Peptide-Based Drug Design

80 Giannakis et al.
and off-scale readings, under which conditions the detector will be saturated and
quantitation is not possible. This will also negatively affect the reproducibility of
the assay. In such cases, consider decreasing the laser intensity value until peaks
are sharp and well resolved (see Note 9).
Calibrate the spectra to ensure reliable mass accuracy. Generate a calibration
equation using calibrants that cover the mass range of interest, and then match
the calibrant and sample matrix. External mass calibration can subsequently be
applied to the relevant spectra.
3.1.2.6. QUANTITATIVE ASSESSMENT OF THE DATA
Relative changes in the concentration of A� can be determined simply by
assessing the differences in peak intensity (signal-to-noise/area under the curve)
between the sample groups. In order to quantitate the amount of A� present in
the sample, a standard curve must be generated. A� standards of known concentration
should be spiked into a control sample, which has been treated in the
exact same manner as the remaining control and test samples, including final
buffer constituents, total protein concentration, storage, etc.
The sensitivity of the assay is peptide specific, consequently it is important to
generate a standard curve for each peptide fragment. Threefold dilutions starting
from 100 to 0.13 nM are a recommended starting point, although depending on
the sensitivity of the assay, it may be necessary to either decrease or increase the
initial starting concentrations. Once the data have been collected, export the peak
information into an Excel spread sheet, including signal-to-noise values/area
under the curve, and use this information to plot the standard curve. From this
curve the R 2 value and slope-intercept equation (y = mx + b) can be generated.
The R 2 value is an indicator of the models “goodness of fit”; it is expressed as
a fraction between 0.0 and 1.0, with high values reflecting a good quality model
(see Note 10). The slope-intercept equation is used to determine the value of the
test sample, where y is the unknown value, x is the signal-to-noise of the sample,
m is the slope, and b is the y-intercept value.
3.2. Capture of Aβ Aggregates on ProteinChip Arrays
3.2.1. Preparation of Aβ Aggregates
1. Dissolve lyophilized A� in hexafluoroisopropanol to a final concentration of
1 mg/mL to induce a monomeric and helical conformation of the peptides.
2. Aliquot 100 �L of peptides into Eppendorf tubes and dry using a speed vacuum
system.
3. As required, resuspend 100-�g aliquots in 50 �L of20mMNaOH and sonicate
for 15 min.
4. Dissolve A� solution into 50 �L of PB and 400 �L ofH2O.