HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M500135-MCP200v1 5/1/172    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Steen, H. Articles by Kirschner, M. W. Search for Related Content PubMed PubMed Citation Articles by Steen, H. Articles by Kirschner, M. W. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 5:172-181, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

Phosphorylation Analysis by Mass Spectrometry

Myths, Facts, and the Consequences for Qualitative and Quantitative Measurements^*

Hanno Steen^,,¶, Judith A. Jebanathirajah^,||,**, John Rush, Nicolas Morrice and Marc W. Kirschner

From the Department of Pathology, Harvard Medical School and Children’s Hospital, Boston, Massachusetts 02115, Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, ^|| Neurobiology Program, Children’s Hospital, Boston, Massachusetts 02115, ^** MDS Sciex, Concord, Ontario L4K 4V8, Canada, Cell Signaling Technology, Beverly, Massachusetts 01915, and MRC Protein Phosphorylation Unit, University of Dundee, Dundee, United Kingdom DD1 5EH

The mass spectrometric analysis of protein phosphorylation is still far from being routine, and the outcomes thereof are often unsatisfying. Apart from the inherent problem of substoichiometric phosphorylation, three arguments as to why phosphorylation analysis is so problematic are often quoted, including (a) increased hydrophilicity of the phosphopeptide with a concomitant loss during the loading onto reversed-phase columns, (b) selective suppression of the ionization of phosphopeptides in the presence of unmodified peptides, and (c) lower ionization/detection efficiencies of phosphopeptides as compared with their unmodified cognates. Here we present the results of a study investigating the validity of these three arguments when using electrospray ionization mass spectrometry. We utilized a set of synthetic peptide/phosphopeptide pairs that were quantitated by amino acid analysis. Under the applied conditions none of the experiments performed supports the notions of (a) generally increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of decreased retention and (b) the selective ionization suppression of phosphopeptides. The issue of ionization/detection efficiencies of phosphopeptides versus their unphosphorylated cognates proved to be less straightforward when using electrospray ionization because no evidence for decreased ionization/detection efficiencies for phosphopeptides could be found.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M.-H. Lee, P. Koria, J. Qu, and S. T. Andreadis JNK phosphorylates {beta}-catenin and regulates adherens junctions FASEB J, November 1, 2009; 23(11): 3874 - 3883. [Abstract] [Full Text] [PDF]

				F. S. Oppermann, F. Gnad, J. V. Olsen, R. Hornberger, Z. Greff, G. Keri, M. Mann, and H. Daub Large-scale Proteomics Analysis of the Human Kinome Mol. Cell. Proteomics, July 1, 2009; 8(7): 1751 - 1764. [Abstract] [Full Text] [PDF]

				S. M. M. Sweet, C. M. Bailey, D. L. Cunningham, J. K. Heath, and H. J. Cooper Large Scale Localization of Protein Phosphorylation by Use of Electron Capture Dissociation Mass Spectrometry Mol. Cell. Proteomics, May 1, 2009; 8(5): 904 - 912. [Abstract] [Full Text] [PDF]

				T. W. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller, and D. C. Johnson Herpesvirus gB-Induced Fusion between the Virion Envelope and Outer Nuclear Membrane during Virus Egress Is Regulated by the Viral US3 Kinase J. Virol., April 1, 2009; 83(7): 3115 - 3126. [Abstract] [Full Text] [PDF]

				V. Zabrouskov, Y. Ge, J. Schwartz, and J. W. Walker Unraveling Molecular Complexity of Phosphorylated Human Cardiac Troponin I by Top Down Electron Capture Dissociation/Electron Transfer Dissociation Mass Spectrometry Mol. Cell. Proteomics, October 1, 2008; 7(10): 1838 - 1849. [Abstract] [Full Text] [PDF]

				J. A. J. Steen, H. Steen, A. Georgi, K. Parker, M. Springer, M. Kirchner, F. Hamprecht, and M. W. Kirschner Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: A quantitative proteomic analysis PNAS, April 22, 2008; 105(16): 6069 - 6074. [Abstract] [Full Text] [PDF]

				E. B. Taylor, D. An, H. F. Kramer, H. Yu, N. L. Fujii, K. S. C. Roeckl, N. Bowles, M. F. Hirshman, J. Xie, E. P. Feener, et al. Discovery of TBC1D1 as an Insulin-, AICAR-, and Contraction-stimulated Signaling Nexus in Mouse Skeletal Muscle J. Biol. Chem., April 11, 2008; 283(15): 9787 - 9796. [Abstract] [Full Text] [PDF]

				M. Marcantonio, M. Trost, M. Courcelles, M. Desjardins, and P. Thibault Combined Enzymatic and Data Mining Approaches for Comprehensive Phosphoproteome Analyses: Application to Cell Signaling Events of Interferon-{gamma}-Stimulated Macrophages Mol. Cell. Proteomics, April 1, 2008; 7(4): 645 - 660. [Abstract] [Full Text] [PDF]

				N. Y. Tan, V. C. Midgley, M. M. Kavurma, F. S. Santiago, X. Luo, R. Peden, R. G. Fahmy, M. C. Berndt, M. P. Molloy, and L. M. Khachigian Angiotensin II-Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1 Circ. Res., February 29, 2008; 102(4): e38 - e51. [Abstract] [Full Text] [PDF]

				S. Sullivan, C. E. Thomson, D. J. Lamont, M. A. Jones, and J. M. Christie In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1 Mol Plant, January 1, 2008; 1(1): 178 - 194. [Abstract] [Full Text] [PDF]

				F. Yang, D. G. Camp II, M. A. Gritsenko, Q. Luo, R. T. Kelly, T. R. W. Clauss, W. R. Brinkley, R. D. Smith, and D. L. Stenoien Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus J. Cell Sci., November 15, 2007; 120(22): 4060 - 4070. [Abstract] [Full Text] [PDF]

				J. Reinders, K. Wagner, R. P. Zahedi, D. Stojanovski, B. Eyrich, M. van der Laan, P. Rehling, A. Sickmann, N. Pfanner, and C. Meisinger Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase Mol. Cell. Proteomics, November 1, 2007; 6(11): 1896 - 1906. [Abstract] [Full Text] [PDF]

				S. Y. Imanishi, V. Kochin, S. E. Ferraris, A. de Thonel, H.-M. Pallari, G. L. Corthals, and J. E. Eriksson Reference-facilitated Phosphoproteomics: FAST AND RELIABLE PHOSPHOPEPTIDE VALIDATION BY {micro}LC-ESI-Q-TOF MS/MS Mol. Cell. Proteomics, August 1, 2007; 6(8): 1380 - 1391. [Abstract] [Full Text] [PDF]

				J. J. Benschop, S. Mohammed, M. O'Flaherty, A. J. R. Heck, M. Slijper, and F. L. H. Menke Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis Mol. Cell. Proteomics, July 1, 2007; 6(7): 1198 - 1214. [Abstract] [Full Text] [PDF]

				G. K. Agrawal and J. J. Thelen Large Scale Identification and Quantitative Profiling of Phosphoproteins Expressed during Seed Filling in Oilseed Rape Mol. Cell. Proteomics, November 1, 2006; 5(11): 2044 - 2059. [Abstract] [Full Text] [PDF]

				S.-L. Wu, J. Kim, R. W. Bandle, L. Liotta, E. Petricoin, and B. L. Karger Dynamic Profiling of the Post-translational Modifications and Interaction Partners of Epidermal Growth Factor Receptor Signaling after Stimulation by Epidermal Growth Factor Using Extended Range Proteomic Analysis (ERPA) Mol. Cell. Proteomics, September 1, 2006; 5(9): 1610 - 1627. [Abstract] [Full Text] [PDF]