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.
authors: Department of Proteomics, Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Blegdamsvej 3B, 2200, Copenhagen, Denmark.
authors: Chiara
authors: C
authors: Francavilla
authors: Omid
authors: O
authors: Hekmat
authors: Blagoy
authors: B
authors: Blagoev
authors: Jesper V
authors: JV
authors: Olsen
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D053000:Analytic Sample Preparation Methods; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012492:Salts / Q000302:isolation & purification; D062211:Spatio-Temporal Analysis
chemical_list: D000596:Amino Acids; D010748:Phosphopeptides; D010750:Phosphoproteins; D012492:Salts