Metabolomic Profiling by CPSI-MS
A full description of the CPSI-MS instrumentation can be found in a prior publication.21,22 After the serum was thaw under ambient conditions, 3 μL serum and 1 μL 4-chlorophenylanine (internal standard, IS) were transferred onto the tip of a conductive polymer to form a dried serum spot for data acquisition. Upon addition of methanol-water (7:3, v/v, 3 μL) onto the dried serum spot for metabolites extraction, high voltage (+4.5 kV) was applied to the conductive polymer tip to trigger the spray ionization process. Data were recorded in the Department of Chemistry, Fudan University using an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose, CA, US.). The full scan range was set at m/z 50-1000 under positive mode. The data acquisition period for each sample lasts for 15 seconds. The intensity of each metabolite ion was normalized with the IS intensity of each sample ([M+Na]+, m/z222.0292). Quality control (QC) samples were prepared by pooling equal volumes of 20 HC and 20 OSCC serum samples. QC samples were analyzed throughout the run to monitor the CPSI-MS system variation. OSCC and HC serum samples were run in alteration with the QC samples evenly inserted into the entire sequence every 30 samples.