Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA033931

Local Sample ID:	GTM6BL1
Subject ID:	SU000636
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000636
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
GTM6BL1	SA033931	FL007465	Glaucomatous	Condition

Collection:

Collection ID:	CO000630
Collection Summary:	The POAG and control TM were procured from cadaver donor eyes or as a surgical specimen(POAG) following institutional review board approved protocols and adherence to the tenets of the Declaration of Helsinki. The TM specimens, during transit, were stored in Optisol(Bausch & Lomb, Rochester, NY, USA) at 48C followed by storage in _808C until time of use. The donors of both sexes, and with a mean age of 56.3+/- 12.7 years for control and 70.1 +/- 11.04 years for glaucoma, were used for these studies. Tissues were sourced from Midwest Eye-Banks, Lions Eye Bank (Miami, FL, USA) and Mundorf Eye Institute (Charlotte, NC, USA). The TM tissue was isolated using published procedures.
Sample Type:	Eye tissue

Treatment:

Treatment ID:	TR000650
Treatment Summary:	No treatment was performed on the samples.

Sample Preparation:

Sampleprep ID:	SP000643
Sampleprep Summary:	The TM tissue was subjected to an alternating cycle of freezing (-80ºC) and thawing (37ºC) for 10 minutes each for five cycles (to break the membrane and release lipids) followed by extraction of lipids using the Bligh and Dyer method. The organic phase, with extracted lipids, was dried in a Speed-Vac (Model 7810014; Labconco, Kansas City, MO, USA). Samples were flushed regularly with argon gas to prevent oxidation.The aqueous phase was subjected to protein quantification using the Bradford method. All extractions and subsequent handling were made using glass vials. A phosphatidylcholine standard (1,2-ditridecanoyl-sn-glycero-3-phosphocholine, molecular mass 649.9; Avanti Polar Lipids, Albaster, AL, USA) was added during tissue homogenization and its recovery was determined to calculate the extraction efficiency of each sample to normalize the recovery efficiency across the samples. Extracted lipids were dried and resuspended in liquid chromatography–mass spectrometry (LC-MS) grade acetonitrile:isopropanol (1:1). A triple quadrupole electrospray mass spectrometer (TSQ Quantum Access Max; Thermo Fisher Scientific, Pittsburgh, PA) was used for analysis of lipids in infusion mode using the TSQ Tune of Xcalibur 2.3 software package. Samples were infused with a flow rate of 10 μl/ min and analyzed for 1.00 min with a 0.500 s scan. Scans typically ranged from 200 m/z to 1,000 m/z unless specified otherwise. The full width at half maximum peak was set at 0.7 and collision gas pressure was set at 1 mTorr. Sheath gas (nitrogen) was set to 20 arbitrary units. Auxiliary gas (argon) was set to 5 arbitrary units. For analyses of the sphingomyelin, sphingoid base, and ceramide classes, the identifications were performed using neutral loss scan (NLS) for m/z 213.2, 48 and 256.2 with collision energies of 50, 18 and 32 V, respectively; except for ceramide (in negative ion mode), all other scans were carried out in positive mode. For sphingoid base-1-phosphate, PIS was performed for product ion m/z of 79.1 in negative ion mode at 24 V collision energy. The spray voltage, ion mode, and collision energies were based on previous studies. The analytical parameters for sphingolipids and ceramides described here are based on standardized collision energy settings as suggested in the recent literature for automated shotgun lipidomics.

Sampleprep ID:

SP000643

Sampleprep Summary:

The TM tissue was subjected to an alternating cycle of freezing (-80ºC) and thawing (37ºC) for 10 minutes each for five cycles (to break the membrane and release lipids) followed by extraction of lipids using the Bligh and Dyer method. The organic phase, with extracted lipids, was dried in a Speed-Vac (Model 7810014; Labconco, Kansas City, MO, USA). Samples were flushed regularly with argon gas to prevent oxidation.The aqueous phase was subjected to protein quantification using the Bradford method. All extractions and subsequent handling were made using glass vials. A phosphatidylcholine standard (1,2-ditridecanoyl-sn-glycero-3-phosphocholine, molecular mass 649.9; Avanti Polar Lipids, Albaster, AL, USA) was added during tissue homogenization and its recovery was determined to calculate the extraction efficiency of each sample to normalize the recovery efficiency across the samples. Extracted lipids were dried and resuspended in liquid chromatography–mass spectrometry (LC-MS) grade acetonitrile:isopropanol (1:1). A triple quadrupole electrospray mass spectrometer (TSQ Quantum Access Max; Thermo Fisher Scientific, Pittsburgh, PA) was used for analysis of lipids in infusion mode using the TSQ Tune of Xcalibur 2.3 software package. Samples were infused with a flow rate of 10 μl/ min and analyzed for 1.00 min with a 0.500 s scan. Scans typically ranged from 200 m/z to 1,000 m/z unless specified otherwise. The full width at half maximum peak was set at 0.7 and collision gas pressure was set at 1 mTorr. Sheath gas (nitrogen) was set to 20 arbitrary units. Auxiliary gas (argon) was set to 5 arbitrary units. For analyses of the sphingomyelin, sphingoid base, and ceramide classes, the identifications were performed using neutral loss scan (NLS) for m/z 213.2, 48 and 256.2 with collision energies of 50, 18 and 32 V, respectively; except for ceramide (in negative ion mode), all other scans were carried out in positive mode. For sphingoid base-1-phosphate, PIS was performed for product ion m/z of 79.1 in negative ion mode at 24 V collision energy. The spray voltage, ion mode, and collision energies were based on previous studies. The analytical parameters for sphingolipids and ceramides described here are based on standardized collision energy settings as suggested in the recent literature for automated shotgun lipidomics.

Combined analysis:

Analysis ID	AN000938
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	TSQ Quantum Access Max
Column	none
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Thermo Quantiva Access Max
Ion Mode	UNSPECIFIED
Units	Peak Area

Chromatography:

Chromatography ID:	CH000669
Instrument Name:	TSQ Quantum Access Max
Column Name:	none
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS000834
Analysis ID:	AN000938
Instrument Name:	Thermo Quantiva Access Max
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	UNSPECIFIED