Methodology article

A novel mass spectrometry-based assay for GSK-3β activity

Erin Bowley^{* 1} , Erin Mulvihill^{* 1} , Jeffrey C Howard¹ , Brian J Pak² , Bing Siang Gan^1,3,4,5 and David B O'Gorman^1,3

¹Cell and Molecular Biology Laboratory, Hand and Upper Limb Centre, Lawson Health Research Institute, St. Joseph's Health Centre, London, Ontario, Canada

²Ciphergen Biosystems International Inc., Fremont, California, USA

³Department of Surgery, University of Western Ontario, London, Ontario, Canada

⁴Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada

⁵Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

author email corresponding author email* Contributed equally

BMC Biochemistry 2005, 6:29doi:10.1186/1471-2091-6-29

Published:	16 December 2005

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.