Mass Spectrometry-Based Protein Identification

Purified proteins bound to streptavidin beads were reduced, alkylated, and digested by sequential addition of trypsin protease (51 (link), 52 (link)). The peptide mixture was desalted using C₁₈ tips and fractionated online using a 75-µm-inner-diameter fritted fused silica capillary column with a 5-µm pulled electrospray tip and packed in house with 15 cm of Luna C₁₈ (2 (link)) 3-µm reversed-phase particles. The gradient was delivered by an easy-nLC 1000 ultrahigh-pressure liquid chromatography (UHPLC) system (Thermo Scientific) (53 (link), 54 (link)). Tandem mass spectrometry (MS/MS) spectra were collected on a Q-Exactive mass spectrometer (Thermo Scientific). Data analysis was performed using the ProLuCID and DTASelect2 implemented in the Integrated proteomics pipeline IP2 (Integrated Proteomics Applications, Inc., San Diego, CA) (55 (link), 56 (link)). Protein and peptide identifications were filtered using DTASelect and required a minimum of two unique peptides per protein and a peptide-level false-positive rate of less than 5%, as estimated by a decoy database strategy (57 (link)). Normalized spectral abundance factor (NSAF) values were calculated as described previously (58 (link)).