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
Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach.
Michael Brad Strader,Nathan C Verberkmoes,David L. Tabb,Heather M. Connelly,John W. Barton,Barry D. Bruce,Dale A. Pelletier,Brian H. Davison,Robert L. Hettich,Frank W. Larimer,Gregory B. Hurst +10 more
TL;DR: A comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement and proteolytic fragment identification to characterize the 70S ribosome from Rhodopseudomonas palustris is presented, which is the most comprehensive protein complex analysis to date that integrates two MS techniques.
Journal ArticleDOI
Tools for analyzing the phosphoproteome and other phosphorylated biomolecules: a review.
TL;DR: This review will focus on sample preparation techniques in the field of phosphoproteomics, but also highlight recent advancements for the analysis of other phosphorylated biomolecules.
Journal Article
Identification of protein phosphorylation sites by a combination of mass spectrometry and solid phase Edman sequencing.
TL;DR: By combining mass spectrometry to identify the phosphopeptide and solid phase Edman degradation to localize the site of phosphorylation, subpmole quantities of phosphopePTides can be successfully characterized.
Journal ArticleDOI
Mining the tumor phosphoproteome for cancer markers.
TL;DR: The potential effect of phosphoproteins as cancer markers in cancer diagnosis and therapeutics is discussed and strategies that might pave the way to high-throughput analysis for routine clinical applications are described.
Journal ArticleDOI
A strategy for large-scale phosphoproteomics and SRM-based validation of human breast cancer tissue samples.
Ryohei Narumi,Tatsuo Murakami,Takahisa Kuga,Jun Adachi,Takashi Shiromizu,Satoshi Muraoka,Hideaki Kume,Yoshio Kodera,Yoshio Kodera,Masaki Matsumoto,Keiichi I. Nakayama,Yasuhide Miyamoto,Makoto Ishitobi,Hideo Inaji,Kikuya Kato,Takeshi Tomonaga,Takeshi Tomonaga +16 more
TL;DR: Large-scale phosphoproteome quantification coupled with SRM-based validation is a powerful tool for biomarker discovery using clinical samples and is well correlated with iTRAQ-based quantification with a few exceptions.
References
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TL;DR: The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database.
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Journal ArticleDOI
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.