Journal ArticleDOI
Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae
Scott B. Ficarro,Mark L. McCleland,P. Todd Stukenberg,Daniel J. Burke,Mark M. Ross,Jeffrey Shabanowitz,Donald F. Hunt,Forest M. White +7 more
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TLDR
In this article, a methodology was proposed to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment, and a total of 216 peptide sequences defining 383 sites of phosphorylation were determined.Abstract:
Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein. Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.read more
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Journal ArticleDOI
Phosphoproteomics strategies for the functional analysis of signal transduction.
Sandra Morandell,Taras Stasyk,Karin Grosstessner-Hain,Elisabeth Roitinger,Karl Mechtler,Guenther K. Bonn,Lukas A. Huber +6 more
TL;DR: Various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates are summarised, including gel‐free methods combining chromatography with highly sensitive MS and recently developed approaches like KESTREL or ’︁chemical genetics’ are summarized.
Journal ArticleDOI
Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism
Sergio de la Fuente van Bentem,Dorothea Anrather,Elisabeth Roitinger,Armin Djamei,Thomas Hufnagl,Andrea Barta,Edina Csaszar,Ilse Dohnal,David Lecourieux,Heribert Hirt +9 more
TL;DR: The application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts suggests that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.
Journal ArticleDOI
Mapping of Phosphorylation Sites by a Multi-Protease Approach with Specific Phosphopeptide Enrichment and NanoLC−MS/MS Analysis
TL;DR: A multi-protease approach that allows sensitive and comprehensive mapping of protein phosphorylation sites was applied to the murine circadian protein period 2 (mPER2), and Titansphere proved to be well suited for the enrichment of a large variety of phosphopeptides.
Journal ArticleDOI
Deciphering metabolic networks
Oliver Fiehn,Wolfram Weckwerth +1 more
TL;DR: Methods to study complex networks of major biochemical steps, i.e. transcripts, proteins, and metabolites, are reviewed with respect to data acquisition, network statistics, and biochemical interpretation.
Journal ArticleDOI
Comparative Proteomic and Transcriptomic Profiling of the Fission Yeast Schizosaccharomyces pombe
TL;DR: Self‐organizing map clustering of large‐scale protein and mRNA data from fission and budding yeast revealed coordinate but not always concordant expression of components of functional pathways and protein complexes, which clustered in groups with similar mRNA–protein ratios.
References
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Journal ArticleDOI
Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography.
Lennart Andersson,Jerker Porath +1 more
TL;DR: Hen egg albumin (ovalbumin) was fractionated into three components of varying phosphate contents and Porcine pepsin was purified in a similar manner to develop purification procedures for phosphoproteins.