Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor

Universitätsklinikum Jena, Jena, Thuringia, Germany
Biochemistry (Impact Factor: 3.19). 03/1999; 38(6):1757-64. DOI: 10.1021/bi982093r
Source: PubMed

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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    • "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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    Proteomics 09/2011; 11(18):3725 - 3742. DOI:10.1002/pmic.201100092 · 3.97 Impact Factor
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    • "Protein identification was carried out as previously described [17]. After visualization with silver staining, electrophoretic spots were excised, destained [18] and then dehydrated with acetonitrile (Baker). "
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    • "Protein identification was mainly carried out by peptide mass fingerprinting (PMF) on Ettan MALDI-TOF Pro mass spectrometer (GE Healthcare) as previously described [24] [25]. "
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