#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