title: The chemoselective one-step alkylation and isolation of thiophosphorylated cdk2 substrates in the presence of native cysteine.
abstract: The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N(6) -(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.
authors.affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Lee
authors.forename: Lucy M
authors.initials: LM
authors.lastname: Elphick
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.forename: Alexandra M E
authors.initials: AM
authors.lastname: Jones
authors.forename: Emma S
authors.initials: ES
authors.lastname: Child
authors.forename: Alexandra A
authors.initials: AA
authors.lastname: Anderson
authors.forename: Laurent
authors.initials: L
authors.lastname: Bonnac
authors.forename: Natsuko
authors.initials: N
authors.lastname: Suwaki
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
authors.forename: Véronique
authors.initials: V
authors.lastname: Gouverneur
authors.forename: David J
authors.initials: DJ
authors.lastname: Mann
mesh_terms: D000478:Alkylation; D002712:Chlorides / Q000737:chemistry*; D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D017553:Phosphorus Compounds / Q000737:chemistry*; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D002712:Chlorides; D010455:Peptides; D017553:Phosphorus Compounds; D051357:Cyclin-Dependent Kinase 2; C043261:thiophosphoryl chloride; D003545:Cysteine