#METABOLOMICS WORKBENCH sbhattacharya_20191114_124121 DATATRACK_ID:1845 STUDY_ID:ST001278 ANALYSIS_ID:AN002119 PROJECT_ID:PR000863 VERSION 1 CREATED_ON November 18, 2019, 3:10 pm #PROJECT PR:PROJECT_TITLE POAG and Control Aqueous Humor IROA Metabolites from Veterans Affairs Patients PR:PROJECT_TYPE untargeted LC-MS/MS IROA metabolomics PR:PROJECT_SUMMARY We performed high-performance liquid chromatography-tandem mass spectrometry PR:PROJECT_SUMMARY (LC-MS/MS) and Isotopic Ratio Outlier Analysis (IROA) data analysis on aqueous PR:PROJECT_SUMMARY humor patient samples. Samples were collected at asynchronous times. PR:INSTITUTE University of Miami PR:DEPARTMENT Ophthalmology, Bascom Palmer Eye Institute PR:LABORATORY Sanjoy K. Bhattacharya PR:LAST_NAME Bhattacharya PR:FIRST_NAME Sanjoy PR:ADDRESS 1638 NW 10th Avenue, Miami, Florida, 33136, USA PR:EMAIL sbhattacharya@med.miami.edu PR:PHONE 3054824103 PR:CONTRIBUTORS Leila Abdelrahman #STUDY ST:STUDY_TITLE POAG and Control Aqueous Humor IROA Metabolites from Veterans Affairs Patients ST:STUDY_TYPE untargeted LC-MS/MS metabolomics IROA ST:STUDY_SUMMARY We performed high-performance liquid chromatography-tandem mass spectrometry ST:STUDY_SUMMARY (LC-MS/MS) analysis of the metabolome of POAG and Control patients. Samples were ST:STUDY_SUMMARY collected asynchronously. ST:INSTITUTE University of Miami ST:DEPARTMENT Ophthalmology, Bascom Palmer Eye Institute ST:LABORATORY Sanjoy K. Bhattacharya ST:LAST_NAME Bhattacharya ST:FIRST_NAME Sanjoy ST:ADDRESS 1638 NW 10th Avenue, Miami, FL 33136 ST:EMAIL SBhattacharya@med.miami.edu ST:PHONE 305-482-4103 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 41 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - S0001_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G27_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0002_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G29_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0003_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G30_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0004_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G31_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0005_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G33_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0006_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G34_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0007_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G35_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0008_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G36_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0009_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G38_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0010_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G39_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0011_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G40_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0012_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G41_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0013_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G42_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0014_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G43_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0015_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G44_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0016_NEG Factor:POAG TQ sample type=Sample; Acquisition method=AHIROA2; Data file=G45_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0017_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N12_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0018_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N14_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0019_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N19_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0020_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N28_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0021_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N29_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0022_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N30_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0023_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N4_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0024_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N55_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0025_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N57_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0026_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N58_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0027_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N59_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0028_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N5_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0029_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N60_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0030_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N61_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0031_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N62_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0032_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N64_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0033_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N65_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0034_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N66_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0035_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N67_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0036_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N68_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0037_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N69_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0038_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N70_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0039_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N72_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0040_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N73_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0041_NEG Factor:Control TQ sample type=Sample; Acquisition method=AHIROA2; Data file=N75_neg_IS.raw SUBJECT_SAMPLE_FACTORS - S0001_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G27_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0002_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G29_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0003_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G30_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0004_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G31_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0005_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G33_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0006_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G34_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0007_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G35_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0008_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G36_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0009_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G38_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0010_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G39_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0011_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G40_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0012_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G41_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0013_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G42_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0014_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G43_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0015_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G44_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0016_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=G45_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0017_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N12_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0018_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N14_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0019_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N19_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0020_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N28_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0021_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N29_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0022_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N30_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0023_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N4_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0024_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N55_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0025_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N57_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0026_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N58_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0027_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N59_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0028_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N5_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0029_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N60_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0030_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N61_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0031_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N62_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0032_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N64_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0033_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N65_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0034_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N66_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0035_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N67_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0036_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N68_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0037_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N69_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0038_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N70_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0039_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N72_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0040_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N73_pos_IS.raw SUBJECT_SAMPLE_FACTORS - S0041_POS Factor:Control TQ sample type=Sample; Acquisition method=AHIROAms2; Data file=N75_pos_IS.raw #COLLECTION CO:COLLECTION_SUMMARY Aqueous Humor samples of VA patients with POAG and Control were taken. CO:SAMPLE_TYPE Eye tissue CO:COLLECTION_METHOD All materials were collected from human donors without identifiers under CO:COLLECTION_METHOD institutional review board exemption/approval. We acquired 58 aqueous humor (AH) CO:COLLECTION_METHOD samples from participants at the Veterans Administration (VA) Medical Center CO:COLLECTION_METHOD (Miami, FL). Patient samples used for 1H-Nuclear Magnetic Resonance (NMR) and CO:COLLECTION_METHOD Isotopic Ratio Outlier Analysis (IROA) consisted of individuals with POAG (n=23) CO:COLLECTION_METHOD and non-POAG controls (n=35). #TREATMENT TR:TREATMENT_SUMMARY No Treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The 41 samples were prepared for isotopic ratio outlier analysis (IROA). SP:SAMPLEPREP_SUMMARY Approximately 800 µL of precipitate solution (8:1:1 Acetonitrile: Methanol: SP:SAMPLEPREP_SUMMARY Acetone) was added to the 50 µL of aqueous humor. The metabolites were vortexed SP:SAMPLEPREP_SUMMARY and incubated at 4 °C for 30 minutes. This was followed by another incubation SP:SAMPLEPREP_SUMMARY period at -20 °C for 1 hour. Each sample was centrifuged at 20,000 x g for 10 SP:SAMPLEPREP_SUMMARY minutes at 4 °C (Beckman Microfuge 18) to form a pellet and 375 µL of SP:SAMPLEPREP_SUMMARY supernatant was collected. The supernatant was dried in a speed vacuum for SP:SAMPLEPREP_SUMMARY approximately 20 minutes or until the sample was fully dry and stored at -20 SP:SAMPLEPREP_SUMMARY °C. The IROA internal standard (IROA-IS, IROA technologies) (U-95% 13C) was SP:SAMPLEPREP_SUMMARY reconstituted in 1.2 mL of LC-MS grade water. The samples were reconstituted in SP:SAMPLEPREP_SUMMARY 25 µL of LC-MS grade water and 10 µL was combined with 20 µL of IROA-IS and SP:SAMPLEPREP_SUMMARY subjected to LC-MS/MS analysis. A long-term reference standard (LTRS) was SP:SAMPLEPREP_SUMMARY reconstituted in 50 µL of LC-MS grade water and subjected to LC-MS/MS as the SP:SAMPLEPREP_SUMMARY first, last, and every 10 samples. Metabolite identification and relative SP:SAMPLEPREP_SUMMARY quantification were performed using Clusterfinder Build 3.1.10 (IROA SP:SAMPLEPREP_SUMMARY Technologies). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Accela 600 CH:COLUMN_NAME ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) CH:FLOW_GRADIENT s 0-40% solvent B over 10 minutes, 40% solvent B over 10-12 minutes, 60% solvent CH:FLOW_GRADIENT B over 12-14 minutes, 95% solvent B over 14-20 minutes,100% solvent B over 20-30 CH:FLOW_GRADIENT minutes CH:FLOW_RATE 350 ul/min CH:COLUMN_TEMPERATURE 40 C CH:SOLVENT_A Water with 0.1% formic acid (FA) CH:SOLVENT_B Acetonitrile with 0.1 % FA #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Both Positive and Negative Ion Mode Metabolite identification and relative MS:MS_COMMENTS quantification were performed using Clusterfinder Build 3.1.10 (IROA MS:MS_COMMENTS Technologies). Thermo raw files were converted into mzxml files before MS:MS_COMMENTS importation into the Clusterfinder software. The manufacturer protocols were MS:MS_COMMENTS followed to recognize IROA peak pairs and determine molecular formulas. MS:MS_COMMENTS Metabolites were identified by comparing retention time, molecular formula and MS:MS_COMMENTS molecular ion m/z with the Mass Spectrometry Metabolite Library of Standards MS:MS_COMMENTS (MSMLS, IROA technologies). MS:MS_RESULTS_FILE ST001278_AN002119_Results.txt UNITS:C12:C13 Ratio Has m/z:Yes Has RT:Yes RT units:Minutes #END