OVERVIEW
Purpose : A matrix-free soft laser desorption/ionization method,
DIUTHAME, was tested for ability to conduct high throughput
screening.
Methods : Acetylcholinesterase (AChE) enzyme functional assays
were performed and monitored by MALDI and DIUTHAME.
Results : AChE reaction time course was successfully monitored
by DIUTHAME with several advantages over MALDI.
AChE assays・Acetylcholinesterase (AChE, Sigma-Aldrich, C3389),
2.5 mU in 40 mM Tris-Cl・Acetylcholine chloride (ACh, Sigma-Aldrich,
A6625), 25 µM in 40 mM Tris-ClMixed 2.5 µL of each aliquoit to make
enzyme reaction assays (no inhibitor was added)
Left to stand for the predefined reaction times then quenched by
1% Formic Ac
MALDI measurementsMatrix: CHCA, 7 mg/mL in MeOH/TFA 0.2% 7:3
(v/v)
Targets were deposited by sandwich method (matrix/sample/matrix,
0.5 µL each)
DIUTHAME measurements
Applied the assays on the top of DIUTHAME-substrate (9ch-type,
0.5 µL each)
All MS measurements were conducted by using Bruker Ultraflex
TOF/TOF equipped with a nitrogen laser (50 Hz), in reflector
mode
The unique characteristics of desorption ionization using
through hole
alumina membrane (DIUTHAME) not only enable imaging mass
spectrometry (IMS) but also are potentially suited for
applications relying on
robotic processing, such as high throughput screening (HTS).
This
expectation was confirmed by monitoring protein functional
activity shown in
DIUTHAME spectra. Acetylcholinesterase (AChE) enzyme reaction
assays
were performed then analyzed by using DIUTHAME.
Scatter charts of peak values in the vicinities of m/z 146 and
m/z 104
reaction time: 0 （without enzyme）
reaction time: 22 minutes
DIUTHAME
Vicinity of m/z 146
Vicinity of m/z 104
MALDI
Vicinity of m/z 146
peaks are unlikely
attributable to
acetylcholine
Vicinity of m/z 104
peaks are not
attributable to
choline
DIUTHAME is interference-free
100
0
%
max (normalized
for each target)
0
Conversion
Conversion =ＩCh
ＩCh + AＩAChＩCh: Peak intensity of choline (m/z 104)
ＩACh: Peak intensity of acetylcholine (m/z 146)
A: Correction factor for ionization efficiencies
Compensate a lower ionization efficiency for choline
than that for acetylcholine
In MALDI, A = 0.16 has been proposed.[1]
In this study, A = 1 to make comparison between
MALDI and DIUTHAME
[1] C. Haslam, et. al.; J. Biomol. Screen. 21(2) 176-186
(2016).
MALDI
0 min 2 min 4 min
6 min 8 min 10 min
14 min 18 min 22 min
DIUTHAME
P H O T O N I S O U R B U S I N E S S
Yasuhide Naito1・Masahiro Kotani2・Takayuki Ohmura2 （The Graduate
School for the Creation of New Photonics Industries
(GPI)1・Hamamatsu Photonics K.K.2）
Introduction
DIUTHAMEMALDIReaction time [minutes] 0 2 4 6 8 10 14 22 18
Acetylcholine (m/z 146)
Choline (m/z 104)
Conversion [%]
0 2 4 6 8 10 14 22 18
DIUTHAMEMALDIminutes 0 2 4 6 8 10 14 22 18
m/z 146
m/z 104
Conv.
0 2 4 6 8 10 14 22 18
For DIUTHAME experiments,
the assays were applied on
the top (hydrophobic) surfaceof a 9ch DIUTHAME-substrate
Mass spectra obtained from AChE assays （reaction time: 2
minutes）
Results
Conclusion
Peak intensities
Conversion
Reaction time
146.0
49
104.0
16
0
1
2
3
4
4x10
Inte
ns.
[a.u
.]
100 105 110 115 120 125 130 135 140 145m /z
MALDI
choline
（product）m/z 104
acetylcholine
（substrate）m/z 146
m/z 122（Tris-derived）
146.0
47
104.0
21
0
200
400
600
800
1000
1200Inte
ns.
[a.u
.]
100 105 110 115 120 125 130 135 140 145m /z
DIUTHAME
choline
（product）m/z 104
acetylcholine
（substrate）m/z 146
m/z 122 （Tris-derived）
Despite of weak signals, DIUTHAME provides good signal-to-noise
ratios
100 110 120 130 140 150
103
0100 110 120 130 140 150
4104
0
m/z m/z
Inte
nsity
Inte
nsity
Monitoring AChE reaction time course:
Heat map representations of target responses and kinetics
plots
Image area: 1.35 mm sq.
Pixel size: 50 µm
Remeasurements of the same targets
(target reuse)
References
DIUTHAME shows more
smooth responses in each
target area than MALDI targets
and brings an improvement in
measurement uncertainty
The enzyme reactions of acetylcholinesterase were
successfully
monitored as conversion of the substrate acetylcholine to
the
product choline with the reaction time course by using
DIUTHAME.
It was found that DIUTHAME has several advantages
(interference
free, reduced uncertainty in measurement, no target
hysteresis)
over MALDI in the measurements of the enzyme reaction
assays.
Because of its high tolerance for additives such as
detergents,
DIUTHAME is highly promising for HTS applications.
DIUTHAME targets have no hysteresis and can repeat measurements
(with shifted irradiation spots)
1. Y. Naito, M. Kotani, T. Ohmura; A novel laser
desorption/ionization method using through
hole porous alumina membranes; Rapid Commun. Mass Spectrom. 32
1851-1858 (2018)
Open access article: https://doi.org/10.1002/rcm.8252
2. DIUTHAME product datasheet, Hamamatsu Photonics K.K.
Download site:
https://www.hamamatsu.com/jp/en/product/type/A13331-3-1/index.html
Surface-assisted LDI processes
schematic of
the membrane
No branches, no side holes,
only vertically aligned straight
through holes (i.d. ~ 200 nm)
are formed in the membrane
Vertical cross-sectional SEM image
samples can be
located underneath
Pt coatingTi frame
bonding
Ø3 Ø3 x 9 Ø18
Basic concept &
device of DIUTHAME
DIUTHAME-substrate has been commercialized as ready-to-use
chips
optical image taken through the membrane
m/z =838
m/z =830
acquired using Waters SYNAPT
m/z
DIUTHAME-IMS
of mouse brain
DIUTHAME has shown
abilities for imaging mass
spectrometry (IMS)
Obtained by K. Kuwata group at WPI-ITbM, Nagoya Univ.
Methods
