Peptide-Based Drug Design

Analysis of Aβ Interactions 75
Analysis of A�–lipid interactions involves performing ultracentrifugation or
size filtration chromatography of vesicles exposed to A�, CD spectroscopy, or
surface plasmon resonance (SPR) technology. Chromatography and ultracentrifugation
require several hours, during which time the peptides can further
aggregate, complicating the interpretation of the data. CD indicates structural
changes to the peptide upon interactions with lipids, but quantitative interpretation
is difficult. SPR allows lipid-binding affinities to be assessed, but
determination of the specific oligomeric species interacting with the lipids
is not possible. Consequently, we have developed novel aggregation lipidbinding
assays using SELDI-TOF MS, in which vesicles are adsorbed to
hydrophobic H50 ProteinChip arrays, forming a supported lipid monolayer
with the hydrophilic heads facing the solution. This assay, unlike conventional
techniques, allows the rapid detection and quantitation of individual oligomeric
species which are bound specifically and directly to the lipid-coated array.
2. Materials
2.1. Capture and Quantitation of Aβ Using Antibody-Coated
ProteinChip Arrays
1. ProteinChip arrays: PS10, PS20 (Bio-Rad Laboratories, Hercules, CA).
2. Ab: Anti-human A� (0.5 mg/mL) in Tris-free buffer (see Table 1 for list of
commonly used Abs).
3. A� peptide standards.
4. Phosphate-buffered saline (PBS): 137 mM NaCl, 10 mM phosphate buffer,
pH 7.4.
5. Deactivation buffer: 0.5 M ethanolamine in PBS, pH 8.0.
6. PBST: PBS, 0.5% Triton X-100.
7. 1 mM hydroxyethyl piperazine ethane sulfonate (HEPES), pH 7.2.
Table 1
Common Ab Used for Detection of A�, Including Information Pertaining to the
Type (mAb/pAb) and Specificity of the Ab (epitope recognition site)
Antibody Type (mAb/pAb) Specificity (Ref.)
4G8 mAb 17-24 aa (25)
6E10 mAb 1-12 aa (25)
G210, G211 mAb C-terminal residues of A�-40 and -42,
respectively (26)
WO2 mAb 4-10 aa (27)
Anti-A� 40 pAb 704-711 aa of APP (28)
Anti-A� 42 pAb 706-713 aa of APP (28)