4.4. Global Protein Quantification

Proteins were quantified relatively as previously described [38]. In short, proteins were extracted from the formalin-fixed paraffin embedded samples and digested using a modified filter aided sample preparation protocol. Peptide samples were chemically labeled with tandem-mass-tag (TMT, Thermo Fisher Scientific, Waltham, MA, USA) for relative quantification, and the 20 fractions for each set from basic reverse phase separation were analyzed with nanoLC on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) operating in MultiNoch MS3 mode. Protein identification and quantification were performed with Proteome Discoverer version 2.4 (Thermo Fisher Scientific) matching against Swissprot Homo sapiens database (January 2021). Differential expression analysis using a two-sample t-test on log2-transformed data was performed using the Perseus software (1.6.15.0) and R. Proteins with a p value < 0.05 and fold change (FC) ≥ 20% were considered differentially expressed.