SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1195

1195

Heap et al.
fluorophore as a downstream consequence of the conversion of ATP to ADP. It therefore provides a generic highthroughput assay for the measurement of kinase enzyme
activity. Screening of full-length GST-tagged human SIK2
was performed using CHKtide as a substrate peptide.
Compounds were stamped into 384-well, low-volume,
black-wall, assay plates (Greiner Bio-one, Stonehouse,
UK) as described above. The assay window was defined by
addition of DMSO in the presence (no effect) or absence
of enzyme (maximum effect) to defined control wells.
Using a Wellmate liquid dispenser (Thermo Scientific,
Hemel-Hempstead, UK), 4 µL of a 2× enzyme solution
(ADP Hunter Plus assay buffer, 1 mM DTT, enzyme) was
dispensed into all wells and preincubated for 20 min at
room temperature. The assay enzyme reaction was initiated
by the addition of 4 µL of a 2× substrate solution (ADP
Hunter Plus assay buffer, 100 µM CHKtide, 200 µM ATP
Gold) such that the final assay concentration of SIK2 was
10 nM. Assay plates were incubated for 30 min at room
temperature, after which the reaction was stopped by the
addition of 4 µL ADP Hunter Plus Reagent A and 8 µL ADP
Hunter Plus Reagent B. Following a further incubation for 30
min at room temperature, resorufin-elicited fluorescence was
measured (ex 550 nm; em 595 nm) using a PHERAstar FS
(BMG Labtech, Aylesbury, UK).

SIK MALDI TOF Assay
In this assay, the appearance of phosphorylated CHKtide
product at m/z 2779.47 was monitored with respect to the
CHKtide substrate at m/z 2700.47 using MALDI TOF mass
spectrometry.
Screening assays were performed with an XRD-384
Automated Reagent Dispenser (Fluid, Nether Alderley,
UK) by dispensing 3 µL of freshly prepared assay buffer (50
mM Tris/HCl, 10 mM magnesium acetate, 2 mM DTT, pH
7.5) and 1 µL of 15 nM SIK2 prepared in assay buffer onto
prepared compound plates followed by a 30-min incubation
at 30 °C. The reaction was then initiated by addition of 1 µL
substrate solution containing a mixture of CHKtide and
ATP to a final concentration of 1 µM and 100 µM, respectively. Plates were then returned to incubation at 30 °C for
30 min before being quenched by addition of 1.2 µL of TFA
to a final concentration of 2%.

MALDI MS Target Spotting
First, 9.04 mg dibasic ammonium citrate was dissolved in 1
mL HPLC-grade water and vortexed to create a 40-mM
solution. Then, 10 mg CHCA was weighed out and prepared to a final volume of 1 mL in 50% HPLC-grade ACN
and 50% water (v/v), 0.1% TFA, and 10 mM dibasic ammonium citrate and was prepared and placed on a shaker for 30
min to aid dissolution before use.

Quenched samples and freshly prepared CHCA matrix
solution were mixed in a 1:1 ratio (v/v) with a Mosquito
nanoliter dispenser in an LVSD plate (TTP Labtech,
Melbourn, Hertfordshire, UK) and spotted with the dried
droplet technique in 200-nL aliquots onto 1536-MTP
AnchorChip targets (Bruker Daltonics). Each sample
was spotted in duplicate and the spotted targets allowed
to air dry before MALDI-MS analysis.
MTP AnchorChip targets were cleaned before each use
by sonication in HPLC-grade isopropanol for 2 min, followed by sonication in 30% acetonitrile and 0.1% TFA for
2 min. Targets were then placed in a clean room and allowed
to ambient dry before use.

MALDI-MS Analysis
A RapifleX MALDI TOF/TOF mass spectrometer (Bruker
Daltonics) equipped with a Smartbeam 3D laser was used
in positive ion mode with Compass 2.0 control for all
data acquisition. Samples were run in automatic mode
(AutoXecute; Bruker Daltonics), acquiring 5000 shots at a
10-kHz frequency per spot in a random walk on spot laser
ablation pattern and M5 Smartbeam Parameter at a 25-µm ×
25-µm scan range. Ionization was achieved using a fixed
laser power of 70% (laser attenuator offset 7%, range 30%)
and detected by the FlashDetector, Bruker, Bremen,
Germany at a detector gain of ×2 in the 2500 to 2800 m/z
mass range. Samples were analyzed in Reflector mode with
optimized voltages for reflector 1 (20.82 kV), reflector 2
(1.085 kV), and reflector 3 (8.8 kV), ion sources (ion source
1, 20 kV, PIE 2.66 kV), lens (11.3 kV), and a pulsed ion
extraction of 200 ns. A novel 10-bit digitizer was used at a
sampling rate of 5.00 GS/s.
Spectra were accumulated with FlexControl software
(v4.0) and processed using FlexAnalysis software (v4.0)
(Bruker Daltonics). Peaks were centroid detected with a
signal-to-noise threshold of 6.00 to detect only the intense
peaks before being processed with a Savitzky-Golay smoothing algorithm (0.05 m/z width, one cycle) and "TopHat" baseline subtraction. External calibration was performed before
each new target in Cubic Enhanced mode with Pepmix II
calibrant (Bruker Daltronics) containing seven peptides:
angiotensin II [M+H]+ = 1046.54, angiotensin I [M+H]+ =
1296.68, substance P [M+H]+ = 1347.74, bombesin [M+H]+ =
1619.82, ACTH clip (1-17) [M+H]+ = 2093.09, ACTH
clip (18-39) [M+H]+ = 2465.20, and somatostatin (28)
[M+H]+ = 3147.47. Internal calibration was performed
using the CHKtide peptide substrate monoisotopic [M+H]+ =
2700.60 Th.

Data Analysis
For enzyme characterization, initial rates in both technologies were determined using time-course experiments under
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
