Peptide-Based Drug Design

74 Giannakis et al.
surface to analyze membrane binding proteins. Proteins retained on the chip
surface are resolved via TOF-MS, which displays the mass-to-charge value and
signal intensities of the proteins (20).
ProteinChip arrays with preactivated surfaces are also available for covalently
coupling of a specific “bait” molecule (protein, DNA, RNA). This allows the
investigation of specific protein interactions such as DNA–protein, receptor–
ligand, and antibody-antigen (Ab-Ag), the latter of which permits the generation
of a standard curve and hence quantitation studies (21).
1.3. Quantitation of Aβ
Traditional methods employed to examine A� processing include enzyme
linked immunosorbant assays (ELISA) and SDS-PAGE/Western blotting.
ELISA generates an average signal of all peptide species bound to the
antibody Ab and hence has poor assay specificity. Analysis of A� by SDS-
PAGE/Western blotting exhibits poor discriminatory power between the variant
forms of the peptide. In addition, molecular weights of peptides cannot be
accurately determined. Hence, using these techniques for the analysis of multiple
A� species can be difficult.
In contrast, ProteinChip technology allows the detailed analysis of A�
fragments and their modifications (22). Peptides are captured on arrays coated
with anti-A� Ab, washed to remove nonspecifically bound fragments, and finally
detected by SELDI-TOF MS. The resolution of the technique allows the discrimination
of peptides of similar mass and modified products, e.g., oxidized peptides
versus native peptides, which differ by only 16 Da. ProteinChip technology has
been used extensively to detect A� fragments in various samples, including cell
culture supernatants, serum, plasma, and cerebrospinal fluid (CSF) (21–23).
1.4. Aβ Aggregation and Interactions with Lipids
Analysis of A� aggregation typically involves performing circular dichoism
(CD) spectroscopy, thioflavin T assays, electron microscopy, SDS-PAGE,
and chromatography. These techniques describe the aggregation of A� in a
general sense without the ability to accurately quantitate the generation of
specific oligomeric species. Photo-induced cross-linking of unmodified proteins
(PICUP) assays have also been used for assessing oligomeric profiles. This
method employs photolysis to induce covalent bonds between transiently
formed oligomers. The PICUP method, however, is prone to artifacts by crosslinking
transitory interactions. In addition, PICUP oligomers are assessed via
SDS-PAGE/Western blotting, the limitations of which have been previously
described. In contrast, SELDI-TOF MS can be used to generate oligomeric
profiles and assess relative changes in stable oligomeric species.