A Methodology for Comprehensive Analysis of Toll-Like Receptor Signaling in Macrophages

摘要: A combination of high-throughput, multiplexed, quantitative methods with computational modeling and statistical approaches is required to obtain system-level understanding biological function. Mass spectrometry (MS)-based proteomics has emerged as a preferred tool for the analysis changes in protein abundance their post-translational modification (PTM) levels at global scale, comparable genomic experiments generating data suitable use mathematical signaling pathways. Here we describe set parallel bottom-up proteomic detect quantify total intracellular proteins, phosphorylation, proteins released by active passive secretion or shedding mechanisms (referred secretome reviewed Makridakis Vlahou, J Proteome 73:2291-2305, 2010) response stimulation Toll-like receptors (TLRs) specific ligands cultured macrophages. The method includes protocols metabolic labeling cells (SILAC: stable isotope amino acids cell culture; Ong et al., Mol Cell Proteomics 1:376-386, 2002), ligand stimulation, lysis media collection, in-gel in-solution digestion phosphopeptide enrichment phosphoproteomics, LC-MS/MS analysis. With these methods, can not only reliably relative TLR components (Sjoelund Res 13:5185-5197, 2014) but also constraints modeling.