Summary of Study ST003278

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 IDST003278
Study TitleMetabolomic analysis of Axon Regeneration in Xenopus laevis Retina
Study SummaryWe profile the metabolite changes in the retina 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 Date2024-02-09
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:SU003398
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
SA354787Blank_NEG3NA Extraction Blank
SA354788Blank_NEG2NA Extraction Blank
SA354789Blank_NEG1NA Extraction Blank
SA354790Blank_POS3NA Extraction Blank
SA354791Blank_POS2NA Extraction Blank
SA354792Blank_POS1NA Extraction Blank
SA354793CTL_27dpi_Ret3_POS2Retina Control
SA354794CTL_27dpi_Ret2_NEG2Retina Control
SA354795CTL_27dpi_Ret2_NEG3Retina Control
SA354796CTL_27dpi_Ret3_POS1Retina Control
SA354797CTL_27dpi_Ret3_NEG3Retina Control
SA354798CTL_27dpi_Ret3_POS3Retina Control
SA354799CTL_27dpi_Ret3_NEG1Retina Control
SA354800CTL_27dpi_Ret3_NEG2Retina Control
SA354801CTL_27dpi_Ret2_POS3Retina Control
SA354802CTL_12dpi_Ret1_POS2Retina Control
SA354803CTL_27dpi_Ret2_NEG1Retina Control
SA354804CTL_12dpi_Ret1_POS1Retina Control
SA354805CTL_27dpi_Ret2_POS2Retina Control
SA354806CTL_12dpi_Ret2_NEG1Retina Control
SA354807CTL_27dpi_Ret2_POS1Retina Control
SA354808CTL_12dpi_Ret2_POS2Retina Control
SA354809CTL_12dpi_Ret2_POS1Retina Control
SA354810CTL_12dpi_Ret1_NEG2Retina Control
SA354811CTL_12dpi_Ret1_NEG1Retina Control
SA354812CTL_12dpi_Ret1_POS3Retina Control
SA354813CTL_12dpi_Ret1_NEG3Retina Control
SA354814CTL_12dpi_Ret2_NEG2Retina Control
SA354815CTL_27dpi_Ret1_NEG1Retina Control
SA354816CTL_12dpi_Ret2_NEG3Retina Control
SA354817CTL_27dpi_Ret1_NEG3Retina Control
SA354818CTL_27dpi_Ret1_NEG2Retina Control
SA354819CTL_12dpi_Ret2_POS3Retina Control
SA354820CTL_27dpi_Ret1_POS3Retina Control
SA354821CTL_27dpi_Ret1_POS2Retina Control
SA354822CTL_27dpi_Ret1_POS1Retina Control
SA354823CX_27dpi_Ret2_POS1Retina Crush
SA354824CX_27dpi_Ret3_NEG1Retina Crush
SA354825CX_27dpi_Ret3_POS3Retina Crush
SA354826CX_27dpi_Ret3_POS2Retina Crush
SA354827CX_27dpi_Ret3_POS1Retina Crush
SA354828CX_27dpi_Ret2_NEG3Retina Crush
SA354829CX_27dpi_Ret2_NEG2Retina Crush
SA354830CX_27dpi_Ret2_NEG1Retina Crush
SA354831CX_27dpi_Ret2_POS3Retina Crush
SA354832CX_27dpi_Ret3_NEG2Retina Crush
SA354833CX_27dpi_Ret3_NEG3Retina Crush
SA354834CX_27dpi_Ret1_NEG3Retina Crush
SA354835CX_12dpi_Ret2_POS1Retina Crush
SA354836CX_12dpi_Ret1_POS1Retina Crush
SA354837CX_12dpi_Ret1_POS2Retina Crush
SA354838CX_12dpi_Ret1_POS3Retina Crush
SA354839CX_12dpi_Ret1_NEG1Retina Crush
SA354840CX_12dpi_Ret1_NEG2Retina Crush
SA354841CX_12dpi_Ret1_NEG3Retina Crush
SA354842CX_27dpi_Ret1_NEG2Retina Crush
SA354843CX_27dpi_Ret2_POS2Retina Crush
SA354844CX_12dpi_Ret2_POS2Retina Crush
SA354845CX_12dpi_Ret2_NEG1Retina Crush
SA354846CX_27dpi_Ret1_NEG1Retina Crush
SA354847CX_27dpi_Ret1_POS3Retina Crush
SA354848CX_27dpi_Ret1_POS2Retina Crush
SA354849CX_27dpi_Ret1_POS1Retina Crush
SA354850CX_12dpi_Ret2_NEG3Retina Crush
SA354851CX_12dpi_Ret2_NEG2Retina Crush
SA354852CX_12dpi_Ret2_POS3Retina Crush
SA354853Pooled_QC2_MS1_Ret_NEG2Retina QC
SA354854Pooled_QC2_MS1_Ret_POS2Retina QC
SA354855Pooled_QC2_MS1_Ret_POS1Retina QC
SA354856Pooled_QC1_MS1_Ret_NEG2Retina QC
SA354857Pooled_QC1_MS1_Ret_NEG1Retina QC
SA354858Pooled_QC2_MS1_Ret_NEG1Retina QC
SA354859Pooled_QC1_MS1_Ret_POS1Retina QC
SA354860Pooled_QC1_MS1_Ret_POS2Retina QC
SA354861Left_Ret_SHAM2_NEG2Retina Sham
SA354862Right_Ret_SHAM2_POS3Retina Sham
SA354863Left_Ret_SHAM1_POS1Retina Sham
SA354864Left_Ret_SHAM1_POS2Retina Sham
SA354865Left_Ret_SHAM1_POS3Retina Sham
SA354866Left_Ret_SHAM1_NEG1Retina Sham
SA354867Left_Ret_SHAM1_NEG2Retina Sham
SA354868Left_Ret_SHAM1_NEG3Retina Sham
SA354869Right_Ret_SHAM2_NEG3Retina Sham
SA354870Right_Ret_SHAM2_NEG2Retina Sham
SA354871Right_Ret_SHAM2_NEG1Retina Sham
SA354872Right_Ret_SHAM2_POS2Retina Sham
SA354873Left_Ret_SHAM2_NEG1Retina Sham
SA354874Left_Ret_SHAM2_NEG3Retina Sham
SA354875Right_Ret_SHAM1_NEG3Retina Sham
SA354876Right_Ret_SHAM1_NEG2Retina Sham
SA354877Right_Ret_SHAM1_NEG1Retina Sham
SA354878Right_Ret_SHAM1_POS3Retina Sham
SA354879Right_Ret_SHAM1_POS2Retina Sham
SA354880Right_Ret_SHAM1_POS1Retina Sham
SA354881Left_Ret_SHAM2_POS1Retina Sham
SA354882Left_Ret_SHAM2_POS2Retina Sham
SA354883Left_Ret_SHAM2_POS3Retina Sham
SA354884Right_Ret_SHAM2_POS1Retina Sham
Showing results 1 to 98 of 98

Collection:

Collection ID:CO003391
Collection Summary:The tissue was removed via dissection from the optic nerve head to the optic chiasm. The retinas 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 retinas were pooled into one tube.
Sample Type:Eye tissue

Treatment:

Treatment ID:TR003407
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:SP003405
Sampleprep Summary:Retina 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 AN005369 AN005370
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:CH004068
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 and held 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:CH004069
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 and held 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:MS005098
Analysis ID:AN005369
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:MS005099
Analysis ID:AN005370
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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