Linked Life Data

12885952

Source:http://linkedlifedata.com/resource/pubmed/id/12885952

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2003-10-28
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1535-9476
pubmed:author
pubmed:issnType
Print
pubmed:volume
2
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1055-67
pubmed:dateRevised
2011-7-11
pubmed:meshHeading
pubmed-meshheading:12885952-Amino Acid Sequence, pubmed-meshheading:12885952-Amino Acid Substitution, pubmed-meshheading:12885952-Animals, pubmed-meshheading:12885952-Base Sequence, pubmed-meshheading:12885952-Binding Sites, pubmed-meshheading:12885952-Chromatography, Affinity, pubmed-meshheading:12885952-Cloning, Molecular, pubmed-meshheading:12885952-DNA, pubmed-meshheading:12885952-Mass Spectrometry, pubmed-meshheading:12885952-Molecular Sequence Data, pubmed-meshheading:12885952-Mutagenesis, Site-Directed, pubmed-meshheading:12885952-Phosphopeptides, pubmed-meshheading:12885952-Phosphorylation, pubmed-meshheading:12885952-Protein-Serine-Threonine Kinases, pubmed-meshheading:12885952-Proteomics, pubmed-meshheading:12885952-Recombinant Proteins, pubmed-meshheading:12885952-Xenopus laevis
pubmed:year
2003
pubmed:articleTitle
Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography.
pubmed:affiliation
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
pubmed:publicationType
Journal Article, In Vitro, Research Support, U.S. Gov't, P.H.S., Research Support, N.I.H., Extramural