Summary of Study ST000616

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 PR000450. The data can be accessed directly via it's Project DOI: 10.21228/M8WC8S This work is supported by NIH grant, U2C- DK119886.

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

Study ID	ST000616
Study Title	Human Aqueous Humor Phospholipids in Control and Glaucomatous eyes
Study Summary	An analysis of phospholipid (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol) classes in human control and glaucomatous aqueous humor (AH). Lipid extraction was performed using a modification of the Bligh and Dyer method, protein concentrations were determined using the Bradford’s method, and select samples were confirmed with Densitometry of PHAST gels. Lipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan (PIS) or neutral ion loss scan (NLS) using appropriate class specific lipid standards in a two-step quantification process. Mass spectrometer data were analyzed using MzMine 2.23.
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	2017-05-31
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:	SP000646
Sampleprep Summary:	Aqueous humor samples were subjected to extraction of lipids using suitable and minimal modification of Bligh and Dyer method. The lower organic phase containing the extracted lipids was isolated and solvent dried with a Speed-Vac (Model 7810014; Labconco, Kansas City, MO). Samples were subsequently flushed with argon gas to prevent oxidation. Proteins recovered from the corresponding upper aqueous phase were quantified using the Bradford method. A subset of protein samples were also subjected to densitometric quantification using bovine serum albumin (BSA) as a standard (amino acid quantified) after electrophoretic separation on a PHAST (GE Healthcare Bio-Sciences AB, Sweden) gel system. We repeated protein estimations using an amino acid analyzer after overnight digestion in hydrochloric acid following previously published protocols. The protein amounts determined using amino acid analyzer were utilized in normalization of lipids per amount of proteins. In order to determine and ensure extraction efficiency, ovine wool cholesterol (molecular mass 386.7; catalog no. 700000; Avanti Polar Lipids, Albaster, AL) was premixed with AH prior to extraction. All extractions and subsequent handling were made using glass vials to avoid contaminating impurities. The quantification lipid standards (all procured from Avanti Polar Lipids, Albaster, AL) we used were ditridecanoyl-sn-glycero-3-phosphocholine (molecular mass 649.89, catalog no. 850340) for PCs, 1,2-dioleoyl-snglycero- 3-phospho-L-serine (molecular mass 810.03, catalog no. 840035) for PSs, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (molecular mass 744.04, catalog no. 850725) for PEs and 1,2-dioleoyl-sn-glycero-3-phospho-(10-myo-inositol) (molecular mass 880.15, catalog no. 850149) for PIs.

Sampleprep ID:

SP000646

Sampleprep Summary:

Aqueous humor samples were subjected to extraction of lipids using suitable and minimal modification of Bligh and Dyer method. The lower organic phase containing the extracted lipids was isolated and solvent dried with a Speed-Vac (Model 7810014; Labconco, Kansas City, MO). Samples were subsequently flushed with argon gas to prevent oxidation. Proteins recovered from the corresponding upper aqueous phase were quantified using the Bradford method. A subset of protein samples were also subjected to densitometric quantification using bovine serum albumin (BSA) as a standard (amino acid quantified) after electrophoretic separation on a PHAST (GE Healthcare Bio-Sciences AB, Sweden) gel system. We repeated protein estimations using an amino acid analyzer after overnight digestion in hydrochloric acid following previously published protocols. The protein amounts determined using amino acid analyzer were utilized in normalization of lipids per amount of proteins. In order to determine and ensure extraction efficiency, ovine wool cholesterol (molecular mass 386.7; catalog no. 700000; Avanti Polar Lipids, Albaster, AL) was premixed with AH prior to extraction. All extractions and subsequent handling were made using glass vials to avoid contaminating impurities. The quantification lipid standards (all procured from Avanti Polar Lipids, Albaster, AL) we used were ditridecanoyl-sn-glycero-3-phosphocholine (molecular mass 649.89, catalog no. 850340) for PCs, 1,2-dioleoyl-snglycero- 3-phospho-L-serine (molecular mass 810.03, catalog no. 840035) for PSs, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (molecular mass 744.04, catalog no. 850725) for PEs and 1,2-dioleoyl-sn-glycero-3-phospho-(10-myo-inositol) (molecular mass 880.15, catalog no. 850149) for PIs.