Prostate Cell Metabolome Profiling

Mass spectrometry-based metabolomics profiling was performed on frozen prostate cell pellets (∼10 million cells). The process of metabolite extraction involved introduction of equimolar mixture of 11 standard compounds dissolved in methanol (Epibrassinolide, [D₃] Testosterone, [¹⁵N] Anthranilic acid, Zeatine, Jasmonic acid, Gibberelic acid, [D₄] Estrone, [¹⁵N]-Tryptophan, [D₄] Thymine, [¹³C] Creatinine and [¹⁵N] Arginine) followed by homogenization of the cells. The homogenate was then subjected to extraction with sequential use of aqueous (chilled water) and organic (chilled methanol and chloroform) solvents in the following ratio 1∶4∶3∶1 (water∶methanol∶chloroform∶water) [28] . The resulting extracts were de-proteinized using a 3 KDa molecular filter (Amicon Ultracel -3K Membrane, Millipore Corporation, Billerica, MA) and the filtrate containing metabolites were dried under vacuum (Genevac EZ-2^plus, Gardiner, NY). Prior to mass spectrometry analysis, the dried extract was resuspended in identical volume of injection solvent composed of water∶methanol (50∶50) with 0.2% acetic acid and subjected to liquid chromatography (LC) mass spectrometry. As additional controls to monitor the profiling process, an equimolar mixture of 11 standard compounds (Epibrassinolide, [D₃] Testosterone, [¹⁵N] Anthranilic acid, Zeatine, Jasmonic acid, Gibberelic acid, [D₄] Estrone, (¹⁵N) Tryptophan, [D₄] Thymine, [¹³C] Creatinine, and [¹⁵N] Arginine) and a characterized pool of mouse liver (extracted in tandem with cell lines) were analyzed along with the cell line samples. Each of these controls, were included multiple times into the randomization scheme such that samples preparation and analytical variability could be constantly monitored. Furthermore, analysis of each cell line was succeeded by at least two blank runs, to prevent any carryover of metabolites between samples. Figure S1 illustrates the reproducibility in the profiling process monitored using the standard mixture described above. Notably the CV for the entire profiling process measured using five independent replicates of the mouse liver extract mentioned above was less than 5%.