SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1198

1198

SLAS Discovery 22(10)

Figure 2. Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) salt-inducible kinase (SIK) kinase assay
specifications. (A) The MALDI TOF spectra of CHKtide in a dilution series. The assay is very sensitive, with 1 fmol still showing high
signal to noise. (B) Linearity of MALDI TOF SIK kinase assay. The assay is linear between 10 and 50 min and over a peptide occupancy
of ~15% to 75%. (C, D) Enzymatic constants determined by MALDI TOF mass spectrometry for CHKtide (C) and adenosine
triphosphate (D) for SIK2. (E) IC50 dose-response curves for five inhibitors against SIK2 as determined by the MALDI TOF assay.

available. Optimization for these specific peptides showed
best results when spotted with CHCA in 50% acetonitrile,
0.1% TFA, and spotting 200-nL sample by a dried droplet
method23 using a Mosquito nanoliter dispenser, which
allowed uniform deposition onto the target and matrix. The
total time taken to spot a full 1536 target was ~30 min,
which is comparable to other MALDI TOF assay spotting
times.22
We next tested which of the two substrates performed
best. The majority of peptides ionize well by MALDI, but it
has been shown in the past that ionization efficiency is
dependent on the gas-phase basicity of peptides.24,25 Thus,
arginine- and lysine-rich peptides tend to have better
responses, and therefore we expected that the CHKtide substrate would perform better as it contains more basic residues that are also more distributed over the whole peptide
sequence. Both peptides were tested for sensitivity, with
CHKtide indeed proving to perform better with a detection
limit of 1 fmol on target with good signal to noise (Fig. 2A)
compared to the NUAK peptide (Suppl. Fig. S1), which
was also detectable at 1 fmol but exhibited a sevenfold
lower signal to noise. Both peptides formed only minimal

salt adducts and oxidized species; therefore, CHKtide was
chosen to be the substrate of choice for this assay.
With the peptide substrate and spotting technique established, the enzymatic reaction was assessed for detection of
the phosphorylated product, as well as the reaction kinetics.
Many buffers of biochemical assays contain components
that are not considered "MS-friendly" as they suppress ionization of the analytes. MALDI TOF has a low tolerance for
some salts such as NaCl, as it often leads to adduct formation, thus making interpretation and quantification of peptide spectra difficult.26 We therefore modified established
kinase assay buffers to be more compatible with MALDI
TOF, guided by a recent investigation into MALDI-MS-
friendly buffers.19 Initial experiments in the ADP Hunter
assay showed that the removal of NaCl from the assay buffer did reduce the signal window but did not alter the rank
order of inhibitors. We therefore removed NaCl from the
assay buffer of the MALDI assay. Final assay conditions
gave a robust and reproducible response of both the phosphorylated and unphosphorylated peptide, as well as being
comparable with the conventional ADP Hunter assay that is
performed in-house.

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