Identification and Structural Characterisation of Carboxy-Terminal Polypeptides and Antibody Epitopes of Alzheimer's Amyloid Precursor Protein Using High-Resolution Mass Spectrometry

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 beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-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 gamma-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 Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-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 Abeta(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. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

“…In order to characterize the possible effect of the sequence environment of an NTresidue, the affinities and specificities of the monoclonal anti-3-NT antibody (legend to Figure 1) used for the proteolytic affinity extraction-MS analysis were assessed by an indirect ELISA method [33], using synthetic Tyrnitrated peptides comprising all tyrosine residues in ECP (Table S1, Figure S7; Supporting Information). The Tyr 33 -nitrated peptide ECP (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) showed substantially higher affinity compared with all other nitrated ECP peptides; in contrast, the Tyr 98 -nitrated peptide which is completely buried in the protein structure, revealed only minimal affinity. These results showed that the affinity differences between the nitrated ECP peptides correlate with a specific sequence environment of the tyrosine residues.…”

“…In contrast, the direct identification of nitrations without affinity-extraction using HPLC-MS, such as in the auto-nitration site of eosinophilperoxidase (EPO) at Tyr 349 required highly purified protein and was only obtained after scrutinous HPLC separation of Figure 3. Identification of the tyrosine nitration site at Tyr −33 in eosinophil-derived neurotoxin by nano-ESI-FTICR-MS of the thermolysine peptide fragmnent, EDN (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). The modification denoted with X corresponds to carbamidomethylation at Cys-37.…”

“…The affinity of the nitrated ECP peptide was found to be completely abolished upon replacing the positively charged residues Arg 28 , Arg 34 , Arg 36 , and Lys 38 by alanine, thus confirming the importance of cationic amino acids in the vicinity to the nitration site for affinity binding. Affinity determinations of the nitrated peptide in comparison to the intact ECP protein using the SAW biosensor provided dissociation constants of approximately 6 nM for the nitrated peptide ECP (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41), and 28 nM for the intact ECP.…”

“…Recent work in our laboratory has focused on the development of combined affinity-mass spectrometry methods for the identification of biomolecular recognition structures, such as protein antigen-epitopes [29][30][31][32][33]. In this CI we describe a general approach for the structural identification of protein nitrations by a combination of proteolytic affinity extraction and ESI mass spectrometry ( Figure 1).…”

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

“…These results and the most recent clinical studies confirming the epitope specificity (ADPD Congress, March 14-17, 2007, Salzburg, Austria) emphasize the therapeutic potential of plaque-specific antibodies. Using epitope excision -mass spectrometry and alanine scanning mutagenesis, we have previously shown that the mouse monoclonal 6E10 antibody, directed against β-amyloid (1-17), recognizes the same short epitope (FRHDSGY) at the N-terminus of Aβ, as did the antibodies resulting from active immunization of transgenic mice with Aβ(1-42) (11,12). Therefore, it is our hypothesis that primary structure details of this plaque-specific antibody (6E10) will provide a better understanding of the antigen recognition process at the molecular level and contribute to the development of more effective vaccines.…”