Summary of Study ST000612

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	ST000612
Study Title	Sphingolipid Analysis of Human Aqueous Humor in Glaucomatous and Control eyes (part I)
Study Summary	This study examined the profiles of sphin¬golipids and ceramides present in the aqueous humor (AH) of human control and POAG donors. Furthermore, we quantitatively compared distinct differences between glaucomatous and age-matched control eyes, identifying potential molecules for further experimentation to determine their biological role in modulating cell behavior. Lipids were identified and ratiometrically quantified in a two-step process using precursor ion scan (PIS) or neutral loss scan (NLS) with appropriate class-specific lipid standards on a TSQ Quantum Access Max mass spectrometer following established procedures. We identified several species of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramides that were common between control and glaucomatous AQH. Some unique lipid species in these classes were also identified in controls but not in glaucoma and vice versa.
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
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:	SP000642
Sampleprep Summary:	Aqueous humor was subjected to lipid extraction using a modified Bligh and Dyer method. Prior to extraction, a fixed amount of a standard (1,2-ditridecanoyl-sn-glycero-3-phosphocholine) was added to determine lipid recovery, ensuring >99% and uniform recovery across all samples analyzed. The organic phase with extracted lipids was flushed with argon gas to prevent oxidation and dried in a Speed-Vac (Model 5301, Brinkmann Instruments Inc., Westbury, NY). The aqueous phase containing proteins was stored at −80 °C for protein quantification. All extractions and subsequent handling were carried out using glass vials to a void contamination from plastics. 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:

SP000642

Sampleprep Summary:

Aqueous humor was subjected to lipid extraction using a modified Bligh and Dyer method. Prior to extraction, a fixed amount of a standard (1,2-ditridecanoyl-sn-glycero-3-phosphocholine) was added to determine lipid recovery, ensuring >99% and uniform recovery across all samples analyzed. The organic phase with extracted lipids was flushed with argon gas to prevent oxidation and dried in a Speed-Vac (Model 5301, Brinkmann Instruments Inc., Westbury, NY). The aqueous phase containing proteins was stored at −80 °C for protein quantification. All extractions and subsequent handling were carried out using glass vials to a void contamination from plastics. 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.