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- Title
A novel mass spectrometry-based assay for GSK-3β activity.
- Authors
Bowley, Erin; Mulvihill, Erin; Howard, Jeffrey C; Pak, Brian J; Bing Siang Gan; O'Gorman, David B
- Abstract
Background: 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. 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. Results: Synthetic 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. Conclusion: We 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.
- Subjects
PROTEOMICS; GENOMICS; PHOSPHORYLATION; MICROBIOLOGICAL assay; LITHIUM chloride
- Publication
BMC Biochemistry, 2005, Vol 6, p29
- ISSN
1471-2091
- Publication type
Article
- DOI
10.1186/1471-2091-6-29