SILAC-Based Temporal Phosphoproteomics
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SILAC-Based Temporal Phosphoproteomics. / Francavilla, Chiara; Hekmat, Omid; Blagoev, Blagoy; Olsen, Jesper V.
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC): Methods and Protocols. ed. / Bettina Warscheid. Vol. 1188 2014. p. 125-48 (Methods in molecular biology (Clifton, N.J.)).Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Education
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TY - CHAP
T1 - SILAC-Based Temporal Phosphoproteomics
AU - Francavilla, Chiara
AU - Hekmat, Omid
AU - Blagoev, Blagoy
AU - Olsen, Jesper V
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
U2 - 10.1007/978-1-4939-1142-4_10
DO - 10.1007/978-1-4939-1142-4_10
M3 - Book chapter
C2 - 25059609
SN - 978-1-4939-1141-7
VL - 1188
T3 - Methods in molecular biology (Clifton, N.J.)
SP - 125
EP - 148
BT - Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)
A2 - Warscheid, Bettina
ER -
ID: 119771428