SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1254

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can work even under extreme conditions (pH, temperature,
and organic solvents).18,19 Generally, the manufacturing of
MIP sorbents involves a preorganization of the target molecule to be imprinted with the functional monomers, followed
by a polymerization step with a cross-linker to lock the
monomers into a polymer network and create the affinity sorbent.20,21 Traditionally, MIP-SPE sorbents have been developed and applied for capture of small molecular targets,22,23
but the development of MIPs for capture of biomolecules has
been growing with the increased scientific understanding of
imprinting.24,25 Currently, MIPs targeting peptides or proteins
cannot match the high affinity of the best antibodies, although
the recent progress in MIP synthesis and the ever-increasing
sensitivity and dynamic range of new analytical instruments
could very well provide ample opportunities for new affinity
SPE assays using MIPs.
In a typical development of a MIP SPE method, several
parameters needs to be screened, comprising those related
to the synthesis of the sorbent, as well as those related to its
use in SPE, that is, load, wash, and elution conditions. The
performance is commonly evaluated by packing the
imprinted polymer (15-50 mg) into SPE cartridges, and
then measuring the recovery of the target compound in the
fractions obtained after cartridge load, wash, and elution.10
Here, an automated, high-throughput SPE screening
method is presented and used for screening of molecularly
imprinted sorbents in order to identify promising candidates.
The method is based on 384-well filter plates and is comparable to the previously presented ISET platform.26 This type of
platform greatly facilitates the screening of different phases
with simultaneous investigation of diverse solvent/buffer conditions. Compared with the ISET, the 384-well filter plate format provides increased bed volume, and thus binding capacity
(needed to enable use of low-affinity materials), and can be
used with analysis techniques other than matrix-assisted laser
desorption/ionization (MALDI), for example, liquid chromatography-electrospray ionization mass spectrometry (LC
ESI-MS). The molecularly imprinted materials used for development and validation were designed to selectively capture a
proteotypic peptide of pro-gastrin-releasing peptide (ProGRP),
NLLGLIEAK, as previously reported.27
Although the 384-well format has been extensively utilized for immunoassays and in vitro receptor binding studies, its use in SPE is relatively new.28 The presented method
has been automated using liquid handling robotics, for the
addition of the different solvents during the SPE process.
The eluents resulting after the SPE experiments were analyzed on two different analytical platforms (ESI-triple
quadrupole and MALDI-Orbitrap), allowing for quantification of NLLGLIEAK and evaluation of the different MIP
sorbents based on recovery and removal of background.
The collected multiplexed experimental data on the
ProGRP-specific MIP and non - imprinted polymer (NIP)
phases provided both mechanistic information concerning

SLAS Discovery 22(10)
analyte binding and an improved protocol for the specific
enrichment of NLLGLIEAK with reduced non - specific
binding.

Materials and Methods
Protein and Peptide Samples
Internal standard (IS) NLLGLIEA[K_13C615N2] (95% purity,
Thermo Fisher Scientific, Braunschweig, Germany) was
diluted according to the AQUA peptide storage and handling
guidelines and stored at −20 °C. Stock solutions of IS and
NLLGLIEAK of 5 µM were used to prepare samples by dilution with 50 mM ammonium bicarbonate (ABC) buffer
solution.
β-Casein (Sigma-Aldrich, St. Louis, MO) was digested
overnight with trypsin (Promega, Madison, WI) in a 1:100
ratio (enzyme-protein) at 37 °C. The digested protein was
diluted with 0.1% trifluoroacetic acid (TFA) and frozen to stop
the digestion process. Stock solutions of 4 µM were used to
prepare samples by dilution with 50 mM freshly prepared
ABC buffer.

µ-SPE in the 384-Filter Plate Format
µ-SPE was performed using 384-well filter plates filled
with MIPs imprinted to capture the ProGRP signature peptide NLLGLIEAK. NIPs were used as controls to assess the
MIP imprinting efficiency.
The MIPs were prepared as described previously with
minor modifications27 (see Supporting Information). Briefly,
PMIP-1 and PMIP-2 were prepared by ethylene glycol dimethacrylate (EDMA) copolymerized with methacrylic acid (MAA),
and PMIP-3 and PMIP-4 polymers were prepared from hydrophobic divinylbenzene (DVB) copolymerized with 2-amino
ethyl methacrylamide (EAMA). For all MIPs, an N-terminally
Z-protected peptide was used as a template for generating the
surface imprint (Suppl. Table S1). The NIPs were prepared by
the same synthetic protocol without the initial addition of the
template molecule. The SPE platform used either MultiScreen
HTS 384-well filter plates from Millipore made of styrene
acrylonitrile (SAN) with polyvinylidene fluoride (PVDF)
membrane/polyester support or AcroPrep 384-well filter plates
from Pall Life Sciences made of polypropylene (SAN) with
glass fiber (borosilicate glass, 1.0 µm) membrane. Thermo
Scientific (Waltham, MA) Nunc plates made of polystyrene
were used as collection plates. A Millipore MultiScreen HTS
Vacuum Manifold coupled to a vacuum pump (0-6 g/cm2,
Nerliens Meszansky, Oslo, Norway) was used to carry out the
SPE protocol. A Biomek 3000 Laboratory Automation
Workstation (Beckman Coulter, Fullerton, CA) was used to
enable automated sample preparation. Control of the robot was
governed directly by Biomek automation software v3.3
(Beckman Coulter).
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
