Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA093346

Local Sample ID:	S0018_NEG
Subject ID:	SU001350
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU001350
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
S0018_NEG	SA093346	FL013366	Control	Factor

Collection:

Collection ID:	CO001344
Collection Summary:	Aqueous Humor samples of VA patients with POAG and Control were taken.
Sample Type:	Eye tissue
Collection Method:	All materials were collected from human donors without identifiers under institutional review board exemption/approval. We acquired 58 aqueous humor (AH) samples from participants at the Veterans Administration (VA) Medical Center (Miami, FL). Patient samples used for 1H-Nuclear Magnetic Resonance (NMR) and Isotopic Ratio Outlier Analysis (IROA) consisted of individuals with POAG (n=23) and non-POAG controls (n=35).

Treatment:

Treatment ID:	TR001365
Treatment Summary:	No Treatment

Sample Preparation:

Sampleprep ID:	SP001358
Sampleprep Summary:	The 41 samples were prepared for isotopic ratio outlier analysis (IROA). Approximately 800 µL of precipitate solution (8:1:1 Acetonitrile: Methanol: Acetone) was added to the 50 µL of aqueous humor. The metabolites were vortexed and incubated at 4 °C for 30 minutes. This was followed by another incubation period at -20 °C for 1 hour. Each sample was centrifuged at 20,000 x g for 10 minutes at 4 °C (Beckman Microfuge 18) to form a pellet and 375 µL of supernatant was collected. The supernatant was dried in a speed vacuum for approximately 20 minutes or until the sample was fully dry and stored at -20 °C. The IROA internal standard (IROA-IS, IROA technologies) (U-95% 13C) was reconstituted in 1.2 mL of LC-MS grade water. The samples were reconstituted in 25 µL of LC-MS grade water and 10 µL was combined with 20 µL of IROA-IS and subjected to LC-MS/MS analysis. A long-term reference standard (LTRS) was reconstituted in 50 µL of LC-MS grade water and subjected to LC-MS/MS as the first, last, and every 10 samples. Metabolite identification and relative quantification were performed using Clusterfinder Build 3.1.10 (IROA Technologies).

Sampleprep ID:

SP001358

Sampleprep Summary:

The 41 samples were prepared for isotopic ratio outlier analysis (IROA). Approximately 800 µL of precipitate solution (8:1:1 Acetonitrile: Methanol: Acetone) was added to the 50 µL of aqueous humor. The metabolites were vortexed and incubated at 4 °C for 30 minutes. This was followed by another incubation period at -20 °C for 1 hour. Each sample was centrifuged at 20,000 x g for 10 minutes at 4 °C (Beckman Microfuge 18) to form a pellet and 375 µL of supernatant was collected. The supernatant was dried in a speed vacuum for approximately 20 minutes or until the sample was fully dry and stored at -20 °C. The IROA internal standard (IROA-IS, IROA technologies) (U-95% 13C) was reconstituted in 1.2 mL of LC-MS grade water. The samples were reconstituted in 25 µL of LC-MS grade water and 10 µL was combined with 20 µL of IROA-IS and subjected to LC-MS/MS analysis. A long-term reference standard (LTRS) was reconstituted in 50 µL of LC-MS grade water and subjected to LC-MS/MS as the first, last, and every 10 samples. Metabolite identification and relative quantification were performed using Clusterfinder Build 3.1.10 (IROA Technologies).

Combined analysis:

Analysis ID	AN002119
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Accela 600
Column	ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	C12:C13 Ratio

Chromatography:

Chromatography ID:	CH001552
Instrument Name:	Thermo Accela 600
Column Name:	ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)
Column Temperature:	40 C
Flow Gradient:	s 0-40% solvent B over 10 minutes, 40% solvent B over 10-12 minutes, 60% solvent B over 12-14 minutes, 95% solvent B over 14-20 minutes,100% solvent B over 20-30 minutes
Flow Rate:	350 ul/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001974
Analysis ID:	AN002119
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Both Positive and Negative Ion Mode Metabolite identification and relative quantification were performed using Clusterfinder Build 3.1.10 (IROA Technologies). Thermo raw files were converted into mzxml files before importation into the Clusterfinder software. The manufacturer protocols were followed to recognize IROA peak pairs and determine molecular formulas. Metabolites were identified by comparing retention time, molecular formula and molecular ion m/z with the Mass Spectrometry Metabolite Library of Standards (MSMLS, IROA technologies).
Ion Mode:	UNSPECIFIED