title: Simultaneous assessment of kinetic, site-specific, and structural aspects of enzymatic protein phosphorylation.
abstract: Protein phosphorylation is a widespread process forming the mechanistic basis of cellular signaling. Up to now, different aspects, for example, site-specificity, kinetics, role of co-factors, and structure-function relationships have been typically investigated by multiple techniques that are incompatible with one another. The approach introduced here maximizes the amount of information gained on protein (complex) phosphorylation while minimizing sample handling. Using high-resolution native mass spectrometry on intact protein (assemblies) up to 150 kDa we track the sequential incorporation of phosphate groups and map their localization by peptide LC-MS/MS. On two model systems, the protein kinase G and the interplay between Aurora kinase A and Bora, we demonstrate the simultaneous monitoring of various aspects of the phosphorylation process, namely the effect of different cofactors on PKG autophosphorylation and the interaction of AurA and Bora as both an enzyme-substrate pair and physical binding partners.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences and Netherlands Proteomics Center, Utrecht University, Padualaan 8, 3584 CH Utrecht (The Netherlands).
authors.forename: Michiel
authors.initials: M
authors.lastname: van de Waterbeemd
authors.forename: Philip
authors.initials: P
authors.lastname: Lössl
authors.forename: Violette
authors.initials: V
authors.lastname: Gautier
authors.forename: Fabio
authors.initials: F
authors.lastname: Marino
authors.forename: Masami
authors.initials: M
authors.lastname: Yamashita
authors.forename: Elena
authors.initials: E
authors.lastname: Conti
authors.forename: Arjen
authors.initials: A
authors.lastname: Scholten
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000818:Animals; D064096:Aurora Kinase A / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D010766:Phosphorylation / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011506:Proteins; D064096:Aurora Kinase A; D017869:Cyclic GMP-Dependent Protein Kinases
keywords: analytical methods; kinase assay; mass spectrometry; protein modifications; protein-protein interactions