We have initiated a study using immuno-affmity technique combined with matrix-assisted laser desorption mass spectrometry to characterize P-Amyloid related peptides. In this study, monoclonal anti-p-amyloid peptide antibody was used as a """"""""fishhook"""""""" to specifically and sensitively capture the target peptide-amyloid related peptides in samples. Amyloid related peptides was first immunoprecipitated by monoclonal Anti-amyloid antibodies. The antibody-antigen complex was precipitated by protein A/Gagarose. After washing away non-specific bonded peptides, the complex was analyzed by matrix-assisted laser desorption mass spectrometry. The sensitivity and quantitative aspects of this method has been carefully evaluated. Cell culture equipment was set up in our laboratory to facilitate cell biological studies and studies of the processing of P-Amyloid in vitro. We have used our method to investigate the processing of P-Amyloid protein in a series of cultured cell lines (e.g.,we have investigated the effects of the S 182 gene mutation on chromosome- 14 on the production and degradation of amyloid P protein) and in the near future plan to extend the investigation to an analysis of amyloid peptides in CSF of patients with Alzheimer's disease. A paper describing these results has been published in the J. Biol. Chem. 272, 43234326, 1997. Currently we are: Analyze the effect of detergents on immunoprecipitation/mass spectrometry analysis of proteins. Compare the effeciency of different detergents on extracting intracellular AP peptides. Optimize the washing condition for removing non-specific boned protein and detergents used in immunoprecipitation.
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