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 fluorescence-based stain with 3,5,7,2′,4′-pentahydroxyflavone (morin hydrate, MH) was designed to stain phosphoproteins in one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Al3+ was applied as a “fixed bridge,” providing an efficient energy transfer channel between phosphoprotein and MH, to produce a strong fluorescent complex for the determination of phosphoprotein. As little 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) could be visualized with a wide linear dynamic range. In comparison with conventional methods, MH stain is a time-saving method that takes just 90 min. It also has good compatibility with routine protein stainings such as Coomassie Brilliant Blue R (CBBR) and SYPRO Ruby for total protein analysis.Analytical Biochemistry 04/2013; 435(1):19–26. · 2.58 Impact Factor
Dataset: Mol. BioSyst., 2011, 7, 2855–2862
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ABSTRACT: A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS-PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al(3+) were employed respectively. The staining method, Fura 2 stain, has sensitivities of 16-32 ng of α-casein and β-casein, 62 ng of ovalbumin, phosvitin and κ-casein using an ultra-violet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other post-staining. Considering the low cost, simplicity and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research. This article is protected by copyright. All rights reserved.Electrophoresis 01/2014; · 3.26 Impact Factor