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MB Sample ID: SA192496

Local Sample ID:	repRGC_over_NN
Subject ID:	SU002129
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

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Sample Preparation:

Sampleprep ID:	SP002135
Sampleprep Summary:	15 µg of total protein from homogenate sample were added to 4 times the volume of acetone at 20°C and incubated at 20°C overnight. Samples were centrifuged at 21,000 g at 4°C for 30 minutes (Thermo Fisher Megafuge 8R). Supernatant was discarded and pellet (very small) was air dried for 10 minutes. The pellet was re-suspended, denatured and reduced with 6 M urea, 10 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature. Following denaturing and reduction, the re-suspended pellet was alkylated with 15 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 minutes while maintained in darkness. Alkylation reaction was quench using 20 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature while kept in darkness. Sample was diluted using 50 mM ammonium bicarbonate to contain 1 M urea. Sample was digested using either trypsin or chymotrypsin (Promega, V5111, V106A) at a 1:30 (w/w) ratio of enzyme to protein. Digestion was incubated overnight at 37°C. Reaction was terminated using 50% formic acid at a 5:100 (v/v) ratio of formic acid to sample volume. Samples were stored at -20°C or immediately desalted. Samples were desalted using the Pierce Graphite Spin Columns (Thermo, 88302) following manufacturer’s recommendations. Samples were then evaporated using the CentriVap Concentrator system (Labconco, Kansas City, MO 64132-2696) and resuspended in 30 µl of protein resuspension solution [2% (v/v) acetonitrile, 0.1% (v/v) formic acid in mass spectrometry grade water]. Lipids were harvested by adding 400 µl of 1:1 v/v methanol/chloroform mix to 200 µl of cell culture lysate. Lysate was vortexed and incubated in ice for 5 minutes, followed by the addition of 350 µl of chloroform. Lysate was centrifuged at 18,000g for 15 minutes at 4ºC. The organic layer containing lipids (bottom layer) was transferred to a new tube and desiccated using the CentriVap Concentrator system. Lipids were resuspended in 50 µl of lipid resuspension solution [50% v/v isopropyl alcohol and 50% v/v acetonitrile].

Sampleprep ID:

SP002135

Sampleprep Summary:

15 µg of total protein from homogenate sample were added to 4 times the volume of acetone at 20°C and incubated at 20°C overnight. Samples were centrifuged at 21,000 g at 4°C for 30 minutes (Thermo Fisher Megafuge 8R). Supernatant was discarded and pellet (very small) was air dried for 10 minutes. The pellet was re-suspended, denatured and reduced with 6 M urea, 10 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature. Following denaturing and reduction, the re-suspended pellet was alkylated with 15 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 minutes while maintained in darkness. Alkylation reaction was quench using 20 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature while kept in darkness. Sample was diluted using 50 mM ammonium bicarbonate to contain 1 M urea. Sample was digested using either trypsin or chymotrypsin (Promega, V5111, V106A) at a 1:30 (w/w) ratio of enzyme to protein. Digestion was incubated overnight at 37°C. Reaction was terminated using 50% formic acid at a 5:100 (v/v) ratio of formic acid to sample volume. Samples were stored at -20°C or immediately desalted. Samples were desalted using the Pierce Graphite Spin Columns (Thermo, 88302) following manufacturer’s recommendations. Samples were then evaporated using the CentriVap Concentrator system (Labconco, Kansas City, MO 64132-2696) and resuspended in 30 µl of protein resuspension solution [2% (v/v) acetonitrile, 0.1% (v/v) formic acid in mass spectrometry grade water]. Lipids were harvested by adding 400 µl of 1:1 v/v methanol/chloroform mix to 200 µl of cell culture lysate. Lysate was vortexed and incubated in ice for 5 minutes, followed by the addition of 350 µl of chloroform. Lysate was centrifuged at 18,000g for 15 minutes at 4ºC. The organic layer containing lipids (bottom layer) was transferred to a new tube and desiccated using the CentriVap Concentrator system. Lipids were resuspended in 50 µl of lipid resuspension solution [50% v/v isopropyl alcohol and 50% v/v acetonitrile].