An ESI‐MS/MS Method for Screening of Small‐Molecule Mixtures against Glycogen Synthase Kinase‐3β (GSK‐3β)
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abstract
AbstractGlycogen synthase kinase‐3β (GSK‐3β) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of γ‐32P‐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 β‐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‐3β. Optimum reaction conditions (level of Mg2+, buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK‐3β and allow for substrate and product quantification through ESI/MS/MS. We show that the MS‐based assay allows for rapid determination of GSK‐3β activity from reaction volumes of ∼40 μL 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‐3β.
