SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1257

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Jagadeesan et al.

- Analytes

Fragmentation

Fragmentation
Selection

ESI

Manifold Collar

Parent Selection

Collection Plate

ESI - MS/MS

Filter Plate

High Voltage

- Analytes

Time of flight separation

- Matrix

Collection Plate

UV - Laser
100
95

Obtained Mass Spectra

90
85
80
75

Sample
+ Matrix

Manifold Base

Relative Abundance

70
65
60
55
50
45
40
35
30
25
20
15
10
5
0

m/z

Standard MALDI plate

MALDI - MS

Figure 2. Schematic representation of the dual (MALDI and ESI)-analysis readout facilitated by the 384-well filter plate SPE method.

the washing buffer on the SPE sample preparation was therefore investigated using increasing ACN% (5, 10, 15, 30, 50,
and 95). Supplemental Figure S6 shows the recovery (R%)
in the elution plotted against the ACN concentration used in
the wash step. As expected, the data showed that a wash buffer with 5% ACN provides a higher recovery than the washes
with higher ACN. This trend is also seen for the NIP, albeit
with a lower recovery in the elution step. This effect can be
explained by considering the nature of the peptide and the
polymer. NLLGLIEAK is a 0.8 kDa proteolytic digest product dominated by a hydrophobic pentapeptide segment (55%
hydrophobic residues); the polymeric sorbents were in this
case based on DVB as the main constituent. In agreement
with our recent report, this leads to a reversed-phase retention
mechanism with a strong non - specific hydrophobic contribution. To suppress this effect, a careful optimization of the
wash conditions is required. Indeed, at an increased ACN%,
that is, at 30% and 50%, more significant imprinting-related
differences in the elution recoveries were observed. However,
as high content of ACN in the wash step resulted in lower
recoveries, there is an obvious trade-off between recovery
and specificity.
A second screening was performed to narrow down the
optimal organic content in the wash step. For this experiment, wash buffers containing 5%, 7.5%, 10%, and 12.5%
ACN were used. Samples of 5 nM NLLGLIEAK peptide in
50 mM ABC buffer were loaded onto the MIP/NIP materials, followed by wash and elution using 80% MeOH/3%
FA. Recovery (R%) of the analyte in the wash and elution

fractions was calculated and plotted against the ACN concentration (Suppl. Fig. S7).
Analysis of the wash fraction (Suppl. Fig. S7A) shows that
using 7.5% ACN as wash buffer leads to 34% of the analyte
loss, whereas usage of 10% ACN as wash buffer increases the
analyte loss to 48%. Based on the recovery observed in the
elution step (Suppl. Fig. S7B), it appears that the most selective MIP retention, for this particular SPE protocol, could be
found when using 7.5%-10% of ACN. At 7.5%, a total recovery of 50% was observed for the MIP, compared with 25% for
its NIP counterpart. At 10% ACN, the recovery was a bit lower
(40%), but the discrimination versus the NIP, showing only
8%, was significantly better. Wash with 12.5% ACN displayed
lower recovery and diminished the affinity of the MIP compared with NIP. The 384-well filter plate-based SPE method
thus provided the same results as the previously reported 1 mL
cartridge for a NLLGLIEAK-specific MIP phase.27

Verification by the MALDI-MS Method
The 384-well filter plate platform can in many situations be
"too fast" for analysis readout with LC-MS; that is, 384 samples would take days to weeks to analyze. Therefore,
MALDI-MS was investigated as an option for rapid readout in
order to achieve a better match between the SPE-screening and
the MS analysis time. While MALDI-MS can easily provide a
qualitative answer, the multiplexed testing is much more useful if quantitative data from the samples can be obtained. The

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