Abstract
BackgroundAs a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Traditional kinase assays rely heavily on the incorporation of γ-P32 radiolabeled isotopes, monoclonal anti-phospho-protein antibodies, or gel shift analysis of substrate proteins. In addition to the expensive and time consuming nature of these methods, the use of radio-ligands imposes restrictions based on the half-life of the radionucleotides and pose potential health risks to researchers. With the shortcomings of traditional assays in mind, the aim of this study was to develop a high throughput, non-radioactive kinase assay for screening Glycogen Synthase Kinase-3beta (GSK-3β) activity.ResultsSynthetic peptide substrates designed with a GSK-3β phosphorylation site were assayed with both recombinant enzyme and GSK-3β immunoprecipitated from NIH 3T3 fibroblasts. A molecular weight shift equal to that of a single phosphate group (80 Da.) was detected by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) in a GSK-3β target peptide (2B-Sp). Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3β used in this assay, this shift was also inhibited by lithium chloride (LiCl), in a dose-dependent manner.ConclusionWe present here a novel method to sensitively measure peptide phosphorylation by GSK-3β that, due to the incorporation of substrate controls, is applicable to either purified enzyme or cell extracts. Future studies using this method have the potential to elucidate the activity of GSK-3β in vivo, and to screen enzyme activity in relation to a variety of GSK-3β related disorders.
Highlights
As a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation
Parent m/z ratio peaks of GSK-3β target synthetic peptides, 2B-Sp, 2B-S, and 2B-A were determined by preparing samples containing untreated peptide substrates for SELDI-TOF-MS analysis
Recombinant GSK-3β induces a shift in the m/z peak of 2BSp Mass to charge ratios of peptide substrates 2B-Sp, 2B-S, and 2B-A incubated with recombinant GSK-3β were analyzed using SELDI-TOF-MS
Summary
As a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Phosphorylation is believed to be the most common protein post-translational covalent modification and is known to occur in the processing of as many as 1/3 of eukaryotic gene products [1]. Functional kinase activity of GSK-3β is important in a variety of biological and biochemical processes and altered GSK-3β activity can contribute to a number of pathological processes including bipolar mood disorder [12,13,14], schizophrenia [15], heart disease [16,17], neurodegeneration [18] Alzheimer's disease [11,19] and diabetes mellitus [11,19,20]. Elucidating the direct activity of GSK-3β phosphorylation activity in vivo is important in contributing to understanding the molecular basis of a variety of disease states
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