An ESl-MS/MS method for screening of small-molecule mixtures against glycogen synthase kinase-3β (GSK-3β)

Glycogen synthose kinose-3 beta (GSK-3 beta) is involved in the hyperphosphorylation of previously phosphoryloted (primed) substrates, and is currently assayed using an approach based on the incorporation of gamma-P-32-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 phosphorylotion site, and thus are only suitable for beta-unphosphorylated substrates. Herein, we have developed an electrosproy-ionization tandem mass spectrometry (ESI-MSIMS) assay to allow for direct detection of a hyperphosphoryloted product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3 beta. Optimum reaction conditions (level Mg2+, buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3 beta and allow for substrate and product quantification through ES/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3 beta activity from reaction volumes of similar to 40 mu L and that it can be used to assess IC50 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-3 beta.