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Simultaneous Assessment of Kinetic, Site‐Specific, and Structural Aspects of Enzymatic Protein Phosphorylation
Author(s) -
van de Waterbeemd Michiel,
Lössl Philip,
Gautier Violette,
Marino Fabio,
Yamashita Masami,
Conti Elena,
Scholten Arjen,
Heck Albert J. R.
Publication year - 2014
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201404637
Subject(s) - autophosphorylation , phosphorylation , protein phosphorylation , enzyme , kinase , biochemistry , chemistry , peptide , substrate (aquarium) , protein kinase a , biophysics , biology , microbiology and biotechnology , ecology
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.