Identification of novel prognostic indicators for oral squamous cell carcinoma based on proteomics

Lysate (1.5% SDS / 100 mm Tris-Cl) was added to samples on ice, mixed well and centrifuged to obtain supernatant. The protein concentration was determined by Bradford assay, and the protein bands were visualized by SDS-PAGE. Enzymatic digestion with trypsin was followed by Sep-Pak C18 desalting. Equal amounts of samples were taken for TMT labeling, which was performed according to the manufacturer's instructions. After equal mixing of the labeled samples, the pooled samples were fractionated by high pH reverse chromatography and checked on the machine. Mass spectrometry data were acquired using a liquid-mass coupled system with an Orbitrap Exploris 480 mass spectrometer in tandem with an EASY-nLC 1200 liquid phase. Mass spectrometric data were searched by MaxQuant (v1.6.6) software and the adopted database searching algorithm was Andromeda. The database used for the search was Swissprot Human (20210312) proteome reference databases.