SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1205

Beeman et al.

1205

Figure 1. Adaptation of c-MET assay to MALDI-MS-compatible buffer conditions. Comparison of different Brij-35, MgCl2, and
HEPES concentrations on the c-MET assay (standard buffer conditions: 20 mM HEPES [pH 7.5], 5 mM MgCl2, 2 mM DTT, 0.01%
Triton X-100). Conversion (%) represents the percentage of substrate (SRC peptide) converted to product (p-SRC peptide) by
c-MET kinase. (A) Influence of Brij-35 concentration vs. 0.01 Triton X-100. (B) Influence of MgCl2 and HEPES salts within standard
buffer conditions containing Brij-35 at 0.01% instead of Triton X-100. (C) Combined buffer conditions from (A) and (B) to compare a
MALDI-MS-compatible buffer (blue) with a formerly used standard buffer (dark gray).

s) and then incubated at 22 °C for 60 min. For kinetic measurements, the ATP concentration was varied from 33.2 µM
[20 µM] to 1667 µM [1000 µM], with incubation times
from 5 to 60 min, respectively. After incubation, the reaction was stopped by adding 7.5 µL of termination buffer
(127 mM HEPES [pH 7.5], 0.01% Brij-35, 40 mM EDTA,
1% DMSO) into each well. The enzyme activity was then
measured using CE. The phosphorylated product and
nonphosphorylated substrate (FITC-SRCtide) were separated and detected with the LabChip 3000 EZ Reader
(PerkinElmer, Waltham, MA) by excitation with a blue
light-emitting diode (LED) and measuring the fluorescence
emission.
MALDI-MS Detection. The assay was performed as
described in Supplemental Figure S3, with the unlabeled
SRCtide peptide as substrate and in a 1536-well format
(1536LDV, Labcyte, Sunnyvale, CA).
The reaction was stopped by adding 2.5 µL of 2% [v/v]
TFA in matrix solution (10 mg/mL 4-HCCA was dissolved
in 80% ACN and 20% water [Baker analyzed Ultra LC/MS
9823-02]) with the Multidrop Combi (Thermo Scientific) to
give an assay reaction to matrix volume ratio of 1:1 and a
final TFA concentration of 1%. This type of dispenser was
used because it operates as a closed system to avoid evaporation of ACN.
Prior to spotting, MALDI targets (polished steel plain
1.0 inserts, Bruker Daltronics 1833280) were cleaned using
the following steps: (1) 100% TFA, (2) water (Baker), and
(3) allowed to dry at room temperature. Immediately after
adding matrix solution, the assay plate was centrifuged at
2000 rpm for 60 s and transferred to the Echo 550 acoustic

liquid handler (Labcyte) as a source plate. The plain steel
MALDI target plate on the 3D printed adapter (Suppl. Fig.
S1) was introduced into the Echo 550 as a destination plate.
Seventy-five nanoliters of the assay and matrix mix was
transferred to the target plate in a 1536-well grid format.
Automated spotting onto the target was performed using the
Echo 550 by either 384- to 1536- or 1536- to 1536-well
transfer.
MALDI Data Acquisition. MALDI time-of-flight (TOF)
mass spectra were collected with either an UltrafleX III
TOF-TOF or a RapifleX TOF Pharma Pulse instrument
(both Bruker Daltronics). FlexControl and FlexAnalysis
or MALDI Pharma Pulse 2.0 (for RapifleX) were used for
acquisition and data analysis. Settings for both instruments
were chosen to achieve the best ratio of sensitivity and
resolution. With the UltrafleX III, spectra were acquired
using an AutoXecute Method. Spectra were recorded in
positive reflector mode for a mass range of 800-2500 m/z
with an initial laser power of 31%. The instruments were
calibrated using the Peptide Calibration Standard II (Bruker
Daltronics, 8222570) and cubic enhanced mode (UltrafleX
III) or quadratic mode (RapifleX), respectively. Data were
acquired by summing up 500 satisfying laser shots in 100
shot steps following the random walk complete sample
mode over each sample spot. For analysis, the mass spectral
peaks representing the isotope pattern of the desired substrate and product peptide were used for analysis. The processing parameters for UltrafleX III were as follows: SNAP
peak detection algorithm, signal-to-noise ratio (S/N) of 6,
Savitzky-Golay smooth with 0.2 m/z width and 1 cycle, and
Top Hat baseline substraction. The processing parameters
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
