Small-molecule extraction from protein samples.

Twenty-microliter volumes of WT and MT S-Trimer samples at 1.21 mg/ml and 1.02 mg/ml, respectively, were transferred to Eppendorf tubes and placed in a heater block at 100°C for 5 min. Samples were then extracted with 100 μl of methanol. The tubes were vortexed and centrifuged at 15,000 rpm and 4°C for 10 min. The supernatant was collected and transferred to glass vials, and 1 μl was injected for liquid chromatography-mass spectrometry (LC-MS) analysis. For oleic acid and linoleic acid reference samples, 10 mM stock solutions of compounds were prepared in dimethyl sulfoxide (DMSO) and diluted to 1 μM by methanol, and 1 μl was injected for the LC-MS analysis.