title: Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Haydon
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Eyers
authors.forename: Lauren D
authors.initials: LD
authors.lastname: Aveline-Wolf
authors.forename: Katheryn A
authors.initials: KA
authors.lastname: Resing
authors.forename: James L
authors.initials: JL
authors.lastname: Maller
authors.forename: Natalie G
authors.initials: NG
authors.lastname: Ahn
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D064127:Aurora Kinases; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
chemical_list: D010748:Phosphopeptides; D011994:Recombinant Proteins; D004247:DNA; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases