title: Detection of low-abundance protein phosphorylation by selective 18O labeling and mass spectrometry.
abstract: Reversible phosphorylation regulates the majority of intracellular networking and pathways. The study of this widely explored post-translational modification is usually challenged by low stoichiometric levels of modification. Many approaches have been developed to overcome this problem and to achieve rigorous characterization of protein phosphorylation. We describe a method for enhanced detection of low-abundance protein phosphorylation that uses selective introduction of (18)O label into phosphorylation sites with H(2)(18)O and mass spectrometric detection. The method was applied to introduce (18)O label into bacterially expressed Aurora A kinase phosphorylation sites and resulted in the representation of phosphorylated peptides as doublets or triplets according to the number of phosphate groups. A total of 28 phosphopeptides were observed by this method.
authors.affiliation: The Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre (MIB), Manchester, United Kingdom.
authors.forename: Waleed M
authors.initials: WM
authors.lastname: Alghamdi
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gaskell
authors.forename: Jill
authors.initials: J
authors.lastname: Barber
mesh_terms: D000595:Amino Acid Sequence; D064127:Aurora Kinases; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D010103:Oxygen Isotopes; D010748:Phosphopeptides; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin