Shotgun Proteomics Workflow for Protein Identification

Shotgun quantitative label-free proteomics was performed in a nanoACQUITY UPLC system (Waters, Milford, MA, USA) coupled with a Xevo Q-TOF G2 mass spectrometer (Waters, Milford, MA, USA), as previously described [23 (link)]. Spectra were processed, and proteins were identified and quantified with Progenesis QI for Proteomics^® (Nonlinear Dynamics; Waters Corporation; version 4.0) using Apex3D (Waters) for peak detection and searching the Swiss-Prot Human proteomic database, using all the peptides for relative quantification. In order to obtain the preliminary protein dataset, the following parameters were considered: trypsin digestion with a maximum of one missed cleavage; variable modification via oxidation (M) and fixed modification via carbamidomethyl (C); false discovery rate (FDR) less than 4%; and mass error less than 20 ppm. In addition, ion-matching requirements were established to select proteins with at least one ion per peptide, three ions per protein, and one peptide per protein. Then, the final list of proteins was reduced to selected proteins identified by at least two unique peptides and proteins whose presence was detected in at least 60% of the samples.
The software CYTOSCAPE version 3.9.0 was used to build networks of molecular interactions between the identified proteins with the aid of the ClueGo and String applications.