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MB Sample ID: SA210057

Local Sample ID:HF_371_S11
Subject ID:SU002272
Subject Type:Other organism
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6N mice
Age Or Age Range:5 weeks
Gender:Male

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Sample Preparation:

Sampleprep ID:SP002278
Sampleprep Summary:Frozen mouse intestinal content samples (n=20, 5 biological replicates for each sample group) were lysed in 500 µl ice-cold lysis buffer (1:1:2, v:v:v, acetonitrile: methanol: ammonium bicarbonate 0.1M - pH 8.0) and sonicated individually using a probe tip sonicator at 50% power (10 pulses). The lysis buffer contained isotopically labeled standards (n=2) to determine sample process variability. Homogenized samples were normalized by weight to the smallest amount of tissue sample such that each sample contained an equal amount of tissue. Proteins were precipitated from individual samples by addition of 800 µL of ice-cold methanol followed by overnight incubation at -80°C. Precipitated proteins were pelleted by centrifugation (15,000 rpm, 15 min) and metabolite extracts were dried down in vacuo and stored at -80°C. Individual samples were reconstituted in 100 μL of reverse phase liquid chromatography reconstitution buffer (acetonitrile/water with 0.1% formic acid, 3:97, v/v) containing isotopically labeled standards (n=2) to assess instrument variability. A pooled quality control (QC) sample was prepared by pooling equal volumes (10 μL) from each individual sample following reconstitution.
Processing Storage Conditions:-80℃
Extraction Method:Following lysis and standard addition, protein precipitation was performed by adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate proteins. The supernatants containing metabolites were dried via speed-vacuum.
Extract Storage:-80℃
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