Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor.
ABSTRACT We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.
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ABSTRACT: A novel fluorescence detection system using a chemosensor for phosphoprotein in gel electrophoretic analysis has been developed. The system employed the alizarin red S -aluminum (III)-appended complex as a fluorescent staining dye to perform the convenient and selective detection of phosphorylated proteins and total proteins in SDS-PAGE, respectively. Therefore, a full and selective map of proteins can be achieved in the same process without resorting to other compatible detection methods. As low as 62.5 ng of α-casein (7 or 8 phosphates) and β-casein (5 phosphates), 125 ng of ovalbumin (2 phosphates) and κ-casein (1 phosphate) can be detected in approximately 135 min, with the linear responses of rigorous quantitation of changes over a 125-4000 ng range. As a result, ARS stain may provide a new choice for selective, economic and convenient visualization of phosphoproteins.Electrophoresis 10/2012; · 3.26 Impact Factor
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ABSTRACT: Proteomic approaches have advanced clinical research towards more reliable, sensitive and specific biological diagnostic markers for diseases. Mood disorders are most difficult to diagnose and very much prevalent in society; hence, their proper diagnosis becomes essential. Despite tremendous research efforts to dissect the neurobiological basis of psychiatric disorders, the diagnosis and evaluation for such diseases is still poor. Biomarker discovery in psychiatry research has been accelerated by proteomic technologies, accepting the challenges in order to meet disease state-related investigations. Proteomics-based research for disease-specific protein signatures is expected to give a new direction in psychiatry research. Therefore, this may become a more powerful tool to predict the development, course and outcome of the disease towards personalized psychiatric ailments. The review discusses the role of proteomics in elucidating mechanisms of psychiatric disorders, current status, prospects, limitations and new possibilities towards a strong diagnostic tool in the clinical laboratory.Expert Review of Proteomics 06/2012; 9(3):249-65. · 3.90 Impact Factor
Dataset: Mol. BioSyst., 2011, 7, 2855–2862