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.
- SourceAvailable from: Francesca Bruzzese
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- "Protein identification was carried out by peptide mass fingerprinting on an Ettan MALDI-TOF Pro (GE Healthcare )   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. "
ABSTRACT: Several solid tumors are characterized by poor prognosis and few effective treatment options, other than palliative chemotherapy in the recurrent/metastatic setting. Epidermal growth factor receptor (EGFR) has been considered an important anticancer target because it is involved in the development and progression of several solid tumors; however, only a subset of patients show a clinically meaningful response to EGFR inhibition, particularly to EGFR tyrosine kinase inhibitors such as gefitinib. We have recently demonstrated synergistic antitumor effect of the histone deacetylase inhibitor vorinostat combined with gefitinib. To further characterize the interaction between these two agents, cellular extracts from Hep-2 cancer cells that were untreated or treated for 24 h with either vorinostat or gefitinib alone or with a vorinostat/gefitinib combination were analyzed using 2-D DIGE. Software analysis using DeCyder was performed, and numerous differentially expressed protein spots were visualized between the four examined settings. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified; some of these were validated by Western blotting. Finally, a pathway analysis of experimental data performed using MetaCore highlighted a relevant relationship between the identified proteins and additional potential effectors. In conclusion, we performed a comprehensive analysis of proteins regulated by vorinostat and gefitinib, alone and in combination, providing a useful insight into their mechanisms of action as well as their synergistic interaction.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 . After visualization with silver staining, electrophoretic spots were excised, destained  and then dehydrated with acetonitrile (Baker). "
ABSTRACT: In chronic heart failure (CHF), peripheral blood mononuclear cells (PBMC) might undergo structural and/or functional alterations as a consequence of the development and progression of the disease. This study was aimed at: (1) assessing the proteome profile of PBMC from Controls and CHF subjects, (2) identifying differentially-expressed proteins in healthy subjects and patients, and (3) analysing the expression of these proteins in patients after heart transplantation. Proteome changes were assessed in PBMC from 8 healthy and 11 end-stage CHF (6 Ischaemic Heart Failure [IHF], 5 Dilated CardioMyopathy [DCM]) subjects by gel electrophoresis, PD-Quest analysis and mass spectrometry. Eighteen proteins were differentially expressed in Controls and CHF patients. However, among CHF patients, these proteins were equally expressed in IHF and DCM subjects. Eleven proteins were found to belong to 4 functional classes (3 cytoskeletal, 4 cell-cycle progression, 2 stress response and DNA repair, 2 energetic metabolism proteins). Changes in three of the differentially-expressed proteins were also confirmed by Western blot and were reversed after heart transplantation. Results demonstrate an altered protein expression profile in PBMC of CHF patients compared to Controls, thus providing a basis for further diagnostic and prognostic tests for CHF.European Journal of Heart Failure 09/2008; 10(8):749-57. DOI:10.1016/j.ejheart.2008.06.003 · 6.58 Impact Factor
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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  . "
ABSTRACT: We identified the proteins involved during apoptosis induced by H2O2 in Saccharomyces cerevisiae, and analyzed the global protein pattern by 2-DE. We analyzed classical parameters of apoptosis such as chromatin condensation, DNA fragmentation, and morphology changes of cells. Exposure of yeast cells to nonphysiological doses of peroxides decreases the expression (or increases degradation) of enzymes involved in protection against oxidative stress. This leads the yeast cells to a reduction of their antioxidant defense and makes the cells more prone to apoptosis. In our data the down expression of peroxiredoxin II and GST I, could induce a perturbation of mitochondrial function with an alteration of permeability of the membrane leading to the mitochondria-mediated apoptosis. Moreover, we identified a new spot of a classical glycolytic enzyme: the glyceraldehyde 3-phosphate dehydrogenase during apoptosis. It is known that GAPDH is an extremely abundant glycolytic enzyme with multiple functions and that its overexpression is evident during apoptosis induced by a variety of stimuli. Our results confirm that it is a major intracellular messenger mediating apoptotic death and that this new spot of GAPDH could be an intracellular sensor of oxidative stress during apoptosis induced by H2O2 in S. cerevisiae.PROTEOMICS 05/2007; 7(9):1434-45. DOI:10.1002/pmic.200600796 · 3.97 Impact Factor