SILAC-Based Temporal Phosphoproteomics

In recent years, thanks to advances in Mass Spectrometry (MS)-based quantitative proteomics, studies on signaling pathways have moved from a detailed description of individual components to system-wide analysis of entire signaling cascades, also providing spatio-temporal views of intracellular pathways. Quantitative proteomics that combines stable isotope labeling by amino acid in cell culture (SILAC) with enrichment strategies for post-translational modification-bearing peptides and high-performance tandem mass spectrometry represents a powerful and unbiased approach to monitor dynamic signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated tyrosines by immunoaffinity and then further enriched for phosphorylated serine/threonine peptides by strong cation exchange in combination with titanium dioxide-beads chromatography. Analysis of enriched peptides on Orbitrap-based MS results in comprehensive and accurate reconstruction of temporal changes of signaling networks.