SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1265

1265

Chandler et al.

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Figure 1. Image representation of the signal intensity of acetylcholine (m/z 146.1 ± 0.2 Da, with white denoting the highest signal
intensity) in the wells of a 1536 plate in the presence of various "buffer" components at different concentrations. Table 1 in
Supplemental Data 6 identifies the "buffer" component and concentration in each well (A1-P24).

as this should not display any suppression of the substrate
ion signal. In the 2D clustered column charts in
Supplemental Data 5, all dilution concentrations are
noted as 100% and individual column data preserved to
allow full examination of the data. Due to the data set
demonstrating a minimum average intensity of 1.0 a.u., an
intensity value >1.0 a.u. was set as the threshold for optimal acetylcholine ionization. In this manner, any intensity
value <1.0 a.u. acquired across the other buffers analyzed,
as well as their corresponding 1-in-2 serial dilutions, was
therefore determined as limiting the acetylcholine signal
within the normalized spectra, thus displaying ion suppression effects.
FlexImaging software was used to generate a MALDI
image of the 1536 average intensity data set, superimposed
onto an optical image of one of the spotted MTP target
inserts (see Fig. 1). Having established the threshold value
for adequate ionization and consequently setting the absolute intensity scale (0-1 a.u.), Figure 1 shows an image representation of the signal intensity of acetylcholine (m/z
146.1 ± 0.2 Da, with white denoting the highest signal
intensity) in the wells of the 1536 plate in the presence of
various "buffer" components at different concentrations.
Table 1 in Supplemental Data 6 identifies the "buffer"
component and concentration in each well (A1-P24). An
obvious ionization efficiency gradient could be observed
for a number of the buffer serial dilutions (Fig. 1). This
indicates that the assumption that ion suppression at high

stock concentrations could be reduced by diluting the buffer
solution within the assay held true.
For all buffers analyzed, an individual 2D clustered column chart was generated to display the degree of correlation between the concentration of buffer and acetylcholine
ion signal intensity (see Suppl. Data 4), as described in the
Methods section. Data sets of particular note are discussed
further.
From the acetylcholine intensity values acquired for the
following assay buffers, it was determined that the highest
concentrations investigated for current screening assays were
well suited for use by MALDI-MS due to all dilution values
displaying an intensity value >1.0 a.u., and thus no alteration
in the use of these assay components would be required to
apply them to MALDI-MS screening: ammonium acetate,
adenosine triphosphate (ATP), β (2-) mercaptoethanol,
bovine serum albumin, dithiothreitol (DTT), formic acid,
glycerol, NADPH, nonessential amino acids (MEM NEAAs),
polyethylene glycol (PEG), potassium chloride, sodium
chloride, sodium pyruvate, sodium orthovandate, Tris(2carboxyethyl)phosphine hydrochloride (TCEP), and trifluoroacetic acid. For these buffer components, almost no
correlation (i.e., no significant change) was observed (r2 <
0.2) between the 1-in-2 serial dilution solutions and the acetylcholine ion signal observed, as shown in the example cluster plot for the assay component, NEAA (Fig. 2). This
reinforces the statement that, for these buffers, the optimal
concentration used within traditional screening techniques

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