Strategies for examination of Alzheimer's disease amyloid precursor protein isoforms.
We describe a proteomics procedure using bioinformatics, immunoprecipitation, two-dimensional gel electrophoresis, Western blotting, in-gel digestion, LC-MS, MALDI-MS, and MS-MS for isolation and identification of amyloid precursor protein (APP) isoforms APP695, APP751, and APP770. Retinoic acid-induced Ntera 2 cell line, derived from a human teratocarcinoma cells, was the in-vitro source of APP. Initial isolation of whole APP was performed by immunoprecipitation, using AB10, 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 stage was separation of whole APP into its isoform components by two-dimensional gel electrophoresis. Because of low APP concentrations, detection by the usual staining methods, for example Sypro Ruby, able to detect low picomole concentrations, did not enable visualisation of the isoforms. Western analysis, however, enabled primary detection of APP, because of the inherent sensitivity of antibodies raised to specific isoform regions. This initial visualization acted as a template for excision of isoforms from 2D gels, which were then subjected to peptide mass mapping. Initial theoretical digestion of each isoform revealed the presence of specific peptides, which were then used as "tags" for isoform detection.
Newton
Jillian R A
JR
Biomedical Research Centre, Sheffield Hallam University, Howard, Sheffield, S1 1WB, UK.
Parkinson
David
D
Clench
Malcolm R
MR
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002853:Chromatography, Liquid; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D020033:Protein Isoforms