SILAC-Based Temporal Phosphoproteomics
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Education
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.
Original language | English |
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Title of host publication | Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) : Methods and Protocols |
Editors | Bettina Warscheid |
Number of pages | 24 |
Volume | 1188 |
Publication date | 2014 |
Pages | 125-48 |
ISBN (Print) | 978-1-4939-1141-7 |
ISBN (Electronic) | 978-1-4939-1142-4 |
DOIs | |
Publication status | Published - 2014 |
Series | Methods in molecular biology (Clifton, N.J.) |
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ISSN | 1064-3745 |
ID: 119771428