#METABOLOMICS WORKBENCH sbhattacharya_20240209_071652 DATATRACK_ID:4635 STUDY_ID:ST003278 ANALYSIS_ID:AN005370 PROJECT_ID:PR002032 VERSION 1 CREATED_ON June 21, 2024, 12:12 pm #PROJECT PR:PROJECT_TITLE Metabolomic Analysis of Axon Regeneration in Xenopus laevis PR:PROJECT_SUMMARY CNS injuries of the anuran amphibian, Xenopus laevis, are uniquely befitted for PR:PROJECT_SUMMARY studying the molecular compositions of neuronal regeneration of retinal ganglion PR:PROJECT_SUMMARY cells (RGC) due to a functional recovery of optic axons disparate to adult PR:PROJECT_SUMMARY mammalian analogues. RGCs and their optic nerve axons undergo irreversible PR:PROJECT_SUMMARY neurodegeneration in glaucoma and associated optic neuropathies, resulting in PR:PROJECT_SUMMARY blindness in mammals. Conversely, Xenopus demonstrates RGC lifetime-spanning PR:PROJECT_SUMMARY regenerative capabilities after optic nerve crush, inciting opportunities to PR:PROJECT_SUMMARY compare de novo regeneration and develop efficient pharmaceutical approaches for PR:PROJECT_SUMMARY vision restoration. Studies revealing metabolome alterations during optic nerve PR:PROJECT_SUMMARY regeneration are sparse and could serve as a solid foundation for these PR:PROJECT_SUMMARY underlying molecular changes. We profile the metabolite changes in the optic PR:PROJECT_SUMMARY tissues of a transgenic line of 1 year old Xenopus laevis Tg(islet2b:gfp) frogs PR:PROJECT_SUMMARY that either had a monocular surgery of either a left optic crush injury (crush) PR:PROJECT_SUMMARY or sham surgery (sham). The matching controls of uninjured right optic nerves PR:PROJECT_SUMMARY were also collected (control). Tg(islet2b:gfp) frogs were allowed to recover for PR:PROJECT_SUMMARY 12 and 27 days post optic nerve crush. Following euthanasia, the tissues were PR:PROJECT_SUMMARY collected for metabolomic analysis. Samples were pooled for each category PR:PROJECT_SUMMARY (crush, sham, and control) at n =3 to obtain sufficient metabolite PR:PROJECT_SUMMARY concentrations for analysis. Metabolites were extracted using a Precellys PR:PROJECT_SUMMARY Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 PR:PROJECT_SUMMARY Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid PR:PROJECT_SUMMARY chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive PR:PROJECT_SUMMARY Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. PR:PROJECT_SUMMARY Metabolites were identified and quantified using Compound Discoverer 3.3 and PR:PROJECT_SUMMARY isotopic internal metabolites standards. PR:INSTITUTE University of Miami PR:DEPARTMENT McKnight - Ophthalmology PR:LABORATORY Bhattacharya Lab PR:LAST_NAME Bhattacharya PR:FIRST_NAME Sanjoy PR:ADDRESS 1638 NW 10th Avenue, Room 706-A, Miami, FL 33136 PR:EMAIL sbhattacharya@med.miami.edu PR:PHONE 3054824103 #STUDY ST:STUDY_TITLE Metabolomic analysis of Axon Regeneration in Xenopus laevis Retina ST:STUDY_SUMMARY We profile the metabolite changes in the retina of a transgenic line of 1 year ST:STUDY_SUMMARY old Xenopus laevis Tg(islet2b:gfp) frogs that either had a monocular surgery of ST:STUDY_SUMMARY either a left optic crush injury (crush) or sham surgery (sham). The matching ST:STUDY_SUMMARY controls of uninjured right optic nerves were also collected (control). ST:STUDY_SUMMARY Tg(islet2b:gfp) frogs were allowed to recover for 12 and 27 days post optic ST:STUDY_SUMMARY nerve crush. Following euthanasia, the tissues were collected for metabolomic ST:STUDY_SUMMARY analysis. Samples were pooled for each category (crush, sham, and control) at n ST:STUDY_SUMMARY =3 to obtain sufficient metabolite concentrations for analysis. Metabolites were ST:STUDY_SUMMARY extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 ST:STUDY_SUMMARY Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were ST:STUDY_SUMMARY analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) ST:STUDY_SUMMARY profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon ST:STUDY_SUMMARY Binary UHPLC LC-MS system. Metabolites were identified and quantified using ST:STUDY_SUMMARY Compound Discoverer 3.3 and isotopic internal metabolites standards. ST:INSTITUTE University of Miami ST:DEPARTMENT McKnight - Ophthalmology ST:LABORATORY Bhattacharya Lab ST:LAST_NAME Bhattacharya ST:FIRST_NAME Sanjoy ST:ADDRESS 1638 NW 10th Avenue, Room 706-A, Miami, FL 33136 ST:EMAIL sbhattacharya@med.miami.edu ST:PHONE 3054824103 #SUBJECT SU:SUBJECT_TYPE Amphibian SU:SUBJECT_SPECIES Xenopus laevis SU:TAXONOMY_ID 8355 SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret1_POS1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret1_POS1.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret1_POS2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret1_POS2.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret1_POS3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret1_POS3.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret1_NEG1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret1_NEG1.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret1_NEG2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret1_NEG2.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret1_NEG3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret1_NEG3.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret2_POS1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret2_POS1.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret2_POS2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret2_POS2.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret2_POS3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret2_POS3.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret2_NEG1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret2_NEG1.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret2_NEG2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret2_NEG2.raw SUBJECT_SAMPLE_FACTORS - CTL_12dpi_Ret2_NEG3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_12dpi_Ret2_NEG3.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret1_POS1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret1_POS1.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret1_POS2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret1_POS2.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret1_POS3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret1_POS3.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret1_NEG1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret1_NEG1.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret1_NEG2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret1_NEG2.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret1_NEG3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret1_NEG3.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret2_POS1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret2_POS1.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret2_POS2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret2_POS2.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret2_POS3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret2_POS3.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret2_NEG1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret2_NEG1.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret2_NEG2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret2_NEG2.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret2_NEG3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret2_NEG3.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret3_POS1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret3_POS1.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret3_POS2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret3_POS2.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret3_POS3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret3_POS3.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret3_NEG1 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret3_NEG1.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret3_NEG2 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret3_NEG2.raw SUBJECT_SAMPLE_FACTORS - CTL_27dpi_Ret3_NEG3 Sample source:Retina | Treatment:Control RAW_FILE_NAME=CTL_27dpi_Ret3_NEG3.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret1_POS1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret1_POS1.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret1_POS2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret1_POS2.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret1_POS3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret1_POS3.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret1_NEG1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret1_NEG1.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret1_NEG2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret1_NEG2.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret1_NEG3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret1_NEG3.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret2_POS1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret2_POS1.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret2_POS2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret2_POS2.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret2_POS3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret2_POS3.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret2_NEG1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret2_NEG1.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret2_NEG2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret2_NEG2.raw SUBJECT_SAMPLE_FACTORS - CX_12dpi_Ret2_NEG3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_12dpi_Ret2_NEG3.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret1_POS1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret1_POS1.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret1_POS2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret1_POS2.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret1_POS3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret1_POS3.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret1_NEG1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret1_NEG1.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret1_NEG2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret1_NEG2.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret1_NEG3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret1_NEG3.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret2_POS1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret2_POS1.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret2_POS2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret2_POS2.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret2_POS3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret2_POS3.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret2_NEG1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret2_NEG1.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret2_NEG2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret2_NEG2.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret2_NEG3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret2_NEG3.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret3_POS1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret3_POS1.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret3_POS2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret3_POS2.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret3_POS3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret3_POS3.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret3_NEG1 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret3_NEG1.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret3_NEG2 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret3_NEG2.raw SUBJECT_SAMPLE_FACTORS - CX_27dpi_Ret3_NEG3 Sample source:Retina | Treatment:Crush RAW_FILE_NAME=CX_27dpi_Ret3_NEG3.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM1_POS1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM1_POS1.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM1_POS2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM1_POS2.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM1_POS3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM1_POS3.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM1_NEG1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM1_NEG1.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM1_NEG2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM1_NEG2.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM1_NEG3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM1_NEG3.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM2_POS1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM2_POS1.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM2_POS2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM2_POS2.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM2_POS3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM2_POS3.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM2_NEG1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM2_NEG1.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM2_NEG2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM2_NEG2.raw SUBJECT_SAMPLE_FACTORS - Left_Ret_SHAM2_NEG3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Left_Ret_SHAM2_NEG3.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC1_MS1_Ret_POS1 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC1_MS1_Ret_POS1.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC1_MS1_Ret_POS2 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC1_MS1_Ret_POS2.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC1_MS1_Ret_NEG1 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC1_MS1_Ret_NEG1.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC1_MS1_Ret_NEG2 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC1_MS1_Ret_NEG2.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC2_MS1_Ret_POS1 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC2_MS1_Ret_POS1.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC2_MS1_Ret_POS2 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC2_MS1_Ret_POS2.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC2_MS1_Ret_NEG1 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC2_MS1_Ret_NEG1.raw SUBJECT_SAMPLE_FACTORS - Pooled_QC2_MS1_Ret_NEG2 Sample source:Retina | Treatment:QC RAW_FILE_NAME=Pooled_QC2_MS1_Ret_NEG2.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM1_POS1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM1_POS1.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM1_POS2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM1_POS2.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM1_POS3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM1_POS3.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM1_NEG1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM1_NEG1.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM1_NEG2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM1_NEG2.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM1_NEG3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM1_NEG3.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM2_POS1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM2_POS1.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM2_POS2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM2_POS2.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM2_POS3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM2_POS3.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM2_NEG1 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM2_NEG1.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM2_NEG2 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM2_NEG2.raw SUBJECT_SAMPLE_FACTORS - Right_Ret_SHAM2_NEG3 Sample source:Retina | Treatment:Sham RAW_FILE_NAME=Right_Ret_SHAM2_NEG3.raw SUBJECT_SAMPLE_FACTORS - Blank_POS1 Sample source:NA | Treatment:Extraction Blank RAW_FILE_NAME=Blank_POS1.raw SUBJECT_SAMPLE_FACTORS - Blank_POS2 Sample source:NA | Treatment:Extraction Blank RAW_FILE_NAME=Blank_POS2.raw SUBJECT_SAMPLE_FACTORS - Blank_POS3 Sample source:NA | Treatment:Extraction Blank RAW_FILE_NAME=Blank_POS3.raw SUBJECT_SAMPLE_FACTORS - Blank_NEG1 Sample source:NA | Treatment:Extraction Blank RAW_FILE_NAME=Blank_NEG1.raw SUBJECT_SAMPLE_FACTORS - Blank_NEG2 Sample source:NA | Treatment:Extraction Blank RAW_FILE_NAME=Blank_NEG2.raw SUBJECT_SAMPLE_FACTORS - Blank_NEG3 Sample source:NA | Treatment:Extraction Blank RAW_FILE_NAME=Blank_NEG3.raw #COLLECTION CO:COLLECTION_SUMMARY The tissue was removed via dissection from the optic nerve head to the optic CO:COLLECTION_SUMMARY chiasm. The retinas were collected at 12- and 27-days post crush and separated CO:COLLECTION_SUMMARY into biological samples. Due to the small tissue and metabolomics resolution CO:COLLECTION_SUMMARY constraints, tissues were pooled to generate higher signal intensities. A total CO:COLLECTION_SUMMARY of 3 retinas were pooled into one tube. CO:SAMPLE_TYPE Eye tissue #TREATMENT TR:TREATMENT_SUMMARY Optic nerves from each transgenic Tg(Islet2b:EGFP-RPL10a) Xenopus laevis frogs, TR:TREATMENT_SUMMARY 3.5 - 5.0 cm in length, underwent monocular surgery of either a left optic crush TR:TREATMENT_SUMMARY injury (crush) or sham surgery (sham). The matching controls of uninjured right TR:TREATMENT_SUMMARY optic nerves were also collected (control). Operated individuals were TR:TREATMENT_SUMMARY anesthetized with 0.05% ethyl 3-aminobenzoate methanesulfonate (Sigma, USA). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Retina remained on dry ice to prevent metabolite degradation while the SP:SAMPLEPREP_SUMMARY metabolite extraction was conducted. Tissues were transferred to 0.5mL Soft SP:SAMPLEPREP_SUMMARY Tissue Lysing Kit Precellys tubes containing beads. Then, 84 µL of chilled 1:1 SP:SAMPLEPREP_SUMMARY MeOH/H2O were added to Precellys tube. Pre-extraction internal standards were SP:SAMPLEPREP_SUMMARY added to the tubes: 5µl of 1mg/ml Caffeine 13C6, 5µl of 1mg/ml D-Glucose 13C6, SP:SAMPLEPREP_SUMMARY 5µl of 1mg/ml Oleic Acid 13C5, and 1µl of 5mg/mL Isoleucine 13C6 to each SP:SAMPLEPREP_SUMMARY sample. Tissues were homogenized using Precellys 24 Touch. Cycle parameters: 2 SP:SAMPLEPREP_SUMMARY cycles: 30 seconds homogenization at 4500 rpm, 10 seconds rest. Homogenate was SP:SAMPLEPREP_SUMMARY transferred to a microcentrifuge tube and centrifuged at 18000xrcf for 20 min at SP:SAMPLEPREP_SUMMARY 4°C. Then, collect supernatant and transfer pellet to Precellys Lysing Kit SP:SAMPLEPREP_SUMMARY tube. Add 84uL of 8:1:1 Acetonitrile/Methanol/Acetone to pellet and add the rest SP:SAMPLEPREP_SUMMARY of the pre-extraction internal standards: 5µl of 1mg/ml Caffeine 13C6, 5µl of SP:SAMPLEPREP_SUMMARY 1mg/ml D-Glucose 13C6, 5µl of 1mg/ml Oleic Acid 13C5, 1µl of 5mg/mL Isoleucine SP:SAMPLEPREP_SUMMARY 13C6. Final pre-extraction internal standards concentrations are 50μg/mL. SP:SAMPLEPREP_SUMMARY Homogenization cycles were repeating using Precellys 24 Touch. Centrifuge as SP:SAMPLEPREP_SUMMARY before and add second supernatant to first round of collected supernatant. SP:SAMPLEPREP_SUMMARY Centrifuge at 1800xrcf for 20 min once more to remove any remaining tissue SP:SAMPLEPREP_SUMMARY debris. Collect supernatant and dry supernatant in Speedvac. Two extraction SP:SAMPLEPREP_SUMMARY blanks were prepared in the same manner as the biological samples. Dried samples SP:SAMPLEPREP_SUMMARY were reconstituted immediately in 0.1% formic acid in 44.75µL of HPLC-MS grade SP:SAMPLEPREP_SUMMARY water. Post-extraction internal standards were added: 25 µl of 5mg/ml SP:SAMPLEPREP_SUMMARY Phenylalanine 13C6, 2.5 µl of .5mg/ml Uracil 13C 15N2, 1.25 µl of 1mg/ml SP:SAMPLEPREP_SUMMARY Arginine 13C6, 1.25 µl of 1mg/ml Serine 13C3 to each sample. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Negative ion mode CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) CH:SOLVENT_A 95% acetonitrile/5% water; 10mM Ammonium Acetate; 0.1% acetic acid CH:SOLVENT_B 50% acetonitrile/50% water; 10mM Ammonium Acetate; 0.1% acetic acid CH:FLOW_GRADIENT The gradient began at 1.0% B for 1 min, then shifted to 95.0% B for 9 minutes, CH:FLOW_GRADIENT then stayed at 95.0% B for 1 min before ramping down quickly to 1.0% B and held CH:FLOW_GRADIENT for 5 minutes. CH:FLOW_RATE 0.5 ml/min CH:COLUMN_TEMPERATURE 35 C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The samples were run using a Q ExactiveTM mass spectrometer coupled to a heated MS:MS_COMMENTS electrospray ionization (HESI) source. The spray voltage was set to 3.50 kV, MS:MS_COMMENTS capillary temperature to 350°C, sheath gas to 55, aux gas to 14, sweep gas to MS:MS_COMMENTS 4, and S-Lens RF Level to 30.0. The mass range was set to 67 – 1000 m/z, MS:MS_COMMENTS resolution 140,000 for full scan and 35,000 for ddMS2. AGC target was set to 1e6 MS:MS_COMMENTS for full scan and 2e5 for ddMS2. The max injection time (IT) was 100 seconds for MS:MS_COMMENTS full scan mode and 50 seconds for ddMS2. The number of microscans was 2, and MS:MS_COMMENTS normalized collision energy (NCE) was set to 20, 35, and 50. Samples were run in MS:MS_COMMENTS both positive and negative ion mode separately. The parameters for negative mode MS:MS_COMMENTS were the same except the spray voltage, which was set to 2.50 kV and capillary MS:MS_COMMENTS temperature to 380°C. Metabolites were identified from their Thermo.RAW scans MS:MS_COMMENTS using Compound DiscovererTM 3.3 software. Extraction blanks were used to MS:MS_COMMENTS determine and correct for reagent effects, allow for the creation of exclusions MS:MS_COMMENTS lists, mark background components, and filters the background components from MS:MS_COMMENTS the results table in Compound DiscovererTM 3.3. Pooled QCs were used for initial MS:MS_COMMENTS compound normalization and identification. All non-identified compounds were MS:MS_COMMENTS removed. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples CTL_12dpi_Ret1_NEG1 CTL_12dpi_Ret1_NEG2 CTL_12dpi_Ret1_NEG3 CTL_12dpi_Ret2_NEG1 CTL_12dpi_Ret2_NEG2 CTL_12dpi_Ret2_NEG3 CTL_27dpi_Ret1_NEG1 CTL_27dpi_Ret1_NEG2 CTL_27dpi_Ret1_NEG3 CTL_27dpi_Ret2_NEG1 CTL_27dpi_Ret2_NEG2 CTL_27dpi_Ret2_NEG3 CTL_27dpi_Ret3_NEG1 CTL_27dpi_Ret3_NEG2 CTL_27dpi_Ret3_NEG3 CX_12dpi_Ret1_NEG1 CX_12dpi_Ret1_NEG2 CX_12dpi_Ret1_NEG3 CX_12dpi_Ret2_NEG1 CX_12dpi_Ret2_NEG2 CX_12dpi_Ret2_NEG3 CX_27dpi_Ret1_NEG1 CX_27dpi_Ret1_NEG2 CX_27dpi_Ret1_NEG3 CX_27dpi_Ret2_NEG1 CX_27dpi_Ret2_NEG2 CX_27dpi_Ret2_NEG3 CX_27dpi_Ret3_NEG1 CX_27dpi_Ret3_NEG2 CX_27dpi_Ret3_NEG3 Left_Ret_SHAM1_NEG1 Left_Ret_SHAM1_NEG2 Left_Ret_SHAM1_NEG3 Left_Ret_SHAM2_NEG1 Left_Ret_SHAM2_NEG2 Left_Ret_SHAM2_NEG3 Pooled_QC1_MS1_Ret_NEG1 Pooled_QC1_MS1_Ret_NEG2 Pooled_QC2_MS1_Ret_NEG1 Pooled_QC2_MS1_Ret_NEG2 Right_Ret_SHAM1_NEG1 Right_Ret_SHAM1_NEG2 Right_Ret_SHAM1_NEG3 Right_Ret_SHAM2_NEG1 Right_Ret_SHAM2_NEG2 Right_Ret_SHAM2_NEG3 Blank_NEG1 Blank_NEG2 Blank_NEG3 Factors Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Control Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Crush Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:QC Sample source:Retina | Treatment:QC Sample source:Retina | Treatment:QC Sample source:Retina | Treatment:QC Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:Retina | Treatment:Sham Sample source:NA | Treatment:Extraction Blank Sample source:NA | Treatment:Extraction Blank Sample source:NA | Treatment:Extraction Blank 1D-chiro-inositol 344113.5112 350063.8669 76481.07157 503860.3164 944642.42 107898.89 1103855.509 866944.4741 126110.2717 4463963.831 363922.878 129313.0671 1116546.267 860557.4147 85433.0998 13023240.54 9505068.565 42162.77042 265778.8126 909678.9273 230385.7134 691777.5572 456534.6693 207359.4799 1017963.581 2991595.804 95534.42919 854510.4121 202529.8813 423177.9603 5049462.044 5797362.55 182087.7465 2407975.552 1064690.358 319901.3992 508184.4805 731215.8574 1740700.694 484288.0421 715450.9 274144.9418 56709.60583 14360355.45 6965738.187 96965.70348 5280.621425 4343.541199 4117.063688 1-Methoxypyrene 342234.984 291986.9507 51603.56663 280068.4745 1099729.644 72972.31964 2220952.596 1864324.782 95118.75496 918246.3115 582172.9138 104473.6903 9757404.926 8701445.509 60638.63011 4565430.876 4664495.618 5136.742653 178905.2156 146860.2903 369943.7345 2171952.733 2475302.222 154439.5606 574157.1993 963531.7872 62337.80493 2622464.112 1859480.157 21317.17601 418869.0042 676080.5123 124625.0729 558672.0352 618918.427 237160.6029 698487.658 640676.043 1257008.481 606691.5415 641650.8731 242981.1759 40211.93505 583579.5249 4055021.13 67366.43047 2721.907268 2099.869871 1721.426061 Allopurinol 106979162.6 85826505.6 497658.0757 115558005.5 39844400.01 513535.2828 47023809.02 67220023.46 371120.4119 15196883.4 131511037.5 535144.662 240043.8744 1034647.685 584731.9231 175623374.2 633540760.4 91294.89771 72615466.07 54578511.83 59367935.96 9090756.057 8187853.662 708287.0203 32101837.32 90374513.87 665830.2494 30798419.94 23492695.42 118973.8997 117018656 161631861.4 461555.7906 65595518.29 205280766 732462.6988 79289265.49 34055329.43 107048453 17552112.41 181827883.1 13279706.17 371304.3491 463723661.5 3789276.275 716374.7802 338795.6322 31038.62356 17904.51949 Didanosine 237680696.4 240619797.8 198191625.1 239344882.5 180430718.4 205684138.7 166211649.7 327096834.4 227880055.3 142994474 291656081.1 591522021.9 65915492.36 38187881.34 158923434.2 20170922.77 25889538.82 28031271.13 153811618.9 100054094 244130256.7 490047328.9 629571875.3 163308801.4 218088551.6 316121687.2 321946589.7 254523278 561947662.6 108807844.3 180569679.1 444414799.2 244573635.9 133309851.7 279725161.6 32094801.91 131887458.3 164581876.2 208534120.7 897684692.3 354589075.7 92288659.76 221466681.7 26593958.83 49168174.26 223816793.8 5567688.299 5011470.511 7685318.06 Imafen 40911825.76 16786048.57 40978477.8 8817005.703 7739301.295 5504404.127 10902977.18 11163099.31 7852781.964 1394259.134 2693111.235 882856.4165 1854276.444 258873.3433 3920534.226 361999.0938 651719.9301 233605.1557 4975983.845 2091898.575 4935317.62 1972791.598 12200514.5 1401077.587 4498701.964 1735290.129 37031553.01 10169038.3 2143902.167 3355968.882 12572146.79 4638215.921 9168636.162 4064958.224 11587515.48 74971.20497 1615006.447 4521303.024 1335566.728 5876436.202 15890140.76 5694636.158 2860706.285 253550.557 213303.8693 5388945.645 178542.8941 166706.4194 177742.2248 indanazoline 30857476.24 21524704.89 29770156.71 6763243.228 7325613.946 5647576.162 10299671.02 9189276.151 5137373.63 1883586.641 10819175.49 1553050.799 2891121.376 247149.8965 3183882.549 5512670.6 4677307.301 177340.2177 4248203.406 1931015.749 3390135.338 9507060.474 1285354.123 1233927.895 31159950.58 3187257.395 33632656.39 1870968.902 4226954.876 852081.2754 1965075.171 2823062.639 7566876.365 3153339.422 3129681.855 176794.4704 1982509.421 3586195.255 2191796.191 5538879.806 5526723.428 4829582.376 2187569.556 212725.5458 266952.5075 3877636.906 934284.7177 224134.7634 199772.0992 meglumine 1112208.689 1580422.135 250375.7924 1782464.699 2088341.514 296801.1216 459874.2094 630929.0634 189388.3962 661462.8177 1601396.827 228447.6132 1134931.01 1330245.296 624655.5275 36458983.1 42832649.62 469982.5845 2239346.34 4696683.575 1103617.535 1298542.055 1058795.85 267487.3779 1055660.865 1214384.336 350352.5974 802025.6382 451752.7357 1217513.829 324390.2951 689047.6221 240665.5728 28114525.95 837849.009 409483.6212 4386065.138 2696044.363 6652741.931 2753489.067 1463060.175 2165853.117 858928.247 41869570.11 67405654.42 362583.336 6347.613242 8711.182979 11813.86497 Myristyl sulfate 370863.3171 560154.6876 126853.6011 906202.8047 232604.9288 163913.6784 272282.3894 270928.6472 161686.4886 659140.162 450912.0617 155328.8969 10753.40283 21441.47785 115183.29 1553720.24 1438758.341 39070.20051 175695.8627 185664.2908 252878.2247 356932.4545 323488.8405 222738.5173 645016.5502 527023.2994 100274.5614 279133.1583 162620.1881 76550.11384 304931.2688 938241.9167 186950.5278 302110.6719 509426.2471 14337.60712 677579.7625 355596.9688 2834295.124 416859.8382 636912.2193 1153239.956 95375.16974 67325.84859 42791.52812 118031.9124 160172.6872 18052.87722 4628.04738 NP-012534 3361693.778 1727419.147 431740.5988 39297843.93 7350343.554 554476.8625 3150530.395 2418076.428 349707.1915 7282663.405 11088899.79 332858.5152 44353.15789 335447.8671 460971.0276 18887778.03 20846178.38 130768.5457 15458310.95 14017858.57 8720637.439 7019430.737 912968.1511 317554.3954 23409984.53 12146747.77 3523794.724 2380689.958 1577755.926 675102.5925 12136243.63 33822508.38 2138969.463 6791770.59 20179089.62 58887.35947 35121390.79 8405992.219 46058304.93 14034588.46 15121125.02 10004299.89 229775.6703 957477.2897 58819.35645 698519.0613 2645739.372 293404.3072 280505.171 tolonidine 25650032.68 35575696.9 21910267 53736246.47 61936770.82 39075765.25 116112396.8 32092967.51 61820752.24 124885843.3 81009174.25 70499623.77 27392208.99 2357081.968 34780726.4 10647255.79 7261304.264 2639709.063 37077704.17 20116294.15 35252663.66 105659805.5 117519529.5 79749671.39 115289788.3 112704103.5 162725738.7 122322793.1 69244092.7 13680296.5 210330887.8 100333594 78471732.04 35765085.03 346625313.2 1885902.18 15384121.79 43791919.59 19363537.93 49554192.99 172318466.7 60059194.68 28478301.05 2584278.114 2895690.414 53295116.69 1325416.667 1181107.548 1141484.235 tramazoline 76478301.79 61873468.88 53540700.72 26762972.32 20698511.98 25088691.2 13978832.51 13728044.34 26130670.28 12264989.5 4407470.577 2633084.302 35237795.03 30348679.61 43159754.15 23272607.56 23811941.49 17433964.01 29660696.84 48293921.77 32917704.82 7640266.395 15067424.53 11102379.77 45151149.14 15642788.12 49885313.15 7237259.128 14322771.06 67534837.12 12403880.97 3130914.524 11206554.8 53695152.56 12433425.42 6546854.065 40131201.56 100671783.5 47510661.06 53909931.46 16089384.53 36314382.18 26616522.93 32691290.45 43859839.12 32684755.38 1821385.244 2622704.759 2769813.344 Xanthine 26892605.52 8203759.383 35791.18769 13828745.49 12344745.91 54456.55618 10189405 2434511.3 66591.61886 20415095.72 6541469.162 92282.20532 52729.97538 80784.27813 77910.00099 47577677.11 23321513.89 122868.1749 2947729.934 1974196.064 1059146.997 2473756.916 1611217.042 69905.47067 7523946.294 18273202.34 76409.12052 3117904.791 4331749.59 19615.99183 34910867.31 18725985.41 57073.41554 8301958.387 22409351.89 87195.97223 6877491.954 12600867.04 9284921.017 10444568.27 26529321.96 1401660.759 105196.9752 6907789.033 4083400.275 227264.532 45092.30591 23856.65493 2802.428855 Zonisamide 449797.8011 532139.5273 156739.3672 11787953.64 9975147.705 219339.8851 518797.9502 734242.0763 176150.5014 1057094.466 2657531.273 173423.0857 86225.7731 18904.51868 142319.6175 1433901.295 322328.3309 34850.71804 391592.7863 108508.4418 515129.1626 595782.9175 399700.4084 279825.5449 3415770.047 825336.5147 188315.274 344894.857 233329.5093 94584.75206 408193.4518 1864092.106 230994.6835 971246.5075 1012331.294 23142.71051 1409179.657 1093024.438 5114767.456 2878956.898 876056.3818 949427.0674 144064.157 905254.0964 240062.6807 162363.212 197908.1826 6822.0159 5463.934664 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Calc. MW m/z RT [min] 1D-chiro-inositol 180.063045 202.058515 5.444 1-Methoxypyrene 232.08886 231.08158 5.684 Allopurinol 136.03749 135.03014 2.696 Didanosine 236.090874 235.083598 5.4368 Imafen 187.110475 186.103135 12.4355 indanazoline 201.126225 200.118865 13.516 meglumine 195.11012 194.10284 4.527 Myristyl sulfate 294.18685 293.17958 0.948 NP-012534 266.15537 265.14809 0.954 tolonidine 209.07151 208.06424 13.735 tramazoline 215.1420833 214.1347 9.996 Xanthine 152.03273 151.02546 3.48 Zonisamide 212.02521 211.01794 0.917 METABOLITES_END #END