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GT4.- Proteomic analysis of cellular responses to protein-aggregation-mediated stress
Perspectives of extension of this common project to a more ambitious proposal to be submitted to funding agencies : Long term Added value of the cooperation : exchange of expertise
Summary of the proposal :
Protein aggregation is related with an increasing number of human diseases.Our purpose is to determine if there is a common response against protein aggregation and to identify the proteins involved.
To achieve this we wanted to compare the proteomic response related with the aggregation of Transthyretin (associated with senile systemic amyloidosis) and amyloid-β-peptide (Alzheimer’s disease) in yeast. The changes at the proteome level will be analyzed by comparing the protein distribution in cells expressing aggregating and non aggregating versions of the protein. Cells would be grown in controlled chemostats and high-throughput differential proteomics analysis will be applied; specifically the 2D-DIGE (Differential in Gel Electrophoresis) methodology will be used. The 2D-DIGE technique requires the use of a scanner with multi-wavelength fluorescence detection, available at ITQB. After the 2D electrophoretic separation, gels will be scanned with a high resolution fluorescent scanner (Fuji Film Fla-5100) at different wavelengths depending on the dye used. Subsequently, images will be analyzed with 2DE software Progenesis Samespots (Nonlinear Dynamics) in order to assess significant differences in protein expression (spot intensities).
Quantitative differences will be evaluated by ANOVA and t-student tests. Cluster and principal component analysis will also be used to verify data quality. The protein spots shown to be differential expressed will be identified using a combined analysis of PMF (Peptide Mass Fingerprint) and tandem mass data, acquired by a MALDI/TOF-TOF mass spectrometer (model 4800, Applied Biosystems) and by a linear ion trap mass spectrometer with electrospray ionization (model LTQ, Thermo-Finnigan) coupled to a micro-RP HPLC (Thermo-Finnigan). Several software for protein identification, ProteinPilot using the Paragon™ algorithm (Applied Biosystems); on-line MASCOT; GPS (Applied Biosystems) and Bioworks™ 3.3.1 (Thermo-Finnigan) will be used in order to achieve better confidence in protein identification.