Quantitative Shotgun Proteomics by RP-LC-ESI-MS/MS

The desalted protein digest was analyzed by RP-LC-ESI-MS/MS in an EASYnLC1000 System, coupled to the Q-Exactive-HF mass spectrometer through the Nano-Easy spray source (Thermo Scientific, Waltham, MA, USA). Peptide identifications were carried out using the Mascotv.2 search engine through the Protein Discoverer Software. A database search was performed against SwissProt. Mascot Scores were adjusted by a percolator algorithm. The acceptance criteria for protein identification were a false discovery rate (FDR) <1% and at least one peptide identified with high confidence (CI > 95%). To determine the abundances of the identified peptides and proteins, a processing free label workflow was initiated in the first step. Finally, the results were normalized to the total amount of the peptides, equaling the total abundance among the different samples. The proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE [27 (link),28 (link)].