SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1212

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specific capture of analytes for high-throughput sample preparation with a throughput of >100,000 compounds per day.13
SAMDI uses self-assembled monolayers on gold arrays in 384
or 1536 formats that are compatible with state-of-the-art
MALDI time-of-flight (TOF) instrumentation and liquid handling. The monolayers are capped with polyethylene glycol for
compatibility with biological molecules and a chemical moiety, such as biotin, to enable capture of a biotinylated-protein
target via immobilized neutravidin. This unique MS capture
approach imparts several advantages for MALDI assay workflows. First, SAMDI simplifies sample preparation and enables
unprecedented purification of up to 1536 samples in <10 s,
while reducing MALDI acquisition time to 1 to 2 s per sample.
This efficient purification of analyte minimizes ion suppression caused by buffer components, such as salts and detergents.
SAMDI also provides uniform monolayers as a foundation for
consistent matrix application onto sample spots in the array.
Furthermore, sample purification can also minimize charging
in the ion optics and significantly reduces source contamination that results in improved robustness.
Although SAMDI technology has been implemented for
label-free screening of compounds that modulate functional
activities of target enzymes,13-16 application to screening
small-molecule binding to protein targets through noncovalent
interactions is unprecedented. Native MS17-19 or affinity selection MS20-22 have enabled MS screening of small-molecule
drug binding. In the former, detecting a mass shift of the protein intact mass under native conditions can indicate the presence of small-molecule binding. Alternatively for the latter, we
infer hit identification from MS detection of the compound
mass after isolation of the protein target incubated with a mixture of compounds under native conditions and subsequent
dissociation of bound compounds using denatured MS. Aside
from lower resolution and sensitivity for native MS, a key
challenge associated with either screening strategy is lower
throughput compared with conventional fluorescence-based
screening assays. Furthermore, affinity selection MS using the
current process of protein isolation employing liquid chromatography or plate-based filtration suffers from low retention of
compounds with rapid off rates.
Here we describe the application of affinity selection MS
of noncovalent compound binders to a protein target using
SAMDI that is amenable to HTS. We demonstrate a workflow and proof of concept for a novel screening format of a
pooled compound mixture using affinity capture and purification by SAMDI paired with the high sampling speed of
MALDI with a 1 kHz laser.

Materials and Methods
Matrix Selection for Small-Molecule Detection
Three representative compounds with molecular weights
(MW) in the 200 to 600 Da range were prepared in a serial

SLAS Discovery 22(10)
dilution ranging from 20 to 0.625 µM compound and tested
with a panel of 13 matrices (Suppl. Table S1; Suppl. Fig.
S1). The compound serial dilutions were diluted 1:1 with
matrix, and 1 µL was spotted to a MALDI target plate.

Protein and Compounds
Biotinylated protein, β-secretase22 (BACE) or Dual Leucine
Zipper Kinase23 (DLK; MAP3K12, Carna Biosciences,
Natick, MA), no protein control, and the desired concentration of small molecule(s) were mixed in buffer containing
20 mM sodium acetate pH 4.4, 0.002% Tween 20, and 0.1%
bovine skin gelatin (BSG; Sigma Aldrich, St. Louis, MO)
for BACE or Tris buffer pH 8, 0.002% Tween 20, and a
range of 1-0.25% DMSO for DLK.23, 24 BSG and Tween
were optimized for the SAMDI process to minimize nonspecific compound binding and spreading on the SAMDI
plate (Suppl. Fig. S2A BSG optimization; Suppl. Fig. 2B
Tween optimization at 0.1% BSG). Compounds with a
range of inhibitory activities were made in-house from historical small-molecule projects and pooled at 10 mM in
DMSO to yield a final assay concentration of 10 µM for
BACE and 5 µM for DLK for each individual compound.

Preparation of Protein with Compounds on
SAMDI Plates
To minimize shot-to-shot variability and ensure quality and
homogeneous signal, the surface of the SAMDI plate should be
saturated with biotinylated-protein. Compounds were bound to
target proteins either in "solution" or "solid" phase (Suppl. Fig.
S3). For solution-phase SAMDI capture, biotinylated-protein
samples were incubated with compound prior to affinity capture on SAMDI plates. Compound titrations of a two-fold dilution series starting at 40 µM compound, eight points, were first
incubated with 1 µM biotinylated BACE in its respective assay
buffer for up to 30 min at room temperature and transferred to
a 384-spot SAMDI array that presents a monolayer of neutravidin immobilized onto a self-assembled monolayer comprising 3% (biotin-ethylene glycol)6-SH and 97% (ethylene
glycol)3-SH and incubated at room temperature for 60 min. In
the solid phase capture format, 1 µM biotinylated BACE protein was captured to the neutravidin-coated SAMDI plate first
for 30 min at room temperature and then washed with assay
buffer; compounds were then added to allow compound binding to the captured protein on the SAMDI plate for 60 min at
room temperature. The solution-phase method was used for
individual compound binding EC50 determination, plate uniformity, and pooled compound experiments. Mixtures of either
1 µM biotinylated BACE or 650 nM biotinylated DLK with 10
µM or 5 µM compound pools, respectively, were incubated in
a humidified chamber at room temperature for up to 30 min
prior to applying 2.5 µL of the solution to a 384-spot SAMDI
array. Surfaces were then washed gently three times with

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