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MB Sample ID: SA093315
Local Sample ID: | S0008_POS |
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 |
---|---|---|---|---|
S0008_POS | SA093315 | 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). |
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 |