SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1256

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SLAS Discovery 22(10)

Solid Phase Extraction

Sample

Wash

Elution

Target analyte
Other materials
MIP

Analysis was performed on a hybrid MALDI LTQ Orbitrap
XL (Thermo Fisher Scientific) instrument. Full MS spectra
for each spot were obtained as the average of 10 mass scans
in positive mode, mass range 700−2000 Da at 60,000 mass
resolution. The selective ion monitoring (SIM) spectra were
obtained in positive mode, mass range 880−990 Da using
60,000 mass resolution. The peak intensities were extracted
using Xcalibur (Thermo Scientific), and the data were analyzed with the statistical software package R.30
The intensity ratio (Intensity Ratio %) of each sample
fraction was calculated from the intensity of the target peptide (NLLGLIEAK) observed in the sample fraction (IEF)
and the intensity of the heavy IS (IIS), according to eq 3:
Intensity Ratio % = ( I EF / I IS ) × 100

(3)

Results and Discussion
The workflow of the method is illustrated in Figure 1,
where the MIPs are prepacked into wells of a 384-well filter
plate; (1) sample is drawn through the SPE bed, and after
washing, (2) the captured analyte is eluted and (3) the collected eluents are analyzed using LC-MS and/or MALDI
for the quantification of recovery and evaluation of the
background removal (i.e., spiked peptides added to the sample) (Fig. 2).

384-Well Filter Plate: SPE Setup and
Development
During SPE, unspecific absorption to surfaces can lead to
sample losses and false results. Therefore, the retention of
analyte contributed by unspecific absorption in the

Figure 1. Schematic representation
of the 384-well filter plates and the
workflow of the SPE step in the
microwell. (A) 384-well filter plate.
(B) SPE sample preparation, where the
sample is transferred to the microwell
loaded with SPE material (1), followed
by a wash step to remove undesired
components (2), and the captured
analytes are then displaced by the
elution step (3).

384-well filter plate setup was investigated. Briefly, 20 µL
of a 5 nM NLLGLIEAK peptide in 50 mM ABC buffer was
loaded into the filter plate wells without any MIP/NIP
phase, followed by running an SPE protocol using 5% ACN
as washing buffer and 80% MeOH + 3% FA as the elution
buffer. The collected eluents were analyzed with LC-MS/
MS for detection of binding of the target peptide. The analysis (Suppl. Fig. S5) showed that approximately 10% of the
loaded peptide was bound to surfaces of the well (membrane absorption, dead volumes in the tip) and recovered
during the elution step. At this quite low concentration (5
nM), the total loss due to adsorption was approximately
25% of the loaded analyte.
Chemical compatibility of the microplates and filter
plates was checked with different conditions. Prolonged
exposure of ACN resulted in warping of MultiScreen HTS
384-well filter plates and cross talk in the collection plates.
This problem was addressed by changing the filter plates to
AcroPrep 384-well filter plates, which are made of polypropylene (SAN) with glass fiber (borosilicate glass, 1.0 µm)
membrane. The latter was inert to the all buffer conditions
used in the described experiments.

High-Throughput Screening of MIPs
Using LC-MS
In the initial development of the 384-well filter plate SPE
platform, only PMIP-4 and its nonimprinted counterpart PNIP-2
were used. These two sorbents had been previously investigated for SPE of the NLLGLIEAK peptide, using a reversedphase-like SPE protocol with a low amount of organic
modifier in the wash solution.27 Thus, the 384-well filter
plate SPE platform should provide the same results as the
previous investigation. The impact of increasing organics in

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