SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1215

1215

B
1.4

Solid Phase

Solution Phase

1.2
1.0
0.8
0.6

+BACE

- BACE

B4 (302 Da)

B3 (406 Da)

B2 (352 Da)

B1 (374 Da)

B4 (302 Da)

B3 (406 Da)

0.0

B2 (352 Da)

0.2

CompoundAUC/ EG3-ThiolAUC

0.4

B1 (374 Da)

CompoundAUC/ EG3-ThiolAUC

VanderPorten et al.

1.2

B1 HTRF IC50 = 6nM
SAMDI EC50 = 80nM

1.0

B2 HTRFIC50 = 29nM
SAMDI EC50 = 150nM

0.8
0.6

B3 HTRFIC50 = 32nM
SAMDI EC50 = 230nM

0.4

B4 HTRFIC50 = 1800nM
SAMDI EC50 = 1600nM

0.2
0.0

-3

-2

-1

Log10 [compound] ( M)

2.0

1.5

1.0

0.5

0.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

384 plate column

Figure 2. Screening feasibility. (A) Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization (SAMDI) smallmolecule capture of solution-phase versus solid-phase complex formation. (B) Dose-response profiles for the individual compounds
using SAMDI are shown. The extrapolated individual compound SAMDI binding EC50 values correlates with β-secretase HTRF IC50
data obtained in a separate experiment. (C) SAMDI plate uniformity of a 384-well array with alternating columns of compound with
and without protein pull down. Comparing screening data in samples with and without protein (averaged signal/noise ratio >400-fold,
22% coefficient of variation) for a given compound pool allows clear identification of hits.

address spot-to-spot variability (possible with faster laser
speed). Optimization of washing procedures such as volume
and timing of wash cycles may also contribute to improvement in CVs. Despite the apparent higher CV relative to the
values typically observed in a functional assay HTS, the hit
selection with this type of binding screens is gated primarily
on a gain of signal above a defined threshold and thus in theory can tolerate higher CV. By comparing the MS signals of
samples with and without protein, we can simultaneously and
reliably determine true hits and eliminate nonspecific binding
interactions. In cases in which compounds may show only
minimal difference between samples with and without protein, additional characterization data defining clear hit-calling criteria prior to a screen will aid in decision making to
include or eliminate these compounds from the hit set.
The EG3 thiol mass also serves as a quality control tool
to assess assay performance over time because it is present
in every well. Tracking this mass over time can show trends
both intra- and interplate to indicate changes in the detection sensitivity for the duration of a screen. This set of data
can also serve as an early indicator to initiate MS source
cleaning to ensure we maintain the highest data quality
throughout the screening campaign. If used in parallel with

control compounds, the normalized data can also inform
potential complications with the automation process during
sample preparation such as incorrect transfer of analytes
from the assay plate to the SAMDI plate.
Overall, uniformity and reproducibility performed well
for the SAMDI small-molecule binding format. Data normalization to EG3 thiol helped minimize signal variation
and improved the %CV. Normalizing data and comparing
the signal difference in the samples with and without protein helps eliminate false-positives and identify true hits for
this proposed HTS format.

Proof-of-Concept Application of SAMDI in Pooled
Compound Binding Screen
For this proof-of-concept experiment, we pooled three compounds with varying inhibitory potencies against BACE
together at a fixed concentration, incubated with biotinylated BACE, captured by SAMDI, and then analyzed in the
same solution-phase capture process described above. We rank
ordered the AUC for each compound showing good correlation with the HTRF activity data (Fig. 3A). Compounds with
more potent biochemical HTRF IC50 values correlated with a

Table of Contents for the Digital Edition of SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017