/50% water; 10mM Ammonium Formate; 0.1% formic acid
Chromatography Type:HILIC
  
Chromatography ID:CH004073
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:MS005102
Analysis ID:AN005373
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:MS005103
Analysis ID:AN005374
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
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