@inbook{15223a9427d448e28e93873e18209383,
title = "SILAC-Based Temporal Phosphoproteomics",
abstract = "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.",
author = "Chiara Francavilla and Omid Hekmat and Blagoy Blagoev and Olsen, {Jesper V}",
year = "2014",
doi = "10.1007/978-1-4939-1142-4_10",
language = "English",
isbn = "978-1-4939-1141-7",
volume = "1188",
series = "Methods in molecular biology (Clifton, N.J.)",
publisher = "Humana Press",
pages = "125--48",
editor = "Warscheid, {Bettina }",
booktitle = "Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)",
}