title: A nascent proteome study combining click chemistry with 2DE.
abstract: To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.
authors.affiliation: Department of Chemistry, A.N. Belozersky Institute for Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Ilya A
authors.initials: IA
authors.lastname: Osterman
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Ustinov
authors.forename: Denis V
authors.initials: DV
authors.lastname: Evdokimov
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Korshun
authors.forename: Petr V
authors.initials: PV
authors.lastname: Sergiev
authors.forename: Marina V
authors.initials: MV
authors.lastname: Serebryakova
authors.forename: Irina A
authors.initials: IA
authors.lastname: Demina
authors.forename: Maria A
authors.initials: MA
authors.lastname: Galyamina
authors.forename: Vadim M
authors.initials: VM
authors.lastname: Govorun
authors.forename: Olga A
authors.initials: OA
authors.lastname: Dontsova
mesh_terms: D001419:Bacteria / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D057930:Click Chemistry; D004917:Erythromycin / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling; D057825:Two-Dimensional Difference Gel Electrophoresis
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D004917:Erythromycin