Summary of Study ST003279

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.

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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003279
Study TitleMetabolomic analysis of Axon Regeneration in Xenopus laevis Tectum
Study SummaryWe profile the metabolite changes in the tectum 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 Date2024-06-05
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

Select appropriate tab below to view additional metadata details:

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:SU003399
Subject Type:Amphibian
Subject Species:Xenopus laevis
Taxonomy ID:8355

Factors:

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

mb_sample_id local_sample_id Sample source Treatment
SA354885Blank_NEG3NA Extraction Blank
SA354886Blank_POS2NA Extraction Blank
SA354887Blank_POS1NA Extraction Blank
SA354888Blank_NEG2NA Extraction Blank
SA354889Blank_POS3NA Extraction Blank
SA354890Blank_NEG1NA Extraction Blank
SA354891CTL_27dpi_Tect1_NEG2Tectum Control
SA354892CTL_27dpi_Tect3_NEG3Tectum Control
SA354893CTL_27dpi_Tect3_NEG2Tectum Control
SA354894CTL_27dpi_Tect3_NEG1Tectum Control
SA354895CTL_27dpi_Tect3_POS3Tectum Control
SA354896CTL_27dpi_Tect3_POS2Tectum Control
SA354897CTL_27dpi_Tect3_POS1Tectum Control
SA354898CTL_27dpi_Tect2_NEG3Tectum Control
SA354899CTL_27dpi_Tect2_NEG2Tectum Control
SA354900CTL_27dpi_Tect2_NEG1Tectum Control
SA354901CTL_27dpi_Tect2_POS3Tectum Control
SA354902CTL_27dpi_Tect2_POS1Tectum Control
SA354903CTL_27dpi_Tect1_NEG3Tectum Control
SA354904CTL_27dpi_Tect2_POS2Tectum Control
SA354905CTL_27dpi_Tect1_NEG1Tectum Control
SA354906CTL_12dpi_Tect2_POS1Tectum Control
SA354907CTL_12dpi_Tect1_POS1Tectum Control
SA354908CTL_12dpi_Tect1_POS2Tectum Control
SA354909CTL_12dpi_Tect1_POS3Tectum Control
SA354910CTL_27dpi_Tect1_POS3Tectum Control
SA354911CTL_12dpi_Tect1_NEG2Tectum Control
SA354912CTL_12dpi_Tect1_NEG3Tectum Control
SA354913CTL_12dpi_Tect1_NEG1Tectum Control
SA354914CTL_12dpi_Tect2_POS2Tectum Control
SA354915CTL_12dpi_Tect2_NEG1Tectum Control
SA354916CTL_12dpi_Tect2_NEG2Tectum Control
SA354917CTL_12dpi_Tect2_NEG3Tectum Control
SA354918CTL_27dpi_Tect1_POS2Tectum Control
SA354919CTL_27dpi_Tect1_POS1Tectum Control
SA354920CTL_12dpi_Tect2_POS3Tectum Control
SA354921CX_27dpi_Tect2_POS1Tectum Crush
SA354922CX_27dpi_Tect1_NEG1Tectum Crush
SA354923CX_27dpi_Tect1_NEG3Tectum Crush
SA354924CX_27dpi_Tect2_NEG1Tectum Crush
SA354925CX_27dpi_Tect2_POS2Tectum Crush
SA354926CX_27dpi_Tect2_POS3Tectum Crush
SA354927CX_27dpi_Tect2_NEG2Tectum Crush
SA354928CX_27dpi_Tect2_NEG3Tectum Crush
SA354929CX_27dpi_Tect1_POS1Tectum Crush
SA354930CX_27dpi_Tect1_POS2Tectum Crush
SA354931CX_27dpi_Tect1_NEG2Tectum Crush
SA354932CX_12dpi_Tect2_NEG3Tectum Crush
SA354933CX_12dpi_Tect1_NEG1Tectum Crush
SA354934CX_12dpi_Tect2_NEG2Tectum Crush
SA354935CX_12dpi_Tect1_POS1Tectum Crush
SA354936CX_12dpi_Tect1_POS2Tectum Crush
SA354937CX_12dpi_Tect1_POS3Tectum Crush
SA354938CX_27dpi_Tect1_POS3Tectum Crush
SA354939CX_12dpi_Tect1_NEG2Tectum Crush
SA354940CX_12dpi_Tect2_POS1Tectum Crush
SA354941CX_12dpi_Tect2_POS2Tectum Crush
SA354942CX_12dpi_Tect2_POS3Tectum Crush
SA354943CX_12dpi_Tect1_NEG3Tectum Crush
SA354944CX_12dpi_Tect2_NEG1Tectum Crush
SA354945Pooled_QC2_MS1_Tect_POS1Tectum QC
SA354946Pooled_QC1_MS2_Tect_POS1Tectum QC
SA354947Pooled_QC1_MS2_Tect_POS2Tectum QC
SA354948Pooled_QC1_MS2_Tect_NEG2Tectum QC
SA354949Pooled_QC2_MS1_Tect_NEG2Tectum QC
SA354950Pooled_QC2_MS1_Tect_POS2Tectum QC
SA354951Pooled_QC2_MS1_Tect_NEG1Tectum QC
SA354952Pooled_QC2_MS2_Tect_POS1Tectum QC
SA354953Pooled_QC2_MS2_Tect_POS2Tectum QC
SA354954Pooled_QC2_MS2_Tect_NEG1Tectum QC
SA354955Pooled_QC2_MS2_Tect_NEG2Tectum QC
SA354956Pooled_QC1_MS1_Tect_NEG2Tectum QC
SA354957Pooled_QC1_MS2_Tect_NEG1Tectum QC
SA354958Pooled_QC1_MS1_Tect_POS2Tectum QC
SA354959Pooled_QC1_MS1_Tect_POS1Tectum QC
SA354960Pooled_QC1_MS1_Tect_NEG1Tectum QC
SA354961Left_Tect_SHAM2_NEG2Tectum Sham
SA354962Right_Tect_SHAM1_POS3Tectum Sham
SA354963Right_Tect_SHAM2_NEG3Tectum Sham
SA354964Right_Tect_SHAM2_NEG2Tectum Sham
SA354965Right_Tect_SHAM2_NEG1Tectum Sham
SA354966Right_Tect_SHAM2_POS3Tectum Sham
SA354967Right_Tect_SHAM2_POS2Tectum Sham
SA354968Right_Tect_SHAM2_POS1Tectum Sham
SA354969Right_Tect_SHAM1_NEG3Tectum Sham
SA354970Right_Tect_SHAM1_NEG2Tectum Sham
SA354971Right_Tect_SHAM1_NEG1Tectum Sham
SA354972Right_Tect_SHAM1_POS2Tectum Sham
SA354973Left_Tect_SHAM2_NEG1Tectum Sham
SA354974Right_Tect_SHAM1_POS1Tectum Sham
SA354975Left_Tect_SHAM2_NEG3Tectum Sham
SA354976Left_Tect_SHAM1_POS2Tectum Sham
SA354977Left_Tect_SHAM1_POS3Tectum Sham
SA354978Left_Tect_SHAM1_NEG1Tectum Sham
SA354979Left_Tect_SHAM1_NEG2Tectum Sham
SA354980Left_Tect_SHAM1_NEG3Tectum Sham
SA354981Left_Tect_SHAM2_POS1Tectum Sham
SA354982Left_Tect_SHAM2_POS2Tectum Sham
SA354983Left_Tect_SHAM2_POS3Tectum Sham
SA354984Left_Tect_SHAM1_POS1Tectum Sham
Showing results 1 to 100 of 100

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
  logo