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An improved method to unravel phosphoacceptors in Ser/Thr protein kinase‐phosphorylated substrates
Author(s) -
Molle Virginie,
Leiba Jade,
ZanellaCléon Isabelle,
Becchi Michel,
Kremer Laurent
Publication year - 2010
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000316
Subject(s) - kinase , phosphorylation , biochemistry , protein kinase a , mycobacterium tuberculosis , chemistry , proteomics , substrate (aquarium) , protein phosphorylation , biology , cyclin dependent kinase 2 , computational biology , microbiology and biotechnology , tuberculosis , medicine , ecology , pathology , gene
Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli , followed by MS analysis in a single‐step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.