In-depth proteomic analysis of secretome and whole cells of mouse astrocyte

In this study, 3 biological replicates with 3 technical replicates for the conditioned media (CM) and the whole cell lysates (WCL) of C8-D1A cell line were analyzed using 108 LC-MS/MS runs. Each peptide sample was separated into 6 fractions using stageTip based High-pH fractionation. The peptide samples were analyzed using LC-MS/MS instrumentation consisting of a Nanoflow Easy-nLC 1000 that was connected to a Q Exactive mass spectrometer through a nanoelectrospray ion source. All raw files were processed in MaxQuant, version 1.3.0.5 and the Andromeda search engine against the IPI mouse database (version 3.87, 59 534 entries), containing both forward and reverse proteins sequences, and common contaminants. MS/MS searches for the secretome and the whole-cell proteome were performed with the following parameters: carbamidomethylation as a fixed modification; oxidation of methionine and protein N-terminal acetylation as variable modifications; a 20-ppm first-search tolerance; a 6-ppm main-search tolerance. Minimum peptide length was set to six residues. The false discovery rate for all peptides, PTM sites, and protein identifications was set to 0.01.