Protocols for label-free LC-MS/MS proteomic analysis

Success in proteomic analysis of biological materials relies on multiple factors including effective and reliable sample preparation techniques, optimized data acquisition parameters, and powerful tools to carry out the subsequent data analysis. In this chapter, we describe the commonly used methodologies for label-free proteomic analysis of biological samples in our group. The label-free workflow is explained in detail from sample preparation to data acquisition and analysis. The sample collection section focuses on the collection of samples from model cell lines (parental vs. drug-resistant counterpart of a cancer cell line), fractionation of cytoplasmic and membrane proteins, extraction of proteins and digestion using the PreOmics iST kit. The following sections focus on parameters used for data acquisition with a liquid chromatography–coupled mass spectrometer (UltiMate 3000 RSLCnano System and Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific)) and data analysis using Proteome Discoverer (Thermo Scientific) and Progenesis QI for Proteomics (Waters).