{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000613","ANALYSIS_ID":"AN000938","VERSION":"1","CREATED_ON":"May 28, 2017, 1:13 pm"}, "PROJECT":{"PROJECT_TITLE":"Sphingolipid Profiling of Human Aqueous Humor and Trabecular Meshwork in Glaucomatous and Control eyes","PROJECT_SUMMARY":"Using a triple quadrupole mass spectrometer and direct infusion lipidomics we identified various sphingolipids present in both the aqueous humor and trabecular meshwork of the human eye and compared them between glaucomatous and control groups.","INSTITUTE":"University of Miami","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy","ADDRESS":"McKnight Vision Research Building, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 1638 NW 10th Avenue, Room 706A, Miami, Florida 33136","EMAIL":"SBhattacharya@med.miami.edu","PHONE":"305-482-4103"}, "STUDY":{"STUDY_TITLE":"Human TM Sphingolipid Analysis (part II)","STUDY_SUMMARY":"We determined the profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide, and their quantitative differences between control and glaucomatous trabecular meshwork (TM) derived from human donors.","INSTITUTE":"University of Miami","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy","ADDRESS":"McKnight Vision Research Building, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 1638 NW 10th Avenue, Room 706A, Miami, Florida 33136","EMAIL":"Sbhattacharya@med.miami.edu","PHONE":"305-482-4103"}, "SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"SGU001", "Sample ID":"NTM1BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"34","Gender":"M","Race":"W","Medical History":"No recorded history found"} }, { "Subject ID":"SGU002", "Sample ID":"NTM2BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"31","Gender":"M","Race":"W","Medical History":"PTSD, Ulcerative colitis, depression, colostomy"} }, { "Subject ID":"SGU003", "Sample ID":"NTM3BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"36","Gender":"M","Race":"W","Medical History":"hypertension, A Fib s/p ablation, hypersholesterolemia, sleep apnea, NIDDM, obesity"} }, { "Subject ID":"SGU004", "Sample ID":"NTM4BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"70","Gender":"F","Race":"W","Medical History":"MI, unremarkable history, vitamins, allergy medication"} }, { "Subject ID":"SGU005", "Sample ID":"NTM5BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"62","Gender":"M","Race":"W","Medical History":"Diabetes, Hypertension, arthritis"} }, { "Subject ID":"SGU006", "Sample ID":"NTM6BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"67","Gender":"F","Race":"W","Medical History":"Cardiac arrest, no known history"} }, { "Subject ID":"SGU007", "Sample ID":"NTM7BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"63","Gender":"M","Race":"W","Medical History":"Pulmonary fibrosis, Diabetes, CAD, Previous incidence of MI"} }, { "Subject ID":"SGU008", "Sample ID":"NTM8BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"68","Gender":"F","Race":"W","Medical History":"Advanced pulmonary emphysema-oxygen dependent"} }, { "Subject ID":"SGU009", "Sample ID":"NTM9BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"50","Gender":"M","Race":"N/A","Medical History":"Severe diabetes, hypercholesterolemia, below knee amputation, spinal stenosis S/P surgery, arterial peripheral bypass, free fibula graft, hip replacement, S/P colostomy, prior seizures due to high blood sugar."} }, { "Subject ID":"SGU010", "Sample ID":"NTM10BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"57","Gender":"M","Race":"W","Medical History":"Thyroid cancer, tracheostomy, PEG tube, Thyroidectomy"} }, { "Subject ID":"SGU011", "Sample ID":"NTM11BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"60","Gender":"M","Race":"N/A","Medical History":"Dyslipidemia, Diabetes, hypertension"} }, { "Subject ID":"SGU012", "Sample ID":"NTM12BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"68","Gender":"M","Race":"B","Medical History":"Prostate cancer, COPD"} }, { "Subject ID":"SGU013", "Sample ID":"NTM13BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"67","Gender":"M","Race":"W","Medical History":"CAD. CABG, HTN, Hypercholesterolemia, Prostate, CA, HIP FX, Illeostomy, Keratosis, Rhinitis"} }, { "Subject ID":"SGU014", "Sample ID":"NTM14BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"59","Gender":"M","Race":"W","Medical History":"Hypertension, high cholesterol"} }, { "Subject ID":"SGU015", "Sample ID":"NTM15BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"54","Gender":"M","Race":"W","Medical History":"Hypertension, Cancer"} }, { "Subject ID":"SGU016", "Sample ID":"NTM16BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"64","Gender":"M","Race":"W","Medical History":"Appendectomy"} }, { "Subject ID":"SGU017", "Sample ID":"NTM17BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"55","Gender":"M","Race":"W","Medical History":"HTN, Renal failure, hypothyroidism, hypercholesterolemia, Chronic obstructive pulmonary disease (COPD), S/P ankle fx repair"} }, { "Subject ID":"SGU018", "Sample ID":"NTM18BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"35","Gender":"F","Race":"W","Medical History":"None recorded"} }, { "Subject ID":"SGU019", "Sample ID":"NTM19BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"74","Gender":"F","Race":"W","Medical History":"MI, HTN, hypercholesterolemia, CHF, Uterine CA, DM, CKD, right great toe amputation, hysterectomy"} }, { "Subject ID":"SGU020", "Sample ID":"NTM20BL1", "Factors":{"Condition":"Control"}, "Additional sample data":{"Age":"52","Gender":"F","Race":"W","Medical History":"DM, HTN, Chronic lymphedema, bilateral cellulitis on legs, emphysema, morbid obesity"} }, { "Subject ID":"SGU031", "Sample ID":"GTM1BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"81","Gender":"F","Race":"W","Medical History":"MMC,beta blocker, PG, steroid, implant"} }, { "Subject ID":"SGU032", "Sample ID":"GTM2BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"72","Gender":"M","Race":"W","Medical History":"Cosopt, PG"} }, { "Subject ID":"SGU033", "Sample ID":"GTM3BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"76","Gender":"M","Race":"B","Medical History":"Cosopt, PG"} }, { "Subject ID":"SGU034", "Sample ID":"GTM4BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"84","Gender":"M","Race":"W","Medical History":"brimonidine, Cosopt, PG, steriod, implant"} }, { "Subject ID":"SGU035", "Sample ID":"GTM5BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"71","Gender":"F","Race":"W","Medical History":"Cosopt"} }, { "Subject ID":"SGU036", "Sample ID":"GTM6BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"78","Gender":"M","Race":"W","Medical History":"beta-blocker,brimonidine"} }, { "Subject ID":"SGU037", "Sample ID":"GTM7BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"81","Gender":"F","Race":"B","Medical History":"beta-blocker,brimonidine, PG"} }, { "Subject ID":"SGU038", "Sample ID":"GTM8BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"85","Gender":"F","Race":"W","Medical History":"brimonidine, Cosopt, PG"} }, { "Subject ID":"SGU039", "Sample ID":"GTM9BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"81","Gender":"M","Race":"W","Medical History":"beta-blocker,brimonidine"} }, { "Subject ID":"SGU040", "Sample ID":"GTM10BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"75","Gender":"F","Race":"W","Medical History":"beta-blocker, PG"} }, { "Subject ID":"SGU041", "Sample ID":"GTM11BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"59","Gender":"F","Race":"W","Medical History":"MMC,beta blocker, brimonidine, PG,"} }, { "Subject ID":"SGU042", "Sample ID":"GTM12BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"67","Gender":"F","Race":"B","Medical History":"Not known"} }, { "Subject ID":"SGU043", "Sample ID":"GTM13BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"59","Gender":"M","Race":"W","Medical History":"beta blocker, brimonidine"} }, { "Subject ID":"SGU044", "Sample ID":"GTM14BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"67","Gender":"M","Race":"W","Medical History":"MMC, Cosopt"} }, { "Subject ID":"SGU045", "Sample ID":"GTM15BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"55","Gender":"M","Race":"W","Medical History":"Cosopt"} }, { "Subject ID":"SGU046", "Sample ID":"GTM16BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"50","Gender":"M","Race":"W","Medical History":"MMC,beta-blocker, CAI, miotic, PG, steriod"} }, { "Subject ID":"SGU047", "Sample ID":"GTM17BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"57","Gender":"F","Race":"W","Medical History":"brimonidine, PG"} }, { "Subject ID":"SGU048", "Sample ID":"GTM18BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"51","Gender":"F","Race":"B","Medical History":"MMC,Cosopt, PG, implant, steroid"} }, { "Subject ID":"SGU049", "Sample ID":"GTM19BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"80","Gender":"M","Race":"W","Medical History":"Xalatan"} }, { "Subject ID":"SGU050", "Sample ID":"GTM20BL1", "Factors":{"Condition":"Glaucomatous"}, "Additional sample data":{"Age":"73","Gender":"F","Race":"W","Medical History":"Timolol, Cosopt"} } ], "COLLECTION":{"COLLECTION_SUMMARY":"The POAG and control TM were procured from cadaver donor eyes or as a surgical specimen(POAG) following institutional review board approved protocols and adherence to the tenets of the Declaration of Helsinki. The TM specimens, during transit, were stored in Optisol(Bausch & Lomb, Rochester, NY, USA) at 48C followed by storage in _808C until time of use. The donors of both sexes, and with a mean age of 56.3+/- 12.7 years for control and 70.1 +/- 11.04 years for glaucoma, were used for these studies. Tissues were sourced from Midwest Eye-Banks, Lions Eye Bank (Miami, FL, USA) and Mundorf Eye Institute (Charlotte, NC, USA). The TM tissue was isolated using published procedures."}, "TREATMENT":{"TREATMENT_SUMMARY":"No treatment was performed on the samples."}, "SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The TM tissue was subjected to an alternating cycle of freezing (-80ºC) and thawing (37ºC) for 10 minutes each for five cycles (to break the membrane and release lipids) followed by extraction of lipids using the Bligh and Dyer method. The organic phase, with extracted lipids, was dried in a Speed-Vac (Model 7810014; Labconco, Kansas City, MO, USA). Samples were flushed regularly with argon gas to prevent oxidation.The aqueous phase was subjected to protein quantification using the Bradford method. All extractions and subsequent handling were made using glass vials. A phosphatidylcholine standard (1,2-ditridecanoyl-sn-glycero-3-phosphocholine, molecular mass 649.9; Avanti Polar Lipids, Albaster, AL, USA) was added during tissue homogenization and its recovery was determined to calculate the extraction efficiency of each sample to normalize the recovery efficiency across the samples. Extracted lipids were dried and resuspended in liquid chromatography–mass spectrometry (LC-MS) grade acetonitrile:isopropanol (1:1). A triple quadrupole electrospray mass spectrometer (TSQ Quantum Access Max; Thermo Fisher Scientific, Pittsburgh, PA) was used for analysis of lipids in infusion mode using the TSQ Tune of Xcalibur 2.3 software package. Samples were infused with a flow rate of 10 μl/ min and analyzed for 1.00 min with a 0.500 s scan. Scans typically ranged from 200 m/z to 1,000 m/z unless specified otherwise. The full width at half maximum peak was set at 0.7 and collision gas pressure was set at 1 mTorr. Sheath gas (nitrogen) was set to 20 arbitrary units. Auxiliary gas (argon) was set to 5 arbitrary units. For analyses of the sphingomyelin, sphingoid base, and ceramide classes, the identifications were performed using neutral loss scan (NLS) for m/z 213.2, 48 and 256.2 with collision energies of 50, 18 and 32 V, respectively; except for ceramide (in negative ion mode), all other scans were carried out in positive mode. For sphingoid base-1-phosphate, PIS was performed for product ion m/z of 79.1 in negative ion mode at 24 V collision energy. The spray voltage, ion mode, and collision energies were based on previous studies. The analytical parameters for sphingolipids and ceramides described here are based on standardized collision energy settings as suggested in the recent literature for automated shotgun lipidomics."}, "CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"None (Direct infusion)","INSTRUMENT_NAME":"TSQ Quantum Access Max","COLUMN_NAME":"none"}, "ANALYSIS":{"ANALYSIS_TYPE":"MS"}, "MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Thermo Quantiva Access Max","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED"}, "MS_METABOLITE_DATA":{ "Units":"Peak Area", 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