Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA242289

Local Sample ID:	33_post
Subject ID:	SU002509
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Sample Preparation:

Sampleprep ID:	SP002515
Sampleprep Summary:	Serum samples were thawed on ice and centrifuged at 2000 g for 10 min to remove particulates and debris. Molecular weight cut-off filters (MWCO, 3 kDa, Millipore Amicon Ultra, Burlington, MA) were prerinsed 3 times with optima water at 14000 g for 20 min. Twenty microliter serum supernatant from each sample was diluted in 380 µl water and added to the filter then centrifuged for 20 min at 14000 g. Flowthrough was collected. Then 400 µL water was added to the filter to rinse the sample. An additional rinse step was applied. All the flowthrough (about 1.2 mL) was combined and dried down in Speedvac and stored at −20 °C until labeling. For normalization, a pooled serum sample from screening normal individuals was also prepared. For labeling of serum samples, 40 µL activated DiLeu reagents were mixed with serum flowthrough dissolved in 10 µL 0.5 M TEAB and shaken for 2 h. After the reaction was quenched, 2.5 µL of each reaction mixture with different DiLeu tags were combined at 1: 1: 1: 1 ratio and mixed well. To compare the relative abundance of each set of 4-plex DiLeu labeled metabolites, one channel from each 4-plex combination was selected and combined with 118-DiLeu tag labeled pooled serum samples. Ten µL of the mixture was taken for drying down and desalted using SCX Ziptips. Samples were dissolved in 15 µL 0.1% FA and 3 µL was injected into LC-MS for data acquisition with 3 technical replicates.

Sampleprep ID:

SP002515

Sampleprep Summary:

Serum samples were thawed on ice and centrifuged at 2000 g for 10 min to remove particulates and debris. Molecular weight cut-off filters (MWCO, 3 kDa, Millipore Amicon Ultra, Burlington, MA) were prerinsed 3 times with optima water at 14000 g for 20 min. Twenty microliter serum supernatant from each sample was diluted in 380 µl water and added to the filter then centrifuged for 20 min at 14000 g. Flowthrough was collected. Then 400 µL water was added to the filter to rinse the sample. An additional rinse step was applied. All the flowthrough (about 1.2 mL) was combined and dried down in Speedvac and stored at −20 °C until labeling. For normalization, a pooled serum sample from screening normal individuals was also prepared. For labeling of serum samples, 40 µL activated DiLeu reagents were mixed with serum flowthrough dissolved in 10 µL 0.5 M TEAB and shaken for 2 h. After the reaction was quenched, 2.5 µL of each reaction mixture with different DiLeu tags were combined at 1: 1: 1: 1 ratio and mixed well. To compare the relative abundance of each set of 4-plex DiLeu labeled metabolites, one channel from each 4-plex combination was selected and combined with 118-DiLeu tag labeled pooled serum samples. Ten µL of the mixture was taken for drying down and desalted using SCX Ziptips. Samples were dissolved in 15 µL 0.1% FA and 3 µL was injected into LC-MS for data acquisition with 3 technical replicates.