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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Using phage display to select antibodies recognizing post-translational modifications independently of sequence context.
John Kehoe,Nileena Velappan,Monica Walbolt,Jytte Rasmussen,Dave King,Jianlong Lou,Kristeene A. Knopp,Peter Pavlik,James D. Marks,Carolyn R. Bertozzi,Andrew Bradbury +10 more
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Identification of Phosphorylation Sites in Protein Kinase A Substrates Using Artificial Neural Networks and Mass Spectrometry
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References
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On target with a new mechanism for the regulation of protein phosphorylation
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TL;DR: Evidence indicates that a novel class of proteins, known as targetting subunits, specifies the location, catalytic and regulatory properties of protein phosphatases and kinases, and thereby plays a key role in ensuring the fidelity ofprotein phosphorylation.
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Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome.
TL;DR: A method for enriching phosphoserine/threonine-containing proteins from crude cell extracts and for subsequently identifying the phosphoproteins and sites of phosphorylation is described, which involves chemical replacement of the phosphate moieties by affinity tags and should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylated or dephosphorylation of serine/Threonine residues.
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.