Einzeltreffer — DigiBib

Protein phosphorylation is among the most common and intensely studied post-translational protein modification. It plays crucial roles in virtually all cellular processes and has been implicated in numerous human diseases, including cancer. Traditional biochemical and genetic methods for identifying and monitoring sites of phosphorylation are laborious and slow and in recent years have largely been replaced by mass spectrometric analysis. Improved methods for phosphopeptide enrichment coupled with faster and more sensitive mass spectrometers have led to an explosion in the size of phosphoproteomic datasets. However, wider application of these methods is limited by equipment costs and the resultant high demand for instrument time as well as by a technology gap between biologists and mass spectrometrists. Here we describe a modified two-step enrichment strategy that employs lysC digestion and step elution from self-packed strong cation exchange (SCX) solid phase extraction (SPE) columns followed by immobilized metal ion affinity chromatography (IMAC) and LC-MS/MS analysis using a hybrid LTQ Orbitrap Velos mass spectrometer. The SCX procedure does not require an HPLC system, demands little expertise, and because multiple samples can be processed in parallel, can provide a large savings of time and labor. We demonstrate this method in conjunction with stable isotope labeling to quantitate peptides harboring >8000 unique phosphorylation sites in yeast in 12h of instrument analysis time and examine the impact of enzyme choice and instrument platform.

Titel:	A solid phase extraction-based platform for rapid phosphoproteomic analysis.
Autor/in / Beteiligte Person:	Dephoure, N ; Gygi, SP
Zeitschrift:	Methods (San Diego, Calif.), Jg. 54 (2011-08-01), Heft 4, S. 379-86
Veröffentlichung:	Duluth, MN : Academic Press ; <i>Original Publication</i>: San Diego : Academic Press, c1990-, 2011
Medientyp:	academicJournal
ISSN:	1095-9130 (electronic)
DOI:	10.1016/j.ymeth.2011.03.008
Schlagwort:	Chromatography, Affinity methods Chromatography, Liquid Fungal Proteins chemistry Isotope Labeling Phosphoproteins chemistry Proteomics methods Tandem Mass Spectrometry Fungal Proteins isolation & purification Phosphoproteins isolation & purification Solid Phase Extraction methods
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, N.I.H., Extramural Language: English [Methods] 2011 Aug; Vol. 54 (4), pp. 379-86. <i>Date of Electronic Publication: </i>2011 Apr 01. MeSH Terms: Fungal Proteins / isolation & purification ; Phosphoproteins / isolation & purification ; Solid Phase Extraction / *methods ; Chromatography, Affinity / methods ; Chromatography, Liquid ; Fungal Proteins / chemistry ; Isotope Labeling ; Phosphoproteins / chemistry ; Proteomics / methods ; Tandem Mass Spectrometry References: Nat Methods. 2007 Mar;4(3):207-14. (PMID: 17327847) ; Electrophoresis. 1999 Dec;20(18):3551-67. (PMID: 10612281) ; Nat Protoc. 2008;3(10):1630-8. (PMID: 18833199) ; Proteomics. 2008 Nov;8(21):4444-52. (PMID: 18972524) ; Mol Cell Proteomics. 2009 Dec;8(12):2759-69. (PMID: 19828875) ; Nat Biotechnol. 2002 Mar;20(3):301-5. (PMID: 11875433) ; Nat Biotechnol. 2006 Oct;24(10):1285-92. (PMID: 16964243) ; Mol Cell Proteomics. 2006 May;5(5):914-22. (PMID: 16452087) ; Anal Chem. 2009 Sep 15;81(18):7757-65. (PMID: 19689114) ; Nat Methods. 2007 Sep;4(9):677-8. (PMID: 17762871) ; Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10762-7. (PMID: 18669648) ; Anal Chem. 2004 Jul 15;76(14):3935-43. (PMID: 15253627) ; Anal Chem. 2003 Feb 1;75(3):663-70. (PMID: 12585499) ; Sci Signal. 2010 Jan 12;3(104):ra3. (PMID: 20068231) ; J Proteome Res. 2008 Apr;7(4):1675-82. (PMID: 18327897) ; Science. 2009 Sep 25;325(5948):1682-6. (PMID: 19779198) ; Anal Chem. 1995 Apr 15;67(8):1426-36. (PMID: 7741214) ; Anal Chem. 2004 Jul 1;76(13):3590-8. (PMID: 15228329) ; J Proteome Res. 2008 Nov;7(11):4756-65. (PMID: 18798661) ; J Biol Chem. 2010 Apr 23;285(17):12803-12. (PMID: 20190278) ; Mol Biosyst. 2009 Oct;5(10):1122-9. (PMID: 19756301) ; Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1488-93. (PMID: 17242355) ; Mol Cell Proteomics. 2009 Aug;8(8):1908-20. (PMID: 19429919) ; Proteomics. 2004 Jun;4(6):1551-61. (PMID: 15174125) ; ACS Chem Biol. 2010 Jan 15;5(1):105-19. (PMID: 20047291) ; Mol Cell Proteomics. 2005 Mar;4(3):310-27. (PMID: 15665377) ; Anal Biochem. 1986 Apr;154(1):250-4. (PMID: 3085541) ; Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5. (PMID: 15302935) ; Cell. 2000 Jan 7;100(1):113-27. (PMID: 10647936) ; Nat Biotechnol. 2005 Jan;23(1):94-101. (PMID: 15592455) ; Cell. 2010 Dec 23;143(7):1174-89. (PMID: 21183079) ; Mol Cell Proteomics. 2002 May;1(5):376-86. (PMID: 12118079) ; Cell. 2006 Nov 3;127(3):635-48. (PMID: 17081983) ; Nat Methods. 2008 Nov;5(11):959-64. (PMID: 18931669) ; J Proteome Res. 2010 Dec 3;9(12):6786-94. (PMID: 20873877) Grant Information: R01 HG003456 United States HG NHGRI NIH HHS; R01 HG003456-07 United States HG NHGRI NIH HHS; HG3456 United States HG NHGRI NIH HHS Substance Nomenclature: 0 (Fungal Proteins) ; 0 (Phosphoproteins) Entry Date(s): Date Created: 20110329 Date Completed: 20120112 Latest Revision: 20211020 Update Code: 20240513 PubMed Central ID: PMC3156280

Klicken Sie ein Format an und speichern Sie dann die Daten oder geben Sie eine Empfänger-Adresse ein und lassen Sie sich per Email zusenden.

BibTeX Citavi, JabRef, u.a.
(Literaturverwaltung)

PDF kein Volltext!
(Merkzettel, Notizen)

RIS Endnote, Citavi u.a.
(Literaturverwaltung)

MODS
(XML zur Weiterverarbeitung)

oder

Wählen Sie das für Sie passende Zitationsformat und kopieren Sie es dann in die Zwischenablage, lassen es sich per Mail zusenden oder speichern es als PDF-Datei.

Gewünschter Zitations-Stil:

oder

Bitte prüfen Sie, ob die Zitation formal korrekt ist, bevor Sie sie in einer Arbeit verwenden. Benutzen Sie gegebenenfalls den "Exportieren"-Dialog, wenn Sie ein Literaturverwaltungsprogramm verwenden und die Zitat-Angaben selbst formatieren wollen.

A solid phase extraction-based platform for rapid phosphoproteomic analysis.