Summary of Study ST000613

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000448. The data can be accessed directly via it's Project DOI: 10.21228/M84W37 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST000613
Study Title	Human TM Sphingolipid Analysis (part II)
Study Summary	We determined the profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide, and their quantitative differences between control and glaucomatous trabecular meshwork (TM) derived from human donors.
Institute	University of Miami
Last Name	Bhattacharya
First Name	Sanjoy
Address	McKnight Vision Research Building, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 1638 NW 10th Avenue, Room 706A, Miami, Florida 33136
Email	Sbhattacharya@med.miami.edu
Phone	305-482-4103
Submit Date	2017-05-26
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	MS(Dir. Inf.)
Release Date	2019-07-17
Release Version	1

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Project:

Project ID:	PR000448
Project DOI:	doi: 10.21228/M84W37
Project Title:	Sphingolipid Profiling of Human Aqueous Humor and Trabecular Meshwork in Glaucomatous and Control eyes
Project Summary:	Using a triple quadrupole mass spectrometer and direct infusion lipidomics we identified various sphingolipids present in both the aqueous humor and trabecular meshwork of the human eye and compared them between glaucomatous and control groups.
Institute:	University of Miami
Last Name:	Bhattacharya
First Name:	Sanjoy
Address:	1638 NW 10th Avenue, Miami, Florida, 33136, USA
Email:	sbhattacharya@med.miami.edu
Phone:	3054824103

Subject:

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

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Condition
SA033897	NTM16BL1	Control
SA033898	NTM14BL1	Control
SA033899	NTM13BL1	Control
SA033900	NTM17BL1	Control
SA033901	NTM19BL1	Control
SA033902	NTM1BL1	Control
SA033903	NTM20BL1	Control
SA033904	NTM12BL1	Control
SA033905	NTM18BL1	Control
SA033906	NTM15BL1	Control
SA033907	NTM5BL1	Control
SA033908	NTM4BL1	Control
SA033909	NTM11BL1	Control
SA033910	NTM2BL1	Control
SA033911	NTM6BL1	Control
SA033912	NTM3BL1	Control
SA033913	NTM7BL1	Control
SA033914	NTM10BL1	Control
SA033915	NTM9BL1	Control
SA033916	NTM8BL1	Control
SA033917	GTM15BL1	Glaucomatous
SA033918	GTM14BL1	Glaucomatous
SA033919	GTM13BL1	Glaucomatous
SA033920	GTM16BL1	Glaucomatous
SA033921	GTM19BL1	Glaucomatous
SA033922	GTM12BL1	Glaucomatous
SA033923	GTM20BL1	Glaucomatous
SA033924	GTM18BL1	Glaucomatous
SA033925	GTM17BL1	Glaucomatous
SA033926	GTM1BL1	Glaucomatous
SA033927	GTM5BL1	Glaucomatous
SA033928	GTM4BL1	Glaucomatous
SA033929	GTM3BL1	Glaucomatous
SA033930	GTM2BL1	Glaucomatous
SA033931	GTM6BL1	Glaucomatous
SA033932	GTM7BL1	Glaucomatous
SA033933	GTM10BL1	Glaucomatous
SA033934	GTM9BL1	Glaucomatous
SA033935	GTM8BL1	Glaucomatous
SA033936	GTM11BL1	Glaucomatous

Showing results 1 to 40 of 40

Showing results 1 to 40 of 40

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