Metabolite extraction from serum and tumor biopsy samples

Aqueous metabolites were extracted from serum (thawed on ice) through addition of 40 μl methanol (precooled on dry ice) to 10 μl of sample. The mixture was vortexed and incubated on dry ice for 20 min, followed by centrifugation (16,000g at 4°C). For tumor samples, two frozen biopsy samples per patient were weighed (~5–10-mg tissue each) and pulverized using a Cryomill (Retsch). The resulting powder was then triturated with ice-cold 40:40:20 acetonitrile:methanol:water w/ 0.5% formic acid (1 ml solvent / 20-mg tissue) followed by neutralization w/ 15% w/v ammonium bicarbonate. Solids were precipitated by centrifugation (16,000g at 4°C). Serum sample supernatants were utilized directly for LC-MS analysis (on HILIC LC-MS); tumor supernatants were either utilized directly (on HILIC LC-MS) or were dried under nitrogen stream and resuspended in water (for reversed-phase LC-MS).