title: An ESI-MS/MS method for screening of small-molecule mixtures against glycogen synthase kinase-3beta (GSK-3beta).
abstract: Glycogen synthase kinase-3beta (GSK-3beta) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of gamma-(32)P-radiolabelled isotopes into substrate peptides. The requirement to detect hyperphosphorylation of a primed substrate poses a particular challenge for development of a high-throughput screening assay, as many current kinase assays are designed to produce a signal in the presence of any phosphorylation site, and thus are only suitable for beta-unphosphorylated substrates. Herein, we have developed an electrospray-ionization tandem mass spectrometry (ESI-MS/MS) assay to allow for direct detection of a hyperphosphorylated product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3beta. Optimum reaction conditions (level of Mg(2+), buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3beta and allow for substrate and product quantification through ESI/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3beta activity from reaction volumes of approximately 40 microL and that it can be used to assess IC(50) values and the site of action of known inhibitors. It also can be used for automated screening of small-molecules mixtures to identify inhibitors of GSK-3beta.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. West, Hamilton, ON, L8N 3Z5, Canada.
authors.forename: Ivan
authors.initials: I
authors.lastname: Partserniak
authors.forename: Geoff
authors.initials: G
authors.lastname: Werstuck
authors.forename: Alfredo
authors.initials: A
authors.lastname: Capretta
authors.forename: John D
authors.initials: JD
authors.lastname: Brennan
mesh_terms: D002021:Buffers; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006006:Glycogen Synthase / Q000737:chemistry / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D020128:Inhibitory Concentration 50; D007700:Kinetics; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D002021:Buffers; D054852:Small Molecule Libraries; D012694:Serine; D006006:Glycogen Synthase; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3