#METABOLOMICS WORKBENCH smcritchie_20190318_093547 DATATRACK_ID:1678 STUDY_ID:ST001150 ANALYSIS_ID:AN001897 PROJECT_ID:PR000769 VERSION 1 CREATED_ON March 19, 2019, 12:08 pm #PROJECT PR:PROJECT_TITLE Metabolomics of a Mouse Model for Retinitis Pigmentosa PR:PROJECT_SUMMARY Retinitis pigmentosa (RP) is a degenerative disease of the retina that affects PR:PROJECT_SUMMARY approximately 1 million people worldwide. There are multiple genetic causes of PR:PROJECT_SUMMARY this disease, for which, at present, there are no effective therapeutic PR:PROJECT_SUMMARY strategies. We utilized broad spectrum metabolomics to identify perturbations in PR:PROJECT_SUMMARY the metabolism of the rd10 mouse, a genetic model for RP. C57BL/6J and rd10 mice PR:PROJECT_SUMMARY were raised in cyclic light followed by either light or dark adaptation at PR:PROJECT_SUMMARY postnatal day (P) 18, an early stage in the degeneration process. Mice raised PR:PROJECT_SUMMARY entirely in the dark until P18 were also evaluated. After euthanasia, retinas PR:PROJECT_SUMMARY were removed and extracted for analysis by ultra-performance liquid PR:PROJECT_SUMMARY chromatography-time of flight-mass spectrometry (UPLC-QTOF-MS). PR:INSTITUTE University of North Carolina at Chapel Hill PR:LABORATORY ERCMRC PR:LAST_NAME Sumner PR:FIRST_NAME Susan PR:ADDRESS NRI, 500 Laureate Way, Kannapolis, NC, 28081, USA PR:EMAIL susan_sumner@unc.edu PR:PHONE 704-250-5066 PR:FUNDING_SOURCE NIDDK U24DK097193 (Sumner, PI); Supplement to NEI R01EY12224 (Weiss, PI) #STUDY ST:STUDY_TITLE Metabolomics of a Mouse Model for Retinitis Pigmentosa ST:STUDY_SUMMARY Retinitis pigmentosa (RP) is a degenerative disease of the retina that affects ST:STUDY_SUMMARY approximately 1 million people worldwide. There are multiple genetic causes of ST:STUDY_SUMMARY this disease, for which, at present, there are no effective therapeutic ST:STUDY_SUMMARY strategies. We utilized broad spectrum metabolomics to identify perturbations in ST:STUDY_SUMMARY the metabolism of the rd10 mouse, a genetic model for RP. C57BL/6J and rd10 mice ST:STUDY_SUMMARY were raised in cyclic light followed by either light or dark adaptation at ST:STUDY_SUMMARY postnatal day (P) 18, an early stage in the degeneration process. Mice raised ST:STUDY_SUMMARY entirely in the dark until P18 were also evaluated. After euthanasia, retinas ST:STUDY_SUMMARY were removed and extracted for analysis by ultra-performance liquid ST:STUDY_SUMMARY chromatography-time of flight-mass spectrometry (UPLC-QTOF-MS). ST:INSTITUTE University of North Carolina at Chapel Hill ST:DEPARTMENT Department of Cell Biology & Physiology ST:LAST_NAME Weiss ST:FIRST_NAME Ellen ST:ADDRESS CB# 7545, 5340B MBRB, 111 Mason Farm Rd. Chapel Hill, NC 27599-7090 ST:EMAIL erweiss@med.unc.edu ST:PHONE 919-966-7683 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN rd10 and C57BL/6J SU:AGE_OR_AGE_RANGE P18 SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratory #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - All_pool_QC1 Condition:QC Pool SUBJECT_SAMPLE_FACTORS - All_pool_QC2 Condition:QC Pool SUBJECT_SAMPLE_FACTORS - All_pool_QC3 Condition:QC Pool SUBJECT_SAMPLE_FACTORS - RD10-RCL-L-100913-RT-1 Condition:RD10-RCL-L SUBJECT_SAMPLE_FACTORS - RD10-RCL-L-110113-RT-1 Condition:RD10-RCL-L SUBJECT_SAMPLE_FACTORS - RD10-RCL-L-110113-RT-2 Condition:RD10-RCL-L SUBJECT_SAMPLE_FACTORS - RD10-RCL-L-110913-RT-1 Condition:RD10-RCL-L SUBJECT_SAMPLE_FACTORS - RD10-RCL-L-110913-RT-2 Condition:RD10-RCL-L SUBJECT_SAMPLE_FACTORS - RD10-RCL-L-111013-RT-1 Condition:RD10-RCL-L SUBJECT_SAMPLE_FACTORS - RD10-RCL-D-100913-RT-2 Condition:RD10-RCL-D SUBJECT_SAMPLE_FACTORS - RD10-RCL-D-110113-RT-3 Condition:RD10-RCL-D SUBJECT_SAMPLE_FACTORS - RD10-RCL-D-110913-RT-3 Condition:RD10-RCL-D SUBJECT_SAMPLE_FACTORS - RD10-RCL-D-111013-RT-2 Condition:RD10-RCL-D SUBJECT_SAMPLE_FACTORS - RD10-RCL-D-111013-RT-3 Condition:RD10-RCL-D SUBJECT_SAMPLE_FACTORS - WT-RCL-L-101513-RT-1 Condition:WT-RCL-L SUBJECT_SAMPLE_FACTORS - WT-RCL-L-101513-RT-2 Condition:WT-RCL-L SUBJECT_SAMPLE_FACTORS - WT-RCL-L-111013-RT-1 Condition:WT-RCL-L SUBJECT_SAMPLE_FACTORS - WT-RCL-L-111013-RT-2 Condition:WT-RCL-L SUBJECT_SAMPLE_FACTORS - WT-RCL-L-111113-RT-1 Condition:WT-RCL-L SUBJECT_SAMPLE_FACTORS - WT-RCL-L-111713-RT-1 Condition:WT-RCL-L SUBJECT_SAMPLE_FACTORS - WT-RCL-D-101513-RT-3 Condition:WT-RCL-D SUBJECT_SAMPLE_FACTORS - WT-RCL-D-111013-RT-3 Condition:WT-RCL-D SUBJECT_SAMPLE_FACTORS - WT-RCL-D-111113-RT-2 Condition:WT-RCL-D SUBJECT_SAMPLE_FACTORS - WT-RCL-D-111113-RT-3 Condition:WT-RCL-D SUBJECT_SAMPLE_FACTORS - WT-RCL-D-111713-RT-2 Condition:WT-RCL-D SUBJECT_SAMPLE_FACTORS - RD10-RD-D-120513-RT-1 Condition:RD10-RD-D SUBJECT_SAMPLE_FACTORS - RD10-RD-D-120513-RT-2 Condition:RD10-RD-D SUBJECT_SAMPLE_FACTORS - RD10-RD-D-120813-RT-1 Condition:RD10-RD-D SUBJECT_SAMPLE_FACTORS - RD10-RD-D-120813-RT-2 Condition:RD10-RD-D SUBJECT_SAMPLE_FACTORS - RD10-RD-D-120813-RT-3 Condition:RD10-RD-D SUBJECT_SAMPLE_FACTORS - RD10-RD-D-120813-RT-4 Condition:RD10-RD-D SUBJECT_SAMPLE_FACTORS - WT-RD-D-011614-RT-1 Condition:WT-RD-D SUBJECT_SAMPLE_FACTORS - WT-RD-D-011614-RT-2 Condition:WT-RD-D SUBJECT_SAMPLE_FACTORS - WT-RD-D-120513-RT-1 Condition:WT-RD-D SUBJECT_SAMPLE_FACTORS - WT-RD-D-120513-RT-2 Condition:WT-RD-D SUBJECT_SAMPLE_FACTORS - WT-RD-D-121213-RT-1 Condition:WT-RD-D SUBJECT_SAMPLE_FACTORS - WT-RD-D-121213-RT-2 Condition:WT-RD-D #COLLECTION CO:COLLECTION_SUMMARY C57BL/6J and rd10 mice were raised under varying light conditions in standard CO:COLLECTION_SUMMARY housing, temperature and access to food and water. Some mice were raised under CO:COLLECTION_SUMMARY normal light/dark cycle at ambient light levels of approximately 80 lux. At 18 CO:COLLECTION_SUMMARY days after birth, mice were exposed to 250 lux for 3 h (light-adapted) followed CO:COLLECTION_SUMMARY by euthanasia using cervical dislocation. The eyes were enucleated and the CO:COLLECTION_SUMMARY retinas dissected in PBS in the light under a microscope, followed by rapid CO:COLLECTION_SUMMARY freezing by dropping them individually into a container filled with liquid CO:COLLECTION_SUMMARY nitrogen. Each set of samples were pooled in liquid nitrogen. Other mice raised CO:COLLECTION_SUMMARY in cyclic light were adapted overnight at P17 in complete darkness CO:COLLECTION_SUMMARY (dark-adapted) before euthanasia at P18. Using infrared goggles and an infrared CO:COLLECTION_SUMMARY microscope, these retinas were processed similarly as light-adapted samples CO:COLLECTION_SUMMARY except that all procedures were performed in the dark. Mice raised in total CO:COLLECTION_SUMMARY darkness were euthanized at P18 and dissected in the dark using infrared goggles CO:COLLECTION_SUMMARY and an infrared microscope. CO:SAMPLE_TYPE Retina #TREATMENT TR:TREATMENT_SUMMARY C57BL/6J and rd10 mice were raised under varying light conditions in standard TR:TREATMENT_SUMMARY housing, temperature and access to food and water. Some mice were raised under TR:TREATMENT_SUMMARY normal light/dark cycle at ambient light levels of approximately 80 lux. At 18 TR:TREATMENT_SUMMARY days after birth, mice were exposed to 250 lux for 3 h (light-adapted) followed TR:TREATMENT_SUMMARY by euthanasia. Other mice raised in cyclic light were adapted overnight at P17 TR:TREATMENT_SUMMARY in complete darkness (dark-adapted) before euthanasia at P18. Mice raised in TR:TREATMENT_SUMMARY total darkness were euthanized at P18. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each sample was homogenized in buffer containing 50:50 acetonitrile:water at a SP:SAMPLEPREP_SUMMARY ratio of 1mg:25 µl tissue:buffer. A 320 µl aliquot of homogenate (12.8 mg SP:SAMPLEPREP_SUMMARY tissue equivalent) was transferred to a tube and centrifuged. The supernatant SP:SAMPLEPREP_SUMMARY was dried and re-suspended in 100 µl 95:5 H2O:methanol for UPLC-TOF-MS SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reversed-Phase Gradient Seperation CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_GRADIENT Time(min) Flow Rate(mL/min) %A %B Curve 1. Initial 0.400 99.0 1.0 6 2. 1.00 CH:FLOW_GRADIENT 0.400 99.0 1.0 6 3. 16.00 0.400 1.0 99.0 6 4. 20.00 0.400 1.0 99.0 6 5. 20.50 CH:FLOW_GRADIENT 0.400 99.0 1.0 6 6. 22.00 0.400 99.0 1.0 6 CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 50 °C CH:COLUMN_PRESSURE 6,000-10,000 psi CH:INJECTION_TEMPERATURE 8 °C CH:INTERNAL_STANDARD L-Tryptophan-d5 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MassLynx 4.1 MS:CAPILLARY_TEMPERATURE 110 °C MS:CAPILLARY_VOLTAGE 1.0 kV MS:COLLISION_ENERGY 4 MS:FRAGMENTATION_METHOD CID MS:HELIUM_FLOW 180 MS:IONIZATION ES- MS:MASS_ACCURACY 5 ppm MS:SOURCE_TEMPERATURE 110 °C MS:DATAFORMAT Continuum MS:DESOLVATION_GAS_FLOW 800 L/Hr MS:DESOLVATION_TEMPERATURE 400 °C MS:RESOLUTION_SETTING 20000 MS:SCAN_RANGE_MOVERZ 50-1000 m/z MS:SCANNING_CYCLE 1.0 s MS:TUBE_LENS_VOLTAGE 75 MS:MS_RESULTS_FILE ST001150_AN001897_Results.txt UNITS:m/z_retention time Has m/z:Yes Has RT:Yes RT units:Minutes #END