Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA093255

Local Sample ID:	S0031_POS
Subject ID:	SU001349
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU001349
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
S0031_POS	SA093255	FL013365	PXE	Treatment1

Collection:

Collection ID:	CO001343
Collection Summary:	All materials were collected from human donors without identifiers under institutional review board exemption/approval. We acquired 31 aqueous humor (AH) samples from participants at the Veterans Administration (VA) Medical Center (Miami, FL). Patient samples used for Isotopic Ratio Outlier Analysis (IROA) consisted of individuals with PEX (n=31).
Sample Type:	Eye tissue
Collection Location:	1201 NW 16th St, Miami, FL 33125

Treatment:

Treatment ID:	TR001364
Treatment Summary:	No Treatment

Sample Preparation:

Sampleprep ID:	SP001357
Sampleprep Summary:	The 31 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.

Sampleprep ID:

SP001357

Sampleprep Summary:

The 31 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.

Combined analysis:

Analysis ID	AN002118
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:	CH001551
Instrument Name:	Thermo Accela 600
Column Name:	ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)
Column Temperature:	40 C
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:	MS001973
Analysis ID:	AN002118
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were ran in positive and negative ion mode Data dependent MS/MS
Ion Mode:	UNSPECIFIED