Strategies for the examination of Alzheimer's disease amyloid precursor protein isoforms.

NEWTON, Jillian R.A. (2004). Strategies for the examination of Alzheimer's disease amyloid precursor protein isoforms. Doctoral, Sheffield Hallam University (United Kingdom)..

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Abstract

The principal aim of this research project has been the utilisation of various proteomic techniques in the investigation of the Alzheimer's disease amyloid precursor protein (APP) isoforms, namely APP[695], APP[751] and APP[770]. One of the most noticeable pathological characteristics of Alzheimer's disease is the presence of neuritic plaques in brain tissue. The chief protein constituent of neuritic plaques is the beta amyloid peptide. This peptide is proteolytically cleaved from APP, as such the interest in APP isoforms is great and a rapid detection method for the presence of each isoform would be a huge advantage to the research effort with regards to the determination and concentration in both diseased and non-diseased states. Two-dimensional gel electrophoresis and peptide mass fingerprinting are two of the most important techniques in the proteomics arena and both are investigated fully in this work. Retinoic acid induced Ntera 2 cells, derived from a human teratocarcinoma cell line, were the in vitro source of APP. Initial isolation of APP was performed by immunoprecipitation, using a monoclonal antibody raised to amino acids 1-17 of the beta-amyloid peptide sequence, which is present in all three alpha secretase cleaved isoforms of interest. The next step was to separate whole APP into its isoform components by two-dimensional gel electrophoresis. The resulting protein spots were then subjected to peptide mass fingerprinting employing the different digest reagents, trypsin, endoproteinase Asp-N and formic acid. Initial distinction between the APP isoforms could be seen upon examination of theoretical in silica digests using the various digest reagents mentioned. The in silica digests revealed peptides unique to each isoform that in theory could be used as indicators of isoform presence.

Item Type: Thesis (Doctoral)
Additional Information: Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 2004.
Research Institute, Centre or Group: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:23
Last Modified: 10 Apr 2018 17:23
URI: http://shura.shu.ac.uk/id/eprint/20769

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