Dual-Channel Enzymatic Inhibition Measurement (DEIM) Coupling Isotope Substrate via Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry

Article abstract of DOI:10.1007/s13361-018-2054-3

A novel dual-channel enzymatic inhibition measurement (DEIM) method was developed to improve the repeatability with light/heavy isotope substrates, producing reliable relative standard deviations (< 3%) by employing acetylcholinesterase (AChE) as the model enzyme. The matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was adapted for enzyme-inhibited method due to its good salt-tolerance and high throughput; meanwhile, dual-channel enzymatic reactions were performed to improve the repeatability of each well. The acetylcholinesterase inhibition measurement was conducted by mixing the quenched enzyme reaction solution of blank group (with heavy isotope as substrate) and experimental group (with light isotope as substrate), of which the inhibition rate might be affected by isotope effects. Hence, inverse study and Km measurement were implemented to validate the method. The inverse study shows similar inhibition rate (68.9 and 70.3%) and the Km of isotope substrates are analogous (0.139 and 0.135?mM), which demonstrated that the novel method is feasible to AChE inhibition measurement. Finally, the method was applied to herb extracts, half of which exhibit inhibition to AChE. The precise dual-channel enzymatic inhibition measurement (DEIM) method could be regarded as a promising approach to potential enzyme inhibitor screening. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13361-018-2054-3

B American Society for Mass Spectrometry, 2018
J. Am. Soc. Mass Spectrom. (2018)
DOI: 10.1007/s13361-018-2054-3
RESEARCH ARTICLE
Dual-Channel Enzymatic Inhibition Measurement (DEIM)
Coupling Isotope Substrate via Matrix-Assisted Laser
Desorption/Ionization Time of Flight Mass Spectrometry
1
,2
1
1
Min Tao, Li Zhang, Yinlong Guo
1
State Key Laboratory of Organometallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Shanghai
Institute of Organic Chemistry, Center for Excellence in Molecular Synthesis, University of Chinese Academy of Sciences, Chinese
Academy of Sciences, 345 Lingling Road, Shanghai, 200032, People’s Republic of China
2
Postdoctoral Programme, Mayinglong Pharmaceutical Group Co. Ltd., Wuhan, People’s Republic of China
Abstract. A novel dual-channel enzymatic inhibi-
tion measurement (DEIM) method was devel-
oped to improve the repeatability with light/
heavy isotope substrates, producing reliable rel-
ative standard deviations (< 3%) by employing
acetylcholinesterase (AChE) as the model en-
zyme. The matrix-assisted laser desorption/
ionization time of flight mass spectrometry
(
MALDI-TOF MS) was adapted for enzyme-
inhibited method due to its good salt-tolerance
and high throughput; meanwhile, dual-channel enzymatic reactions were performed to improve the repeatability
of each well. The acetylcholinesterase inhibition measurement was conducted by mixing the quenched enzyme
reaction solution of blank group (with heavy isotope as substrate) and experimental group (with light isotope as
substrate), of which the inhibition rate might be affected by isotope effects. Hence, inverse study and Km
measurement were implemented to validate the method. The inverse study shows similar inhibition rate (68.9
and 70.3%) and the Km of isotope substrates are analogous (0.139 and 0.135 mM), which demonstrated that the
novel method is feasible to AChE inhibition measurement. Finally, the method was applied to herb extracts, half
of which exhibit inhibition to AChE. The precise dual-channel enzymatic inhibition measurement (DEIM) method
could be regarded as a promising approach to potential enzyme inhibitor screening.
Keywords: Dual-channel enzymatic inhibition measurement, Isotope substrate, Repeatability, Enzyme kinetics
Received: 21 April 2018/Revised: 20 July 2018/Accepted: 4 August 2018
enzyme prodrug therapy, etc. [1, 2]. As the most widely used
Introduction
therapeutic approaches, enzyme inhibitor has been under heat-
ed development for the treatment of various diseases, such as
tumor, hepatitis, and HIV. In vitro tests are conducted to
explore the influence of inhibitors on enzymes (e.g., inhibition
type, enzymatic inhibition rate, and inhibitory concentration),
paving the way for in vivo assays. Therefore, numerous ana-
lytical methods for the measurement of enzyme activity and
inhibition thrived to meet the precise, sensitive, fast demand for
inhibitor screening, comprising spectrophotometric method
[3], electrochemical method [4], and mass spectrometric (MS)
method [5–10].
lmost all metabolic processes need enzyme catalysis. Due
to their vital role in all aspects of life, the malfunction or
lack of enzymes leads to severe pathologic conditions in or-
ganisms, and some clinical treatments act on enzyme,
encompassing enzyme replacement therapy, enzyme inhibitor,
A
Electronic supplementary material The online version of this article (https://
doi.org/10.1007/s13361-018-2054-3) contains supplementary material, which
is available to authorized users.
Over the past decades, tremendous mass spectrometric
methods have been used to measure enzyme activity and screen
Correspondence to: Li Zhang; e-mail: zhangli7488@sioc.ac.cn, Yinlong Guo;
e-mail: ylguo@sioc.ac.cn

M. Tao et al.: DEIM Coupling Isotope-Substrate via MALDI-MS
inhibitors [11–13]. As opposed to traditional spectrophotomet-
ric method requiring fluorescent labeling or chromogenic re-
agent, mass spectrometry shows m/z of substrate, product, and
inhibitor directly offering more flexibility in assays. Moreover,
mass spectrometry has a natural advantage that each m/z has a
corresponded intensity or area, which is easy for quantitative
calculation.
kinetics assay. Moreover, dual-channel enzymatic inhibition
measurement (DEIM) method was applied to measure the
IC50 of donepezil hydrochloride and analyze the crude extrac-
tion of herbs for potential inhibitor screening. This method is
anticipated to broaden perspectives for precise measurement of
enzyme inhibition and inhibitor screening.
Since the invention of matrix-assisted laser desorption/
ionization mass spectrometry (MALDI-MS), it was successful-
ly applied not only to small or large molecule detection [14, 15]
but also to enzymatic reaction monitoring [16, 17]. Particularly,
MALDI-MS is most suitable to study the kinetics of enzyme
reactions and for screening purposes because of its salt-toler-
ance, high throughput, and simultaneous detection of multiple
assay components [18]. In assaying enzyme reactions,
MALDI-FT-MS [19, 20], thin-layer chromatography/UV hy-
phenated with MALDI-TOF-MS [21], and intensity-fading
MALDI-TOF-MS [22] were reported.
Experimental
Chemicals and Reagents
Acetylcholinesterase from Electrophorus electricus (Type VI-
S, EC 3.1.1.7, 374 U/mg), α-cyano-hydroxycinnamic acid
(CHCA), disodium hydrogenphosphate (Na HPO ), and sodi-
2 4
um dihydrogenphosphate (NaH PO ) were purchased from
Sigma Chemical Co. (St Louis, Missouri, USA). Potassium
iodide (KI), potassium carbonate (K CO ), dichloromethane,
2
4
2
3
However, poor repeatability is an inevitable limitation for
MALDI because of the non-uniform nature of the crystalliza-
tion of the matrix/analyte complex. As a consequence, for a
parallel of analyte/matrix sample dropped in different wells, the
detector response of each well is remarkably variable, which is
disadvantageous for quantification. To circumvent this prob-
lem, several sample drying methods have been developed to
form uniform and fine crystals that provide superior signal
repeatability, for instance, deposition of droplets by micro-
dispenser [23, 24], preparation with sandwich methods [25],
fast evaporation methods using highly volatile solvents [26,
and chloroform were from Shanghai Chemical Reagent Cor-
poration (Shanghai, China). Neostigmine bromide, donepezil
hydrochloride, 2-bromoethyl acetate, diethylamine,
iodomethane, and iodomethane-d were purchased from Alad-
3
din Reagent Co., Ltd. (Shanghai, China). The ultrapure water
used in this paper was purified by a Milli-Q water purification
system (Millipore Corp., Bedford, MA, USA). Methanol, ace-
tonitrile and ethanol were of HPLC-grade quality and were
purchased from Merk (Darmstadt, Germany).
2
7], and addition of glycerol in matrix [28]. Alternatively,
Synthesis of Light-Isotope/Heavy-Isotope Substrates
sample preparation methods without conventional matrix were
also reported to improve signal repeatability: solvent-free sam-
ple preparation [29, 30], ionic liquid matrix [31, 32], carbon
nanotube matrix [33], and prestructured sample supports such
as SALDI [34, 35] and DIOS [36–39]. For the reliable quanti-
fication of enzymatic reactions via MALDI-MS, application of
an appropriate internal standard was developed [40, 41] and
measurement of substrate-to-product ratio also had good results
Synthesis of 2-(diethylamino) ethyl acetate: to a solution of
diethylamine (8.78 g, 0.12 mol, 2.4 equiv), K CO (8.3 g,
2
3
0.06 mol, 1.2 equiv), and KI (100 mg, 0.6 mmol) in CH CN,
3
2-bromoethyl acetate was added (8.35 g, 0.05 mol, 1 equiv).
The mixture was refluxed for 4 h. The resulting mixture was
filtered and concentrated. Vacuum distillation gave 3 (6.89 g,
0.043 mol, 86.6% yield) as yellow liquid.
[
42, 43]. In analytical chemistry, ratio metric method is widely
Synthesis of N,N-diethyl-N-methyl-aminoethyl acetate io-
dide: 2-(diethylamino) ethyl acetate (500 mg, 3.14 mmol, 1
equiv) was dissolved in chloroform in a Schlenk tube. The
used to obtain accuracy and precision, through which a broad
range of substances can be detected. For instance, bisphenol A
can be measured by dual-ratiometric fluorescent measurements
with excellent repeatability between 1.09 and 7.38% (RSD, n =
CH I (0.5 mL, 8 mmol, 2.5 equiv) was added dropwise at 0
3
°C. The solution was stirred for 4 h at 50 °C. Removal of
3
) [44]. However, enzymatic inhibition measurement through
solvents and excess CH I by reduced evaporation gave N,N-
3
dual-ratio metric method has not been reported.
diethyl-N-methyl-aminoethyl acetate iodide (0.95 mg,
3.14 mmol, 100% yield) as yellow liquid (see Supporting
Information Fig. S3).
In this work, dual-channel enzymatic inhibition measure-
ment (DEIM) method was employed to improve the well-to-
well and sample-to-sample repeatability. Acetylcholinesterase
Synthesis of N,N-diethyl-N-methyl-d -aminoethyl acetate
3
(
AChE) was selected as the model enzyme for inhibition mea-
iodide: 2-(diethylamino) ethyl acetate (500 mg, 3.14 mmol, 1
equiv) was dissolved in chloroform in a Schlenk tube. The
surement. Isotope substrates, ACh-N (Et) (short for N,N-
2
diethyl-N-methyl-aminoethyl acetate iodide) and ACh-N
CD I (0.5 mL, 8 mmol, 2.5 equiv) was added dropwise at 0
3
(
Et) -d (short for N,N-diethyl-N-methyl-d -aminoethyl acetate
°C. The solution was stirred for 4 h at 50 °C. Removal of
2
3
3
iodide), were synthesized by two steps to implement dual-
channel enzymatic reaction. With low RSD (< 3%) of parallel
wells, the novel method manifested great repeatability. The
method was further validated by inverse study and enzyme
solvents and excess CH I by reduced evaporation gave N,N-
3
diethyl-N-methyl-d -aminoethyl acetate iodide (0.96 mg,
3
3.14 mmol, 100% yield) as yellow liquid (see Supporting
Information Fig. S4).

M. Tao et al.: DEIM Coupling Isotope-Substrate via MALDI-MS
Materials Preparation
reason for dropping sample of one EP tube to three different
wells is that the final value should be the average of different
wells in order to decrease random errors.
Stock solutions of neostigmine bromide, donepezil hydrochlo-
ride were prepared in deionized water at a concentration of
0
.1 mg/mL. Stock solutions of AChE were prepared and dilut-
ed by 10 mmol/L Na HPO -NaH PO buffer (pH = 7.4) to
2
4
2
4
Enzyme Kinetics Assay AChE solution (50 μL, 0.1 U/mL)
was incubated with light-isotope substrate (50 μL, 0.2 mg/mL)
at 36 °C. The mixture was quenched at 3, 6, 9, 12, 15, 20, 30,
2
0 U/mL, and were stored at − 20 °C in dark. Working
solutions were obtained by appropriate mixture and dilution
of the stock. The light-isotope/heavy-isotope substrates solu-
40, 50, 60, and 75 min by the addition of acetonitrile (200 μL)
tions (0.05 mg/mL) were prepared with Na HPO -NaH PO
2
4
2
4
to draft the progress curve. To fully demonstrate the accuracy
of the linear correlation between conversion and time before
buffer freshly. The matrix solution was prepared by adding
1
5
0 mg of CHCA to 1 mL of ethanol/water solution (V∶V =
0∶50).
5
5
min, experiments of time data acquisitions are added below
min.
The dry powder (1 g) of Phellodendron chinense Schneid.
A series of light-isotope/heavy-isotope substrates in differ-
was extracted in ethanol (70%, 20 mL × 3) via ultrasonic for
0 min at 35 °C. The extracted solution were filtered, concentrat-
ent concentrations (0.025, 0.05, 0.1, 0.2, 0.4 mg/mL, 50 μL)
were incubated with AChE solution (0.1 U/mL, 50 μL) at 36
°C to calculate the Michaelis constants of different isotope
substrates.
3
ed, and reconstituted in H O to give stock solution. The stock
2
solution was diluted by 1000 times to afford final aqueous
solution used in enzyme reaction for potential inhibitor screening.
The fresh leaves (0.2 g) of 21 different herbs were extracted
in acetonitrile (50% in water, 20 mL × 3) via ultrasonic for
Simultaneous Enzymatic Catalysis Study A solution with the
same concentration of light-isotope substrate (0.05 mg/mL)
and heavy-isotope substrate (0.05 mg/mL) was prepared;
3
0 min at 35 °C. The extracted solution were filtered, concen-
trated, and reconstituted in H O to give stock solution. The
2
1
0 μL of the solution was mixed with 10 μL of enzyme
stock solution was diluted by 10,000 times to afford final
aqueous solution (0.1 mg/L) to be used in enzyme reaction
for inhibition measurement.
solution (0.1 U/mL). The simultaneous enzymatic catalysis
was quenched with acetonitrile. The quenched solution was
mixed with matrix and dropped to wells.
Validation of Novel Method
Isotope Substrate Inverse Study All reactions were conducted
simultaneously in Eppendorf plastic (EP) tubes (see Supporting
Information Fig. S5). Each group was carried out in triplicate
Comparison Between Traditional Method
and Novel Method
(
in three EP tubes). In group A, AChE solution (50 μL, 0.1 U/
All reactions were conducted simultaneously in Eppendorf
plastic (EP) tubes (see Supporting Information Fig. S6). In
group A where reaction was carried out in one EP tube, AChE
solution (50 μL, 0.1 U/mL) was incubated with water (50 μL)
for 5 min at 36 °C, then light-isotope substrate (50 μL, 0.05 mg/
mL) was added and incubated for 20 min at 36 °C. In group B
where nine parallel reactions (in nine EP tubes) were carried
out, AChE solution (50 μL, 0.1 U/mL) was incubated with
neostigmine bromide (50 μL, 5 μg/L or 2 μg/L or 1 μg/L) for
5 min at 36 °C, and then light-isotope substrate (50 μL,
0.05 mg/mL) was added and incubated for 20 min at 36 °C.
In group C where reaction was carried out in one EP tube,
AChE solution (50 μL, 0.1 U/mL) was incubated with water
(50 μL) for 5 min at 36 °C, and then heavy-isotope substrate
(50 μL, 0.05 mg/mL) was added and incubated for 20 min at 36
°C. The reaction was quenched by acetonitrile (200 μL). For
traditional method, on the one hand, 1 μL of solution in group
A was dropped to nine different wells to explore the repeat-
ability of well-to-well; on the other hand, 1 μL of solution in
each EP tube of group B was dropped to three different wells,
as a result, 27 (3 × 9) wells was detected in group B to explore
the repeatability of sample-to-sample. For novel method which
needs blending, 20 μL of solution in the EP tube of group C
was mixed with 20 μL of solution in each EP tube of group B to
form a new group M containing nine parallel EP tubes, and
mL) was incubated with water (50 μL) for 5 min at 36 °C, then
light-isotope substrate (50 μL, 0.05 mg/mL) was added and
incubated for 20 min at 36 °C. In group B, AChE solution
(
(
50 μL, 0.1 U/mL) was incubated with neostigmine bromide
50 μL, 5 μg/L) for 5 min at 36 °C, then heavy-isotope
substrate (50 μL, 0.05 mg/mL) was added and incubated for
0 min at 36 °C. In group C, AChE solution (50 μL, 0.1 U/mL)
2
was incubated with water for 5 min at 36 °C, and then heavy-
isotope substrate (50 μL, 0.05 mg/mL) was added and incu-
bated for 20 min at 36 °C. In group D, AChE solution (50 μL,
0
5
0
The reaction was quenched by acetonitrile (200 μL). For Mix 1,
triplicate of group A was mixed to one EP tube, and then 50 μL
of the mixture in the new-formed EP tube was blended with
.1 U/mL) was incubated with neostigmine bromide (50 μL,
μg/L) for 5 min at 36 °C, then light-isotope substrate (50 μL,
.05 mg/mL) was added and incubated for 20 min at 36 °C.
5
0 μL of solution in each EP tube of group B to form H1, H2,
and H3. For Mix 2, triplicate of group C was mixed to one EP
tube, and then 50 μL of the mixture in the new-formed EP tube
was blended with 50 μL of solution in each EP tube of group D
to form H4, H5, and H6. Finally, each sample of H1, H2, H3,
H4, H5, and H6 was dropped to 3 different wells and 18 (3 × 6)
wells were formed. Before being dropped to well, all samples
were mixed with CHCA matrix (sample:matrix = 1:5). The

M. Tao et al.: DEIM Coupling Isotope-Substrate via MALDI-MS
Figure 1. Progress curves of enzyme-catalyzed reaction. The initial substrate concentration was 0.2 mg/mL; the reaction was
quenched at 3, 6, 9, 12, 15, 20, 30, 40, 50, 60, and 75 min by the addition of acetonitrile (200 μL); to fully demonstrate the accuracy of
the linear correlation between conversion and time before 5 min, experiments of time data acquisitions are added below 5 min
then 1 μL of the mixture in each EP tube of group M was
dropped to three different wells to explore the repeatability of
sample-to-sample except that one tube in group M was dropped
to nine different wells to explore the repeatability of well-to-
well. Before being dropped to well, all samples were mixed
with CHCA matrix (sample: matrix = 1:5).
were described above. In group A, AChE solution (50 μL,
0.1 U/mL) was incubated with water (50 μL) for 5 min at 36
°C, and then heavy-isotope substrate (50 μL, 0.05 mg/mL) was
added and incubated for 20 min at 36 °C. In group B, AChE
solution (50 μL, 0.1 U/mL) was incubated with inhibitor
(50 μL) for 5 min at 36 °C, and then light-isotope substrate
(
°
5
50 μL, 0.05 mg/mL) was added and incubated for 20 min at 36
C. The reaction was quenched by 200 μL of acetonitrile;
0 μL of solution in each group was mixed and the mixture
Application
Various donepezil hydrochloride concentrations (0.1, 1, 2, 10,
was dropped to three wells (see Supporting Information
Fig. S7). Before being dropped to well, all samples were mixed
with CHCA matrix (sample:matrix = 1:5). To fully
5
0, 100, 500, 1000, and 2000 nmol/L) were prepared. Extrac-
tion of Phellodendron chinense Schneid. and 21 other herbs
Figure 2. Spectrum of simultaneous enzymatic catalysis. Appearing simultaneously in one spectrum, the peaks of isotope
substrates and isotope products show that isotope substrates have similar hydrolysis rate. (In the spectrum, m/z = 177.2 and
+
174.2 are peaks of isotope substrates; m/z = 135.2 and 132.2 are peaks of isotope products; m/z = 122.2 is peak of [Tris+H]
because the enzyme powder was lyophilized in Tris-HCl buffer before reconstitution)

M. Tao et al.: DEIM Coupling Isotope-Substrate via MALDI-MS
Table 1. Repeatability of Conversion Rate/Inhibition Rate in Traditional Method
a
b
Neostigmine concentration (μg/L)
Statistic
Well-to-well
Sample-to-sample
5
2
1
μg/L
μg/L
μg/L
AVE (%)
RSD (%)
AVE (%)
RSD (%)
AVE (%)
RSD (%)
78.3
4.5
49.0
6.1
52.1
5.0
68.0
5.6
49.3
7.1
29.8
6.5
a
To explore the well-to-well repeatability of traditional method, the quenched solution of blank group was dropped to nine different wells, and the mean was calculated
by averaging the conversion rates of nine wells. As a result, the RSD in the column of well-to-well stands for repeatability of conversion rate (n = 9)
b
There are nine parallel EP tubes in the inhibition group, and then quenched solution of each EP tube was dropped to three different wells to calculate the average
conversion rate of each EP tube. With the nine parallel average conversation rates of inhibition group and the blank group average conversation rate, nine parallel
inhibition rates could be calculated. The mean and RSD was calculated by averaging the nine parallel inhibition rates. Hence, the RSD in the column of sample-to-
sample stands for repeatability of inhibition rate (n = 9)
demonstrate the repeatability of the novel method in real-world
samples, extraction of Clerodendrum trichotomum. Thunb.
was adopted to perform method comparison study through
the identical experimental procedure described in BComparison
between traditional method and novel method^ section (see
Supporting Information Fig. S6).
Sample of each EP tube was dropped to three different wells
to calculate the average as the final value except that some
sample was dropped to nine different wells to explore the
repeatability of well-to-well.
The inhibition curve was plotted using GraphPad Prism 5
software (Mountain View, CA), and the IC50 value was calcu-
lated by the software.
MALDI-TOF Analysis
All data were acquired via Axima performance, Shimadzu
Biotech Lauchpad with an air-cooled Nd:YAG laser
Results and Discussion
Isotope Substrate Design and Analysis-Condition
Optimization
(
337 nm). The spectrum of each well was accumulated in
multiples of two laser shots and 199 dots in total with auto
movement of the stainless steel target. The operation was
conducted with reflection mode, 20 kV of accelerating voltage
and 5 Hz of laser frequency. The TOF was calibrated with
internal standard method by using the peaks derived from
CHCA. The acquisition mass range was 50–500.
The abundance of peaks of substrates and products could be
used to calculate the conversion rate (C) (see Supporting Infor-
mation Fig. S1).
In the beginning, N-(2-acetyloxyethyl)-pyridinium bromide
was chosen as the substrate. However, the low hydrolysis rate
made it difficult to conduct the catalytic process (see
Supporting Information Fig. S1). In order to insure the affinity
to AChE, the design of isotope substrates were based on the
natural substrate. It is fortunate that the Km of ACh-N (Et) is
2
close to natural substrate [45]. On the other hand, the prepara-
tion of ACh-N (Et) and ACh-N (Et) -d was quite feasible
2
2
3
The inhibition rate (I, %) was calculated as follows:
with high purity and excellent yield. One challenge with
MALDI-TOF for such assays with low MW substrates and
products is the interference from matrix ions. By changing
carbon chain from natural substrate, the novel isotope substrate
can easily distinguish itself from CHCA matrix; the signals of
the substrates and products are strong enough to outstrip those
I ð%Þ ¼ ½ðCo−CiÞ=CoŠ  100%
(Co is the conversion rate of blank group and Ci is the
conversion rate of inhibition group)
Table 2. Repeatability of Inhibition Rate in Novel Method
Neostigmine concentration (μg/L)
a
b
Statistic
Well-to-well
Sample-to-sample
5
2
1
μg/L
μg/L
μg/L
AVE (%)
RSD (%)
AVE (%)
RSD (%)
AVE (%)
RSD (%)
68.1
2.2
48.9
2.5
29.8
2.8
69.9
1.7
50.0
2.5
29.8
3.0
a
To explore the well-to-well repeatability of novel method, one of the EP tube in group M (see Supporting Information Fig. S6) was dropped to nine wells. In the novel
method, since the substrates and products appeared simultaneously in one spectrum, each spectrum has a correspondent inhibition rate. By averaging nine inhibition
rates calculated with nine spectrums, the well-to-well RSD of inhibition rate could be determined (n = 9)
b
There are nine parallel EP tubes in group M (see Supporting Information Fig. S6), and the mixture of each EP tube was dropped to three wells except one EP tube of
which the mixture was dropped to nine wells to explore the well-to-well repeatability (see a). By averaging three inhibition rates of three wells, each EP tube has an
average inhibition rate. Then the mean and RSD was calculated by averaging the nine parallel inhibition rates to exhibit the repeatability of sample-to-sample (n = 9)

M. Tao et al.: DEIM Coupling Isotope-Substrate via MALDI-MS
the isotope effects [46]. On the other hand, even if the chemical
bond including a heavy isotope is not involved in the rate-
limiting step, it may affect the catalysis rate for the change of
polarity, dipole moment, molar volume, electron donation, Van
der Waal’s forces, lipophilicity, and protein binding. In novel
method where inhibition (I) was calculated by Clight (light-
isotope substrate was added to inhibition group) and Cheavy
(heavy-isotope substrate was added to blank group), the differ-
ence of enzyme catalysis rate between the heavy-isotope sub-
strate and light-isotope substrate can certainly bias the calcula-
tion of inhibition. As a result, it is imperative to validate the
novel method.
In the inverse study, the inhibition of Mix 1 is 68.9% and the
inhibition of Mix 2 is 70.3%. The similar inhibitions of two
mixtures indicate that the novel method can be an alternative of
traditional method. A further validation was conducted by
measuring the Michaelis constant of light-isotope/heavy-iso-
tope substrates.
Figure 3. Plot of the inhibition efficiency of donepezil hydro-
chloride toward AChE. The concentration of light-isotope/
heavy-isotope substrates were 0.05 mg/mL and AChE concen-
tration was fixed at 0.1 U/mL
Firstly, a full progress curve was drawn to select quench
time at which enzyme velocity is constant by monitoring the
conversion rate as a function of time (Figure 1). The curve fits
linear correlation well before 5 min and the time point was
determined as 5 min. It is noteworthy that taking conversion
rate as the ordinate rather than product concentration is not only
convenient but also accurate [35].
of matrix ions. Overall, we chose ACh-N (Et) and ACh-N
2
(
Et) -d as the isotope substrates. Generally speaking, with low
2 3
MW substrates and products, there are many ways to eliminate
the interference from matrix in the spectrum, such as altering
carbon chain or isotope group to change the MW, adding
groups which have higher responses in MALDI-TOFMS or
simply changing the matrix.
To start our optimization, we should select an appropriate
matrix. CHCA gave a more homogeneous crystallization and
better quantitative results than DHB. The optimal ratio was
determined as 5:1 (matrix:sample).
Different initial enzyme velocities were detected at a
wide range of substrate concentrations (from 0.14 to
2
.30 mM). By transforming enzyme velocity (V) and sub-
strate concentration (S) to the reciprocal, these data was
fitted to Lineweaver-Burk plot to calculate Km (see
Supporting Information Figs. S8 and S9). The Km of
light-isotope substrate is 0.139 mM and the Km of heavy-
isotope substrate is 0.135 mM. The difference of Km
between light-isotope and heavy-isotope substrates was so
little that there was no problem of calculating inhibition
Validation of Novel Method
A chemical bond formed by a heavy isotope and another atom
will be different from the same bond between the light isotope
and that atom. Once breaking this chemical bond is the rate-
limiting step in an enzyme-catalyzed reaction, the catalysis
process of the molecule with the heavy isotope is slower than
that of the molecule with the light isotope, which is known as
with Clight and Cheavy
.
The result of simultaneous enzymatic catalysis indicated
that AChE gave no discrimination to light-isotope substrate
Figure 4. The mass spectrum for screening inhibitors from crude Phellodendron chinense Schneid. extracts. The active ingredient,
Majarine (exact mass = 336.1230), can be shown in the spectrum. (In the spectrum, m/z = 177.2 and 174.2 are peaks of isotope
substrates; m/z = 135.2 and 132.2 are peaks of isotope products; m/z = 190.1, 212.0, and 379.1 are peaks of CHCA matrix)

M. Tao et al.: DEIM Coupling Isotope-Substrate via MALDI-MS
Table 3. Inhibition Activity of Crude Plant Extracts
Number
Plant
Part
Inhibition rate (%)
a
Control
Phellodendron chinense Schneid.
Podocarpus macrophyllus var. maki
Sambucus williamsii Hance
Podocarpus nagi (Thunb.) Zoll. et Mor. ex Zoll.
Pittosporum tobira (Thunb.) Ait
Pittosporum tobira (Thunb.) Ait
Vitex negundo L.var. Cannabifolia (Sieb. et Zucc.) Hand.-Mazz.
Flueggea suffruticosa (Pall.) Baill.
Boehmeria nivea (L.) Gaud.
Bark
Leaf
Leaf
Leaf
Leaf
Fruit
Leaf
Leaf
Leaf
Leaf
Flower
Leaf
Fruit
Leaf
Flower
Leaf
Flower
Leaf
Leaf
Leaf
Leaf
Leaf
Leaf
Root
Root
Fruit
9.98
0.63
1.98
10.86
1.56
8.00
8.08
9.86
0.24
33.91
2.31
12.49
16.69
4.92
1
2
3
4
5
6
7
8
9
Clerodendrum trichotomum. Thunb.
Yulania denudata (Desr.) D. L. Fu
Euonymus maackii Rupr.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Euonymus maackii Rupr.
Fagopyrum dibotrys (D.Don) Hara
Fagopyrum dibotrys (D.Don) Hara
Carpesium abrotanoides Linn.
Carpesium abrotanoides Linn.
Rubus corchorifolius L. f.
Eriobotrya japonica (Thunb.) Lindl.
Morus alba L.
Osmanthus fragrans Lour
5.87
1.69
12.87
44.70
0.20
17.27
3.78
30.16
13.14
17.00
11.63
6.61
Ginkgo biloba L.
Saxifraga stolonifera (L.) Meerb.
Astragalus membranaceus (Fisch.) Bge.
Polygonatum odoratum (Mill.) Druce
Forsythia suspensa (Thunb.) Vahl
a
Phellodendron chinense Schneid. was chosen as the positive control because it contains a well-known AChE inhibitor, Majarine, of which the content is not less than
3.0% stipulated by Pharmacopoeia of the People’s Republic of China. The process of extracting Phellodendron chinense Schneid. and enzyme inhibition experiment
was conducted simultaneously with other plants, forming final aqueous solution (0.1 mg/L), in which the amount of Majarine was estimated to be close to 3 μg/L
and heavy-isotope substrate because the reduction of substrate
and generation of product are equal (Figure 2).
Phellodendron chinense Schneid. (Figure 4.), both of which
are reported to exhibit AChE inhibition [47, 48] and serve as
positive control in the research. The IC50 of donepezil hydro-
chloride calculated by novel method was 8.88 nM, which is
similar to 6.7 nM [47]. Therefore, the novel method is appli-
cable for complex inhibition experiment. The extract of tradi-
tional Chinese medicine affects the activity of acetylcholines-
terase with an inhibition of 89.6% with a conspicuous peak of
Majarine (exact mass = 336.1230) (Figure 4), the representa-
tive active ingredient in Phellodendron chinense Schneid. [48],
which exhibited that the novel method can be applied to the
screening of potential inhibitor in herbs.
It is noteworthy that many clinical available drugs have
come or been derived from natural molecules [49], because
of the prominent chemical and functional diversity of biomol-
ecules existing in organisms. In this work, as many as 25 herb
extracts were tested via dual-channel enzymatic inhibition
measurement (DEIM) with positive control Phellodendron
Comparison Between Traditional/Novel Method
Even though the repeatability remains to be improved, tradi-
tional method is widely used in MALDI enzyme inhibition
study where the blank group and inhibition group was dropped
to different wells (Table 1). In order to improve the repeatabil-
ity, a novel method was developed. In this method, the blank
group and inhibition group were mixed together and dropped to
one well so that four peaks appeared in one spectrum (Table 2).
It is apparent that the repeatability of novel method was im-
proved dramatically since the RSD was reduced by half.
Application of Novel Method
The capability of novel method was evaluated with pure chem-
ical compound of donepezil hydrochloride (Figure 3.) and
Table 4. Repeatability Comparison of Clerodendrum trichotomum. Thunb.
a
b
Method
Statistic
Well-to-well
Sample-to-sample
DEIM
AVE (%)
RSD (%)
AVE (%)
RSD (%)
34.4
3.0
53.1
5.6
34.5
2.9
33.5
7.0
Traditional method
a
The well-to-well RSD in DEIM stands for repeatability of inhibition rate (n = 9), while the well-to-well RSD in traditional method stands for repeatability of
conversion rate (n = 9)
b
The sample-to-sample RSD in both DEIM and traditional method stand for repeatability of inhibition rate (n = 9)

M. Tao et al.: DEIM Coupling Isotope-Substrate via MALDI-MS
6
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chinense Schneid. in the same concentration, about half of
which showed inhibition activity (Table 3). To fully demon-
strate the repeatability of the novel method in real-world sam-
ples, extraction of Clerodendrum trichotomum. Thunb. was
adopted to perform method comparison study and the result
shows great priority to DEIM (Table 4).
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Conclusions
In this study, a dual-channel enzymatic inhibition measurement
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inhibitor screening by functionalized magnetic carbonaceous micro-
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(
DEIM) coupling isotope substrate method was developed via
MALDI-TOF-MS by using AChE as model enzyme. The
novel method had solved the problem of poor repeatability of
MALDI, with RSD below 3%. A two-step synthesis of light
and heavy isotope substrates was designed, which is similar in
ionization process and easy to distinguish in spectrum. The
enzyme kinetics of light-isotope substrate and heavy-isotope
substrate are almost equivalent, insuring the accuracy of inhi-
bition calculation. This method was successfully applied to
IC50 measurement of donepezil hydrochloride. We employed
this method to crude herb extracts and half of which exhibit
inhibition to AChE. Overall, dual-channel enzymatic inhibition
measurement (DEIM) is a precise, sensitive and straightfor-
ward method for fast screening of biological molecules even in
crude extracts without the need for complicated sample prepa-
ration. Importantly, while AChE was used in this work, this
method can be extended to other types of enzymatic assays
with well-designed substrates.
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Acknowledgements
The authors acknowledge the financial support from National
Key Research and Development Program of China (Grant No.
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