#METABOLOMICS WORKBENCH smcritchie_20180211_103656 DATATRACK_ID:1313 STUDY_ID:ST000929 ANALYSIS_ID:AN001523 PROJECT_ID:PR000643 VERSION 1 CREATED_ON February 26, 2018, 3:58 pm #PROJECT PR:PROJECT_TITLE Metabolomics Analysis of Aqueous Humour in the Presence of Glaucoma Drainage PR:PROJECT_TITLE Devices PR:PROJECT_TYPE Metabolomics of the aqueous humor of rabbits with Ahmed Glaucoma Valve PR:PROJECT_TYPE implantation. PR:PROJECT_SUMMARY Aqueous humor samples for metabolomics experiment were generated in the PR:PROJECT_SUMMARY following manner: Eight adult male white rabbits were given seven days to PR:PROJECT_SUMMARY acclimate to their environment. On day eight, an Ahmed Glaucoma Valve was PR:PROJECT_SUMMARY implanted into the left eye of every rabbit. The un-operated right eye served as PR:PROJECT_SUMMARY control. One rabbit was lost during the course of the study. After adequate PR:PROJECT_SUMMARY anesthesia, aqueous humor samples were taken from both eyes of each rabbit PR:PROJECT_SUMMARY before surgery (A samples), and at 27 (B samples), 52 (C samples) and 153 days PR:PROJECT_SUMMARY (IV samples) after surgery. PR:INSTITUTE Tulane University School of Medicine PR:DEPARTMENT Ophthalmology PR:LAST_NAME Ayyala PR:FIRST_NAME Ramesh S. PR:ADDRESS 1430 Tulane Ave., New Orleans, LA 70112 PR:EMAIL rayyala@tulane.edu PR:PHONE 504-998-2261 #STUDY ST:STUDY_TITLE Metabolomics Analysis of Aqueous Humour in the Presence of Glaucoma Drainage ST:STUDY_TITLE Devices ST:STUDY_TYPE Metabolomics Evaluation of Aqueous Humor Samples from Left (treated) and Right ST:STUDY_TYPE (Control) Eyes of Rabbits. ST:STUDY_SUMMARY Metabolomic experiments were carried out on aqueous humor samples provided by ST:STUDY_SUMMARY Ramesh Ayyala. Through this collaboration, we propose to study aqueous humor ST:STUDY_SUMMARY from control and experimental rabbits to better understand metabolic differences ST:STUDY_SUMMARY in the aqueous humor samples. ST:INSTITUTE University of North Carolina at Chapel Hill ST:DEPARTMENT Nutrition Research Institute ST:LABORATORY ERCMRC ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 500 Laureate Way, Kannapolis, NC, 28081, USA ST:EMAIL susan_sumner@unc.edu ST:PHONE 7042505066 ST:NUM_GROUPS 9 ST:TOTAL_SUBJECTS 56 ST:NUM_MALES 56 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Oryctolagus cuniculus SU:TAXONOMY_ID 9986 SU:GENDER Male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - C_IV-58R_POS Eye:Right Eye | Treatment:control- 153 days post-valve SUBJECT_SAMPLE_FACTORS - C_IV-59R_POS Eye:Right Eye | Treatment:control- 153 days post-valve SUBJECT_SAMPLE_FACTORS - C_IV-60R_POS Eye:Right Eye | Treatment:control- 153 days post-valve SUBJECT_SAMPLE_FACTORS - C_IV-61R_POS Eye:Right Eye | Treatment:control- 153 days post-valve SUBJECT_SAMPLE_FACTORS - C_IV-62R_POS Eye:Right Eye | Treatment:control- 153 days post-valve SUBJECT_SAMPLE_FACTORS - C_IV-64R_POS Eye:Right Eye | Treatment:control- 153 days post-valve SUBJECT_SAMPLE_FACTORS - C_IV-65R_POS Eye:Right Eye | Treatment:control- 153 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-58-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-59-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-60-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-61-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-62-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-63-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-64-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-65-C_POS Eye:Right Eye | Treatment:control- 52 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-X58-B_POS Eye:Right Eye | Treatment:control- 27 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-X59-B_POS Eye:Right Eye | Treatment:control- 27 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-X60-B_POS Eye:Right Eye | Treatment:control- 27 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-X61-B_POS Eye:Right Eye | Treatment:control- 27 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-X62-B_POS Eye:Right Eye | Treatment:control- 27 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-X64-B_POS Eye:Right Eye | Treatment:control- 27 days post-valve SUBJECT_SAMPLE_FACTORS - C_R-X65-B_POS Eye:Right Eye | Treatment:control- 27 days post-valve SUBJECT_SAMPLE_FACTORS - Day153_IV-58L_POS Eye:Left Eye | Treatment:153 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day153_IV-59L_POS Eye:Left Eye | Treatment:153 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day153_IV-60L_POS Eye:Left Eye | Treatment:153 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day153_IV-61L_POS Eye:Left Eye | Treatment:153 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day153_IV-62L_POS Eye:Left Eye | Treatment:153 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day153_IV-64L_POS Eye:Left Eye | Treatment:153 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day153_IV-65L_POS Eye:Left Eye | Treatment:153 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day27_L-X58-B_POS Eye:Left Eye | Treatment:27 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day27_L-X59-B_POS Eye:Left Eye | Treatment:27 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day27_L-X60-B_POS Eye:Left Eye | Treatment:27 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day27_L-X61-B_POS Eye:Left Eye | Treatment:27 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day27_L-X62-B_POS Eye:Left Eye | Treatment:27 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day27_L-X64-B_POS Eye:Left Eye | Treatment:27 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day27_L-X65-B_POS Eye:Left Eye | Treatment:27 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-58-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-59-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-60-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-61-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-62-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-63-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-64-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - Day52_L-65-C_POS Eye:Left Eye | Treatment:52 days post-valve implantation SUBJECT_SAMPLE_FACTORS - L_Pool_A1_POS Eye:Pool from aqueous humor from treated (left) eyes before valve implantation. | Treatment:pre-valve implantation SUBJECT_SAMPLE_FACTORS - L_Pool_A2_POS Eye:Pool from aqueous humor from treated (left) eyes before valve implantation. | Treatment:pre-valve implantation SUBJECT_SAMPLE_FACTORS - L_Pool_A3_POS Eye:Pool from aqueous humor from treated (left) eyes before valve implantation. | Treatment:pre-valve implantation SUBJECT_SAMPLE_FACTORS - Pool_1_POS Eye:Total Pool | Treatment:Whole Study QC Pool _1 SUBJECT_SAMPLE_FACTORS - Pool_2_POS Eye:Total Pool | Treatment:Whole Study QC Pool _2 SUBJECT_SAMPLE_FACTORS - Pool_3_POS Eye:Total Pool | Treatment:Whole Study QC Pool _3 SUBJECT_SAMPLE_FACTORS - Pool_4_POS Eye:Total Pool | Treatment:Whole Study QC Pool _4 SUBJECT_SAMPLE_FACTORS - Pool_5_POS Eye:Total Pool | Treatment:Whole Study QC Pool _5 SUBJECT_SAMPLE_FACTORS - Pool_6_POS Eye:Total Pool | Treatment:Whole Study QC Pool _6 SUBJECT_SAMPLE_FACTORS - R_Pool_A1_POS Eye:Pool from aqueous humor from control (right) eyes before surgery in the left eye. | Treatment:pre-valve implantation SUBJECT_SAMPLE_FACTORS - R_Pool_A2_POS Eye:Pool from aqueous humor from control (right) eyes before surgery in the left eye. | Treatment:pre-valve implantation SUBJECT_SAMPLE_FACTORS - R_Pool_A3_POS Eye:Pool from aqueous humor from control (right) eyes before surgery in the left eye. | Treatment:pre-valve implantation #COLLECTION CO:COLLECTION_SUMMARY Aqueous humor was collected from both eyes before surgery, then at one month, CO:COLLECTION_SUMMARY two months, and five months post operatively. After adequate anesthesia the eye CO:COLLECTION_SUMMARY was prepped with 5% betadine solution and then covered with a sterile drape. A CO:COLLECTION_SUMMARY sterile 27G needle was inserted into the anterior chamber through the clear CO:COLLECTION_SUMMARY cornea of the para-limbal area. Approximately 0.1 ml of aqueous humor was CO:COLLECTION_SUMMARY acquired in each collection cycle. The samples were immediately placed over an CO:COLLECTION_SUMMARY ice bath then transferred to a freezer for storage at -70ºC. CO:SAMPLE_TYPE aqueous humour #TREATMENT TR:TREATMENT_SUMMARY A Glaucoma Drainage Device was inserted into the left eye of each of Eight TR:TREATMENT_SUMMARY Rabbits. Aqueous humor was collected from the left (treated) eye and right TR:TREATMENT_SUMMARY (control) eye before implantation and at several time points after surgey. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Whole Study Pooled QC Thawed aqueous humor samples on ice. Whole study pooled QC SP:SAMPLEPREP_SUMMARY samples were created by combining 10 µL aliquot from each of the study samples SP:SAMPLEPREP_SUMMARY (Time Points B, C and D) into a 2 mL Lo-Bind eppendorf tube. The QC pooled SP:SAMPLEPREP_SUMMARY sample was then vortexed for 30 seconds. Time Point A Pools Aqueous humor SP:SAMPLEPREP_SUMMARY samples were collected from both, right and left, eyes and provided for SP:SAMPLEPREP_SUMMARY metabolomics analysis. Aqueous humor from the right eyes from experiment A were SP:SAMPLEPREP_SUMMARY combined into a 2 mL Lo-Bind Eppendorf tube to make a right eye pool from SP:SAMPLEPREP_SUMMARY experiment A. This same procedure was repeated to make a pool from the left eye SP:SAMPLEPREP_SUMMARY aqueous humor from experiment A. The two time point A pools were vortexed for 30 SP:SAMPLEPREP_SUMMARY seconds. Sample Preparation To make the study samples, aqueous humor (30 µL) SP:SAMPLEPREP_SUMMARY was transferred from their original tubes into 2.0 mL Lo-Bind Eppendorf tubes. SP:SAMPLEPREP_SUMMARY Six whole study pooled QC samples of 30 µL each were created along with SP:SAMPLEPREP_SUMMARY three-30 µL time point A pool from right eye and three-30 µL time point A pool SP:SAMPLEPREP_SUMMARY from left eye aqueous humor. For all samples (study, whole study pooled QC and SP:SAMPLEPREP_SUMMARY time point A samples) 400 µL Protein Precipitation Buffer with internal SP:SAMPLEPREP_SUMMARY standard (0.025 mg/mL of Tryptophan-d5 in methanol) was added to each tube and SP:SAMPLEPREP_SUMMARY vortexed. The samples were then centrifuged at 16,000 rcf for 4 min. A 350 µL SP:SAMPLEPREP_SUMMARY aliquot of the supernatant from each sample was transferred into pre-labeled 2.0 SP:SAMPLEPREP_SUMMARY mL LoBind eppendorf tube and stored at -80 °C for one hour followed by a drying SP:SAMPLEPREP_SUMMARY step on a lyophilizer. The residue was reconstituted in 100 µL of SP:SAMPLEPREP_SUMMARY reconstitution buffer (95:5 Water:Methanol, v/v) and mixed on a multiple tube SP:SAMPLEPREP_SUMMARY vortexer for 10 min at 5,000 rpm. Then, the samples were centrifuged at room SP:SAMPLEPREP_SUMMARY temperature for 4 min at 16,000 rcf, and the supernatants were transferred to SP:SAMPLEPREP_SUMMARY autosampler vials for data acquisition. SP:SAMPLEPREP_PROTOCOL_ID Protocol: RTI-RCMRC-LCMS-SA-018 Version: 1.0 SP:SAMPLEPREP_PROTOCOL_FILENAME 18 AP-Williamson-Aqueous humor-RTI-RCMRC-LCMS-SA-018_v1.0.docx SP:PROCESSING_METHOD Protein Precipitation followed by Metabolite Extraction and Lyophilization SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Cold Methanol with Internal Standard SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION 95:5 Water:Methanol SP:SAMPLE_SPIKING L-Tryptophan-d5 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reversed-Phase Gradient Seperation CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity 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 2. 1.00 0.400 CH:FLOW_GRADIENT 99.0 1.0 3. 16.00 0.400 1.0 99.0 4. 20.00 0.400 1.0 99.0 5. 20.50 0.400 99.0 1.0 CH:FLOW_GRADIENT 6. 22.00 0.400 99.0 1.0 CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 8 °C CH:SOLVENT_A Water with 0.1% formic acid CH:SOLVENT_B Methanol with 0.1% formic acid CH:INTERNAL_STANDARD L-Tryptophan-d5 CH:ANALYTICAL_TIME 22 min CH:WEAK_WASH_SOLVENT_NAME 10% MeOH CH:WEAK_WASH_VOLUME 1000 µL CH:STRONG_WASH_SOLVENT_NAME 80% MeOH CH:STRONG_WASH_VOLUME 1000 µL CH:TARGET_SAMPLE_TEMPERATURE 8 °C CH:SAMPLE_LOOP_SIZE 10 µL CH:SAMPLE_SYRINGE_SIZE 100 µL CH:RANDOMIZATION_ORDER Yes #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME ERCMRC AN:OPERATOR_NAME James Carlson AN:DETECTOR_TYPE TOF AN:SOFTWARE_VERSION MassLynx 4.1 AN:ACQUISITION_DATE 2/13/2015-2/14/2015 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:ACQUISITION_DATE 2/13/2015-2/14/2015 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 100 °C MS:DATAFORMAT Continuum MS:DESOLVATION_GAS_FLOW 400 L/Hr MS:DESOLVATION_TEMPERATURE 400 °C MS:RESOLUTION_SETTING 18000 MS:SCAN_RANGE_MOVERZ 50-1000 m/z MS:SCANNING_CYCLE 1.0 s MS:TUBE_LENS_VOLTAGE 75 MS:MS_RESULTS_FILE ST000929_AN001523_Results.txt UNITS:Compound Normalised Abundance #END