Intracellular Metabolite Profiling by CE-TOFMS

Intracellular metabolites were measured by CE-TOFMS (Agilent Technologies, Palo Alto, CA, USA) as previously described^{24 (link),50 (link),51 (link)}. In brief, cells were washed twice with 5% (w/v) mannitol (FUJIFILM Wako Pure Chemical Industries) and dissolved in 600 µL of methanol containing internal standards (25 μM each of methionine sulfone, ethane sulfonic acid, and d-Camphor-10-sulfonic acid). The homogenate was mixed with 200 μL of Milli-Q water and 400 μL of chloroform. After centrifugation, the separated methanol–water layer was ultra-filtrated through a Millipore 5-kDa cutoff filter (Millipore, Bedford, MA, USA) to remove proteins. The filtrate was lyophilized, dissolved in 25 μL of Milli-Q water containing internal standards (200 μM each of 1,3,5-benzenetricarboxylic acid and 3-aminopyrrolidine), and analyzed using CE-TOFMS. The raw data were processed with MasterHands^{52 (link)}. Metabolite identities were assigned by matching their m/z values and migration times with those of standard compounds.