Proteomic Analysis of Biological Samples

The label-free proteomic analysis was performed in a nanoACQUITY UPLC system (Waters, Milford, MA, USA) coupled to a Xevo Q-TOF G2 mass spectrometer (Waters, Milford, MA, USA). The nanoACQUITY UPLC system was equipped with a Trap Columm (100 Å, 5 μm, 180 μm × 200 mm) and a HSS T3 M-Class type column (analytical column 75 μm × 150 mm; 1.8 μm) (Waters, Milford, MA, USA). The reading and identification of peptides was performed using the ProteinLynx GlobalServer software (PLGS) version 3.03 (Waters, Milford, MA, USA), as previously described [63 (link)]. The PLGS software, applying the Monte-Carlo algorithm, was used to determine the difference in protein expression between the groups, considering p < 0.05 for downregulated proteins and 1 − p > 0.95 for upregulated proteins. The identification of proteins was performed by downloading UniProt databases. Then, bioinformatics analyses were performed using Cytoscape^® 3.6 (Java^®) with the ClusterMarker plugin for the PPI network, and for the determination of the biological process groups based on Gene Ontology annotations, we used the ClueGO plugin [64 (link)].