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
Phosphoproteomic approaches to elucidate cellular signaling networks.
Katrin Schmelzle,Forest M. White +1 more
TL;DR: The combination of multiple approaches, coupled with phenotypic response measurements, computational modeling and biochemical manipulations, will ultimately reveal the mechanistic regulation of signaling networks.
Book ChapterDOI
The CCR4–NOT Complex Plays Diverse Roles in mRNA Metabolism
Clyde L. Denis,Junji Chen +1 more
TL;DR: The known biochemical roles of the individual components and of the CCR4-NOT complex are described with particular emphasis on the two known functions of the complex, repression of TFIID action and deadenylation of mRNA.
Journal ArticleDOI
Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis.
Sergio de la Fuente van Bentem,Dorothea Anrather,Ilse Dohnal,Elisabeth Roitinger,Edina Csaszar,Jos Joore,Joshua Buijnink,Alessandro Carreri,Celine Forzani,Zdravko J. Lorković,Andrea Barta,David Lecourieux,Andreas Verhounig,Claudia Jonak,Heribert Hirt +14 more
TL;DR: The data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.
Journal ArticleDOI
Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis
Houjiang Zhou,Ruijun Tian,Mingliang Ye,Songyun Xu,Shun Feng,Chensong Pan,Xiaogang Jiang,Xin Li,Hanfa Zou +8 more
TL;DR: The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins, and Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed.
Journal ArticleDOI
Application of Mass Spectrometry in Proteomics
TL;DR: Advances in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins.
References
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Journal ArticleDOI
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