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Submitted on June 9, 2003
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309
Corresponding Author: Natalie.Ahn{at}Colorado.edu
Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using LC-ESI-MS/MS, 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, Ser12, Thr21, Thr103, Ser116, Thr122, Tyr155 and Thr294, as well as the previously identified regulatory sites, Ser53, Thr295 and Ser349. 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, Lys169Arg, still retains 10% of activity of the wild-type enzyme in vitro, along with occupancy of Thr295 and Ser12. However, mutation of Asp281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Due to 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.
Revised on July 26, 2003
Accepted on July 27, 2003
Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography
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