Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA033876

Local Sample ID:	NAH8BL3
Subject ID:	SU000635
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Select appropriate tab below to view additional metadata details:

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.