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: M300054-MCP200v1 2/10/1055    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 Haydon, C. E. Articles by Ahn, N. G. Search for Related Content PubMed PubMed Citation Articles by Haydon, C. E. Articles by Ahn, N. G. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 2:1055-1067, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

Identification of Novel Phosphorylation Sites on Xenopus laevis Aurora A and Analysis of Phosphopeptide Enrichment by Immobilized Metal-affinity Chromatography ^*

Claire E. Haydon^,, Patrick A. Eyers^,¶, Lauren D. Aveline-Wolf^,, Katheryn A. Resing, James L. Maller^,¶ and Natalie G. Ahn^,,||

From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, the ^¶ Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, and Howard Hughes Medical Institute, Chevy Chase, Maryland 20815

Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using liquid chromatography-electrospray ionization tandem mass spectrometry, 14 phosphorylation sites were identified on Xenopus laevis His₆-Aurora A, a highly conserved regulator of centrosome maturation and cell division. These included seven novel phosphorylation sites, Ser-12, Thr-21, Thr-103, Ser-116, Thr-122, Tyr-155, and Thr-294, as well as the previously identified regulatory sites, Ser-53, Thr-295, and Ser-349. The identification of these novel phosphorylation sites will be important for future studies aimed at elucidating the mechanisms of Aurora A regulation by phosphorylation. Furthermore, we demonstrate that a "kinase-inactive" mutant of Aurora A, K169R, still retains 10% of activity of the wild-type enzyme in vitro along with occupancy of Thr-295 and Ser-12. However, mutation of Asp-281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Because of the abundance of phosphorylated residues on His₆-Aurora A, we found this protein to be an ideal tool for the characterization of immobilized metal-affinity chromatography (IMAC) as a method for phosphopeptide enrichment from complex mixtures. We present a detailed analysis of the binding and elution properties of both the phosphopeptides and unphosphorylated peptides of His₆-Aurora A to Fe³⁺-IMAC before and after methyl esterification. Moreover, we demonstrate a significant difference in enrichment of phosphopeptides when different resins are used for Fe³⁺-IMAC and characterize the strengths and limitations of this methodology for the study of phosphoproteomics.

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

This article has been cited by other articles:

				S. P. Mirza and M. Olivier Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry Physiol Genomics, October 8, 2008; 33(1): 3 - 11. [Abstract] [Full Text] [PDF]

				N. Sugiyama, T. Masuda, K. Shinoda, A. Nakamura, M. Tomita, and Y. Ishihama Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications Mol. Cell. Proteomics, June 1, 2007; 6(6): 1103 - 1109. [Abstract] [Full Text] [PDF]

				J. Lee, Y. Xu, Y. Chen, R. Sprung, S. C. Kim, S. Xie, and Y. Zhao Mitochondrial Phosphoproteome Revealed by an Improved IMAC Method and MS/MS/MS Mol. Cell. Proteomics, April 1, 2007; 6(4): 669 - 676. [Abstract] [Full Text] [PDF]

				M. O. Collins, L. Yu, H. Husi, W. P. Blackstock, J. S. Choudhary, and S. G. N. Grant Robust Enrichment of Phosphorylated Species in Complex Mixtures by Sequential Protein and Peptide Metal-Affinity Chromatography and Analysis by Tandem Mass Spectrometry Sci. Signal., August 23, 2005; 2005(298): pl6 - pl6. [Abstract] [Full Text] [PDF]

				P. A. Eyers, J. Liu, N. R. Hayashi, A. L. Lewellyn, J. Gautier, and J. L. Maller Regulation of the G2/M Transition in Xenopus Oocytes by the cAMP-dependent Protein Kinase J. Biol. Chem., July 1, 2005; 280(26): 24339 - 24346. [Abstract] [Full Text] [PDF]

				G. Lominadze, M. J. Rane, M. Merchant, J. Cai, R. A. Ward, and K. R. McLeish Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils J. Immunol., June 1, 2005; 174(11): 7257 - 7267. [Abstract] [Full Text] [PDF]

				D. L. Satinover, C. A. Leach, P. T. Stukenberg, and D. L. Brautigan Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein PNAS, June 8, 2004; 101(23): 8625 - 8630. [Abstract] [Full Text] [PDF]

				P. A. Eyers and J. L. Maller Regulation of Xenopus Aurora A Activation by TPX2 J. Biol. Chem., March 5, 2004; 279(10): 9008 - 9015. [Abstract] [Full Text] [PDF]