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
Citations
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ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry.
Richard D. LeDuc,Gregory K. Taylor,Yong Bin Kim,Thomas E. Januszyk,Lee H. Bynum,Joseph V. Sola,John S. Garavelli,Neil L. Kelleher +7 more
TL;DR: Sequence databases from across the phylogenetic tree are supported, with a new database strategy of 'shotgun annotation' used to assist characterization of wild-type proteins.
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Multidimensional separation of peptides for effective proteomic analysis.
TL;DR: This manuscript provides a review of two- and three-dimensional peptide separation strategies combined with MS for the analysis of complex peptide mixtures.
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RNA Polymerase I Structure and Transcription Regulation.
TL;DR: The crystal structure of Pol I from the yeast Saccharomyces cerevisiae at 2.8 Å resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II.
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Protein microarrays for multiplex analysis of signal transduction pathways
TL;DR: A multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells is developed.
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Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry.
TL;DR: Using conventional tandem mass spectrometry-based sequencing technology and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome conclude that ETD identifies a larger number of unique phosphopeptides than CAD, more frequently localizes the phosphorylation site to a specific residue, and sequences whole classes of phosphopePTides previously unobserved.
References
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Journal ArticleDOI
An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.
TL;DR: The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database.
Journal ArticleDOI
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Journal ArticleDOI
On target with a new mechanism for the regulation of protein phosphorylation
Michael J. Hubbard,Philip Cohen +1 more
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.