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Analysis of Aβ Interactions 73<br />
are then converted into mature fibrils, which adopt cross-�-pleated sheets (9)<br />
and eventually fibrils (10). Interestingly, it is the intermediate products, rather<br />
than the mature fibrils that have been shown to be the most neurotoxic (8,10,11).<br />
The mechanisms by which A� induce toxicity include (1) oxidative stress<br />
(12), (2) direct disruption of membrane integrity (13), and(3) alteration in<br />
Ca 2+ homeostasis (14). Oxidative stress is initiated by binding interactions<br />
between A� and metal ions (Cu 2+ ,Fe 3+ ) via histidine residues 6, 13, and 14<br />
(15). This interaction leads to the reduction in the oxidation state of metal<br />
ions and generation of H2O2 with the subsequent generation of free radicals<br />
that induce oxidative damage, including lipid peroxidation and the subsequent<br />
disruption of the cellular membrane. Methionine 35 (Met35) of A� is also<br />
thought to play a critical role since oxidized Met35 A� products have been<br />
found in postmortem AD plaques (16). Furthermore, peptides lacking Met35<br />
have a decreased capacity to reduce Cu 2+ and generate H2O2 (17). A� has<br />
also been demonstrated to directly interact with membrane lipids to form cation<br />
selective channels (18). It is thought that these channels disrupt ion homeostasis<br />
(18), leading to an accumulation of intracellular Ca 2+ levels and the subsequent<br />
induction of apoptosis (19).<br />
The study of A� and the mechanisms described, including (1) production<br />
of toxic A� fragments, (2) generation of aggregates, and (3) interactions<br />
with membranes is essential to further understand the pathology of disease.<br />
ProteinChip R○ technology is a method that facilitates the analysis of all these<br />
processes. Information acquired from such analysis will not only help to further<br />
elucidate the precise role of A� in AD, but may have wider implications for<br />
the development of an anti-A� therapeutic that blocks the toxic effects of the<br />
molecule. Furthermore, this technology provides a valuable means to assess the<br />
effectiveness of therapeutic intervention. This chapter will describe ProteinChip<br />
technology, its advantages over traditional techniques, and how it can be used<br />
for the analysis of the mechanisms described.<br />
1.2. ProteinChip Technology<br />
ProteinChip technology employs surface enhanced laser desorption/<br />
ionization time-of-flight mass spectrometry (SELDI-TOF MS), which combines<br />
two well-established methods of solid phase chromatography and TOF-MS into<br />
an integrated platform (20). The proprietary ProteinChip arrays distinguish this<br />
technology from other MS-based systems. The arrays possess chromatographic<br />
surfaces including hydrophobic, hydrophilic, anion exchange, cation exchange,<br />
and immobilized-metal affinity and are utilized to enrich for subsets of the<br />
proteome with common biochemical properties. Furthermore, we have adapted<br />
this technology to create a synthetic solid phase membrane layer on the array
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