Functional Proteomics Analysis of Signal Transduction Pathways of the Platelet-Derived Growth Factor β Receptor †

Universitätsklinikum Jena, Jena, Thuringia, Germany
Biochemistry (Impact Factor: 3.02). 03/1999; 38(6):1757-64. DOI: 10.1021/bi982093r
Source: PubMed


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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    • "Protein identification was carried out by PMF on an Ettan MALDI-TOF Pro (GE Healthcare), as previously described [22,23]. Electrophoretic spots from SYPRO Ruby stained gels were mechanically excised by an Ettan Spot Picker (GE Healthcare), destained in 2.5 mM ammonium bicarbonate and 50% acetonitrile, and dehydrated in acetonitrile. "
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    Full-text · Article · Oct 2011 · Current Proteomics
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    • "Protein identification was carried out by peptide mass fingerprinting on an Ettan MALDI-TOF Pro (GE Healthcare ) [17] [18] or by peptide sequencing by MS/MS on an ESI-ion trap LCQ DECA IT mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Details of all procedures used have been included in the Supporting Information. "
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