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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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:	SU000635
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
SA033857	NAH16BL3	Control
SA033858	NAH14BL3	Control
SA033859	NAH13BL3	Control
SA033860	NAH17BL2	Control
SA033861	NAH19BL3	Control
SA033862	NAH1BL3	Control
SA033863	NAH20BL3	Control
SA033864	NAH12BL3	Control
SA033865	NAH18BL2	Control
SA033866	NAH15BL3	Control
SA033867	NAH5BL3	Control
SA033868	NAH4BL3	Control
SA033869	NAH11BL4	Control
SA033870	NAH2BL3	Control
SA033871	NAH6BL3	Control
SA033872	NAH3BL3	Control
SA033873	NAH7BL4	Control
SA033874	NAH10BL3	Control
SA033875	NAH9BL3	Control
SA033876	NAH8BL3	Control
SA033877	GAH15BL3	Glaucomatous
SA033878	GAH14BL3	Glaucomatous
SA033879	GAH13BL3	Glaucomatous
SA033880	GAH16BL3	Glaucomatous
SA033881	GAH19BL3	Glaucomatous
SA033882	GAH12BL3	Glaucomatous
SA033883	GAH20BL3	Glaucomatous
SA033884	GAH18BL3	Glaucomatous
SA033885	GAH17BL4	Glaucomatous
SA033886	GAH1BL3	Glaucomatous
SA033887	GAH5BL3	Glaucomatous
SA033888	GAH4BL3	Glaucomatous
SA033889	GAH3BL3	Glaucomatous
SA033890	GAH2BL3	Glaucomatous
SA033891	GAH6BL3	Glaucomatous
SA033892	GAH7BL4	Glaucomatous
SA033893	GAH10BL3	Glaucomatous
SA033894	GAH9BL3	Glaucomatous
SA033895	GAH8BL3	Glaucomatous
SA033896	GAH11BL3	Glaucomatous

Showing results 1 to 40 of 40

Showing results 1 to 40 of 40

Collection:

Collection ID:	CO000629
Collection Summary:	Aqueous humor from POAG and control human subjects (n=20 each totaling 40 samples) were procured following institutional review board approved protocols and adhering to the tenets of the Declaration of Helsinki. AH samples were stored at −80 °C until time of use. The mean age of donors was 71.2 +/- 10.2 and both genders were included in these studies.
Sample Type:	Eye tissue

Treatment:

Treatment ID:	TR000649
Treatment Summary:	No treatment was applied to the groups.

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.

Combined analysis:

Analysis ID	AN000937
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:	CH000668
Instrument Name:	TSQ Quantum Access Max
Column Name:	none
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS000833
Analysis ID:	AN000937
Instrument Name:	Thermo Quantiva Access Max
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	UNSPECIFIED