A novel mass spectrometry-based assay for GSK-3beta activity.

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 gamma-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-3beta) activity. RESULTS: Synthetic peptide substrates designed with a GSK-3beta phosphorylation site were assayed with both recombinant enzyme and GSK-3beta 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-3beta target peptide (2B-Sp). Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3beta 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-3beta 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-3beta in vivo, and to screen enzyme activity in relation to a variety of GSK-3beta related disorders.

Beta-catenin signaling in fibroproliferative disease.

BACKGROUND: Beta-catenin has been historically recognized as both an intermediate in the "canonical Wnt signaling pathway" and as a component of functional adherens junctions. MATERIALS AND METHODS: Cellular accumulation of beta-catenin levels can result in transactivation of gene transcription and cellular proliferation during normal cellular and disease development. Recent evidence has identified beta-catenin in an additional role as a component of cutaneous wound healing. RESULTS: This finding is in keeping with previous observations that post-translational modifications of beta-catenin that are associated with its cytoplasmic accumulation are frequently observed in fibroproliferative diseases with characteristics of dysregulated wound healing. These diseases include hypertrophic scar formation, aggressive fibromatoses, Lederhose disease, and Dupuytren's contracture (DC). CONCLUSIONS: While its precise roles in disease initiation and progression remain to be explored, this review highlights our current knowledge of beta-catenin regulation and describes some potential upstream mediators of beta-catenin accumulation and signaling in fibroproliferative disease.

Identification of protein biomarkers in Dupuytren's contracture using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS).

BACKGROUND: To study the protein expression profiles associated with Dupuytren's contracture (DC) to identify potential disease protein biomarkers (PBM) using a proteomic technology--Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). METHODS: Normal and disease palmar fascia from DC patients were analyzed using Ciphergen's SELDI-TOF-MS Protein Biological System II (PBSII) ProteinChip reader. Analysis of the resulting SELDI-TOF spectra was carried out using the peak cluster analysis program (BioMarker Wizard, Ciphergen). Common peak clusters were then filtered using a bootstrap algorithm called SAM (Significant Analysis of Microarrays) for increased fidelity in our analysis. RESULTS: Several differentially expressed low molecular weight (<20 kDa) tissue proteins were identified. Spectra generated using both ZipTipC18 aided Au array and WCX2 array based SELDI-TOF-MS were reproducible, with an average peak cluster mass standard deviation for both methods of <1.74 x10(-4). Initial peak cluster analysis of the SELDI spectra identified both up-(14) and down-(3)regulated proteins associated with DC. Further analysis of the peak cluster data using the bootstrap algorithm SAM identified three disease-associated protein features (4600.8 Da, 10254.5 Da, and 11405.1 Da) that were elevated (5.45, 11.7, and 4.28 fold, respectively, with a 0% median false discovery rate). CONCLUSION: SELDI-TOF-MS identified three potential low molecular weight tissue protein markers (p4.6DC, p1ODC, p11.7DC) for DC. The ability of SELDI-TOF-MS to resolve low molecular weight proteins suggests that the method may provide a means of deciphering the biomarker-rich low molecular weight region of the human proteome. Application of such novel technology may help clinicians to focus on specific molecular abnormalities in diseases with no known molecular pathogenesis, and uncover therapeutic and/or diagnostic targets.