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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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.