Ultra 3000 uhplc

UHPLC/Q-Orbitrap system: Thermo Fisher Scientific’s Ultra 3000 UHPLC (USA), Thermo Fisher Scientific’s Q Exactive high-resolution mass spectrometer (USA). BX7200HP desktop ultrasonic cleaner from Shanghai Xinmiao Medical Equipment Manufacturing Co., Ltd.; AL104 balance (with an accuracy of 0.0001) from Mettler Toledo Shanghai Co., Ltd., Switzerland; VORTEX-GENIE 2 vortex oscillator from SITM (USA); Hitachi’s CF16RN Centrifuge (Japan); Heraeus Fresco 17 high-speed refrigerated centrifuge from Thermo Fisher Scientific (USA); Ultrapure water meter from MilliPore (USA).

A proteomic analysis was performed on the individual FFPE patient samples. In short, proteins were extracted from the deparaffinized biopsies, digested into peptides, and dissolved in 0.1% formic acid for LC-MS analysis. Samples were analyzed on a commercial C18 column (Acclaim PepMap RSLC, 75 μm × 50 cm, Thermo Scientific) using an Ultra3000 UHPLC connected to Exploris 480 mass spectrometer (Thermo Scientific). The generated MS/MS spectra were searched against the Uniprot Human database (https://www.uniprot.org) using the SEQUEST searching engine in the Proteome Discoverer 2.4 software (PD, Thermo Scientific, Waltham, MA, USA). Label-free quantification was carried out only for proteins with two or more unique peptide matches. Protein ratios were calculated as the median of all peptide hits belonging to a protein. Quantitative precision was expressed as the protein ratio variability.