Functional Proteomics Analysis of Signal Transduction Pathways of the Platelet-Derived Growth Factor β 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: Luca Bini
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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. "
ABSTRACT: Pulmonary Langerhans-cell histiocytosis (PLCH) is a rare interstitial lung disease characterized by clusters of Langerhans cells, organized in granulomas, in the walls of distal bronchioles. It is a diffuse lung disease related to tobacco smoking but otherwise of unknown etiopathogenesis. In this study we used a proteomic approach to analyze BAL protein composition of patients with PLCH and of healthy smoker and non-smoker controls to obtain insights into the pathogenetic mechanisms of the disease, to study the effect of cigarette smoking on susceptibility to PLCH and to identify potential new biomarkers. Two-dimensional electrophoresis and image analysis revealed proteins that were differently expressed (quantitatively and qualitatively) in the three groups of subjects. The proteins were identified by mass spectrometry and have various functions (antioxidant, proinflammatory, antiprotease) and origins (plasma, locally produced, etc.). Many, such as protease inhibitors (human serpin B3) and antioxidant proteins (glutathione peroxidase and thioredoxin) are already linked to PLCH pathogenesis, whereas other proteins have never been associated with the disease. Interestingly, numerous proteolytic fragments of plasma proteins (including kininogen-1 N fragments and haptoglobin) were also identified and suggest increased proteolytic activity in this inflammatory lung disease. Differences in protein expression were found between the three groups and confirmed by Principal Component Analysis (PCA). Analysis of BAL proteomes of PLCH patients and of smoker and non-smoker controls also proved to be useful for researching the pathogenetic mechanisms and for identifying biomarkers of this rare diffuse lung disease.Journal of Clinical Bioinformatics 11/2011; 1(1):31. DOI:10.1186/2043-9113-1-31
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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 · 3.81 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 · 6.53 Impact Factor