Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high-resolution mass spectrometry

Eur. J. Mass Spectrom. 11, 547–556 (2005)
DOI: 10.1255/ejms.722

Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer’s amyloid precursor protein using high-resolution mass spectrometry

Xiaodan Tian,^a* Roxana Cecal,^a JoAnne McLaurin,^b Marilena Manea,^a, Raluca Stefanescu,^a Sandra Grau,^c Mona Harnasch,^c Sarah Amir,^c Michael Ehrmann,^c Peter St George- Hyslop,^b Markus Kohlmann^a,d and Michael Przybylski^a
^aDepartment of Chemistry, Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany
^bCentre for Research on Neurodegenerative Diseases, University of Toronto, Canada
^cSchool of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom
^dPresent Address: Aventis Pharma, Structural & Physical Chemistry, Building G838, Lab. 202, D-65926 Frankfurt am Main, Germany

ABSTRACT:

Alzheimer’s disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is β-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, β-and γ-secretase acting at the N- and C-terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the γ-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Aβ42 has been recently shown to be effective to inhibit and disaggregate Aβ-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Aβ(4–10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS.

Keywords: Alzheimer’s disease, amyloid precursor protein, amyloid peptide, C99, amyloid plaque specific antibody, epitope identification, structural characterisation, high resolution FT-ICR-MS

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