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
Phosphoproteome analysis of Drosophila melanogaster embryos.
TL;DR: A well-established phosphopeptide enrichment and identification strategy including the combination of strong cation exchange Chromatography, immobilized metal affinity chromatography, and high-accuracy mass spectrometry instrumentation is used to study phosphorylation in developing Drosophila embryos.
Journal ArticleDOI
Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques
TL;DR: It is shown that the inclusion of various detergents can enhance the efficiency of this enrichment method, as phosphopeptides that otherwise adhere to plastic surfaces can be efficiently solubilized and subsequently purified.
Journal ArticleDOI
MAPKAP Kinase 2 Phosphorylates Tristetraprolin on in Vivo Sites Including Ser178, a Site Required for 14-3-3 Binding
Carol A. Chrestensen,Melanie J. Schroeder,Jeffrey Shabanowitz,Donald F. Hunt,Jared W. Pelo,Mark T. Worthington,Thomas W. Sturgill +6 more
TL;DR: In vitro and in vivo phosphorylation sites in Tristetraprolin are identified using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc linked to magnetic beads).
Journal ArticleDOI
The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis.
Alexey Shapiguzov,Björn Ingelsson,Iga Samol,Charles Andrès,Felix Kessler,Jean-David Rochaix,Alexander V. Vener,Michel Goldschmidt-Clermont +7 more
TL;DR: In this paper, the authors identify a phosphatase of Arabidopsis thaliana called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII).
Journal ArticleDOI
Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.
W. Andy Tao,Ruedi Aebersold +1 more
TL;DR: Advances instable isotope protein labeling and recent biological studies that used stable isotope based quantitative proteomics techniques are reviewed.
References
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Journal ArticleDOI
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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.