Rapid detection of irreversible acetylcholineasterase inhibitor by mass spectrometry assay

Article abstract of DOI:10.1002/cjoc.201200250

Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) for screening irreversible AChE inhibitors. Due to its good salt-tolerance and low sample consumption, MALDI-FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE-inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC-MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non-AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI-FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.

Full text of DOI:10.1002/cjoc.201200250

FULL PAPER
DOI: 10.1002/cjoc.201200250
Rapid Detection of Irreversible Acetylcholineasterase
Inhibitor by Mass Spectrometry Assay
Cai, Tingtinga(蔡婷婷)
Liang, Chenb(梁晨)
Zhang, Li*,a(张立)
Wang, Rongb(汪蓉)
Fu, Defengd(傅得锋)
Zhao, Wushengc(赵武生)
Zhang, Yurong*,b(张玉荣)
Guo, Yinlonga(郭寅龙)
a Shanghai Mass Spectrometry Center, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences,
Shanghai 200032, China
b Shanghai Institute of Forensic Science, Shanghai 200083, China
c Jiangsu Institute of Forensic Science, Nanjin, Jiangsu 210024, China
d Zhejiang Institute of Forensic Science, Hangzhou, Zhejiang 310009, China
Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix-assisted laser de-
sorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) for screening irreversible AChE inhibi-
tors. Due to its good salt-tolerance and low sample consumption, MALDI-FTMS could facilitate rapid detection,
especially detection in real application. AChE activity was determined through calculating abundance of substrate
and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) con-
centrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibi-
tions still kept good correlation with concentration of OPs. Finally, this AChE-inhibited method was applied to
screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of dece-
dents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following de-
tecting in GC-MS/MS, the possible death reason of these three decedents was confirmed, and another decedent ac-
tually died for sumicidin, a non-AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by de-
tecting enzyme activity in MALDI-FTMS provided fast and accurate analysis results and excluded another toxicants
not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.
Keywords acetylcholinesterase, irreversible inhibitor, organophosphorous pesticide, mass spectrometry, rapid
detection
Introduction
sensitive methods for detecting the irreversible AChE
inhibitors.
Acetylcholinesterase (AChE) is an enzyme that hy-
drolyzes the neurotransmitter acetylcholine, producing
choline and an acetate group. It is mainly found at
neuromuscular junctions and cholinergic nervous sys-
tem. AChE has a very high catalytic activity and plays
an important role in mammalian physiological proc-
ess.[1]
AChE activity would be inhibited by various com-
pounds reversibly or irreversibly. The irreversible inhi-
bition takes place via the covalent reaction of the serine
hydroxyl (OH) in the enzyme active site.[2-4] Recently,
owing to the extensive use of pesticides, those usual
irreversible AChE inhibitor including organophospho-
rous (OP) and carbamate pesticides exist anywhere in
our life, which threaten human health indiscernibly and
seriously. Therefore, it is necessary to develop fast and
In order to screen AChE inhibitors, the most com-
monly used methods have been developed about meas-
uring AChE activity. It is reported that many assays can
be used to indicate the existence of AChE inhibitors via
detecting enzyme activity, such as thin layer chroma-
tography,[5,6] fluorometric detection,[7,8] chemometrics
assays,[9,10] capillary electrophoresis[11] and biosen-
sors.[12,13] Through detecting the decrease amount of
substrate acetylthiocholine (ATCh) and the increase of
product thiocholine (TCh) from enzymatic reaction,
AChE activity is determined. Up to the present time,
mass spectrometry with various ionization modes and
detector techniques has been applied to the identifica-
tion of enzyme inhibitors.[14-20] The known OP com-
pounds, carbamate pesticides and some alkaloids can be
*
E-mail: zhangli7488@sioc.ac.cn, yr_zhang@126.com; Tel.: 0086-021-54925314; Fax: 0086-021-54925314; Tel.: 0086-021-22028901; Fax:
0086-021-65443131
Received March 14, 2012; accepted May 3, 2012; published online XXXX, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200250 or from the author.
Chin. J. Chem. 2012, XX, 1—5
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Cai et al.
FULL PAPER
－20 ℃. The matrix solution was prepared fresh daily
by adding 150 mg of DHB to 1 mL of ethanol/water
solution (V∶V＝50∶50), and 1% CF3COOH was
added.
separated and quantitatively analyzed directly, while the
unknown inhibitors become aware via detecting the de-
crease of enzymatic hydrolysis product.[21-25] In high
performance liquid chromatography (HPLC), the novel
enzymatic microreactor is involved, which realizes the
online detection and fast analysis.[26,27] However, the
complex matrixes such as whole blood are apt to per-
plex the detection results. Besides, most of methods
need tedious pretreatment in order to maintain the nor-
mal work of instruments.
Matrix-assisted laser desorption/ionization Fourier
transform mass spectrometry (MALDI-FTMS) has also
attracted great attention in studying enzyme activity and
enzyme inhibitor.[28-30] Due to its salt-tolerance and low
consumption, MALDI-FTMS has advantages in dealing
with complex matrix and achieving high-throughput
screening. In this work, we applied MALDI-FTMS to
detect the whole blood from four poisoning decedents
and healthy persons. It turned out that whole blood of
three decedents with low AChE activity showed these
persons died for irreversible AChE inhibitors such as
OP pesticides and another decedent whole blood show
equal AChE activity with healthy persons. In further
investigation by GC-MS/MS method, three OP pesti-
cides were detected in three decedents with low enzyme
activity and sumicidin (not AChE inhibitor) existed in
the decedent with normal enzyme activity. These results
confirmed the judgment of enzymatic method. This en-
zyme-inhibited method based on MALDI-FTMS shows
new perspectives for rapid and accurate response to
AChE inhibitor.
Whole blood samples preparation
Five whole blood samples were collected from
Shanghai Institute of Forensic Science. Taking 2—3 mL
of each blood sample in a 10 mL glass tube and adding
5 mL CHCl3/CH2Cl2 (V∶V＝1∶1) solution, the mix-
ture was agitated and extracted with a blender for 1 min.
Then extracted solutions (5 mL) were dried via air
blow-down techniques, and 500 µL of each sample was
centrifuged for 5 min at 10000 r/min. The supernatants
(100 µL) were diluted by 10 times since AChE activity
in human whole blood was too high to detect in a short
time. The whole blood dilutions were collected as ace-
tylcholinesterase source.
Measurement of AChE and reaction conditions
Enzyme reactions were carried out in eppendorf
plastic (EP) tubes. AChE solution (10 µL) and 10 µL of
inhibitors (different pesticides solutions) were added
and incubated for 30 min at 37 ℃ with 10 µL of ul-
trapure water as control group. Then, the substrate
－
1
(ATCh, 5 mmol•L , 10 µL) was added in the reaction
for 20 min at 37 ℃ until acetonitrile (30 µL) was added
to enzyme system and quenched the reaction.
Each whole blood dilution (10 µL) and substrate so-
lution (10 µL) were incubated in an EP tube for 10 min
at 37 ℃ until acetonitrile (30 µL) was added and
quenched the reaction. The relative blank group was
performed as whole blood (10 µL) being quenched by
acetonitrile (30 µL) firstly, then the substrate ATCh
solution was added.
Experimental
Reagents and materials
Acetylcholinesterase (AChE) from electrophorus
MALDI-FTMS conditions and analysis
－
1
electricus (Type VI-S, EC 3.1.1.7, 426 U•mg ), ace-
tylthiocholine iodide, 2-amino-2-(hydroxymethyl)-1,3-
propanediol (Tris) and 2,5-dihydroxybenzoic acid (DHB)
were purchased from Sigma Chemical Co. (St Louis,
MO). All the organophosphorus pesticides were from
Shanghai Institute for Food and Drug Control (Shanghai,
China). The ultrapure water was purified by a Milli-Q
water purification system (Millipore Corp., Bedford,
MA, USA). Methanol and acetonitrile were of HPLC-
grade quality from Merk (Darmstadt, Germany). Poly-
ethylene glycol-200 (PEG-200), PEG-400, hydrochloric
acid (HCl) and trifluoroacetic acid (CF3COOH), were
from Shanghai Chemical Reagent Corporation (Shang-
hai, China).
Experiments were conducted using an Ionspec 4.7 T
HisRes MALDI-FTMS (Ionspec, Irvine, CA, USA).
The external Ionspec MALDI ion source used an
air-cooled Nd:YAG laser (355 nm, New Wave Research,
Fremont, CA) with a gradient filter for adjusting the
UV-laser power. Ions, generated from a MALDI source,
were transferred via a quadruple ion guide to the capaci-
tive coupled closed cylindrical cell. The intensity of
MALDI-laser irradiation was varied between 35% and
45% as needed. The laser irradiation pulse time was set
at 50 ms. For low-mass region, the quadruple guide had
an applied voltage of 30 V (base to peak) at a frequency
of 725 kHz. The mass spectrometry was calibrated with
PEG-200 or PEG-400 for each test. The acquisition
mass-to-charge (m/z) range was 80—200.
Materials preparation
The matrix solution was deposited on the stainless
steel target to produce a microcrystalline layer of matrix,
followed by sample deposition on the preformed matrix
layer. These wet sample spots were getting dried slowly
to produce a microcrystalline layer which was homoge-
neous crystals of matrix and samples, and was analyzed
AChE solutions were prepared and diluted by 50
mmol•L－ Tris-HCl buffer (pH 8.0) to 5 U•mL , and
－
1
1
were stored at －20 ℃ in dark. The substrate acetyl-
－
1
thiocholine (ATCh) solutions (5 mmol•L ) were pre-
pared with Tris-HCl buffer freshly. All of the OP pesti-
cides were made by fresh ultrapure water and stored at
2
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Chin. J. Chem. 2012, XX, 1—5

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay
by MALDI-FTMS.
could be used for calculating enzyme activity (ex-
The abundances of peaks reflected the content of
compounds. The conversion (C) was calculated as fol-
lows:
pressed as conversion of substrate), which make it pos-
sible to assess following enzyme inhibition. In order to
obtain reliable results about enzyme activity, the data
reproducibility of substrate conversion had been inves-
tigated. The percentage relative standard deviation
(RSD) values in dot, dot-to-dot and sample-to-sample
were evaluated and all of RSD values were within 6%,
which confirmed the accuracy of quantitative analysis
of enzyme activity.
C＝[[TCh]/([ATCh]+[TCh])]×100%
where [TCh] is replaced by the aboundance of TCh.
[ATCh] is replaced by the aboundance of remaining
ATCh as well as the fragment of ATCh. The inhibition
(I) was calculated as follows:
I＝[(C0－Ci )/C0 ] 100%
where C0 is the conversion rate of ATCh to TCh with
ultrapure water as control group. Ci is the conversion
rate of ATCh to TCh inhibited by OP pesticide.
GC-MS/MS analysis
GC-MS/MS analysis was performed on an Agilent
5975 tandem quadrupole mass spectrometer coupled
with the Agilent 6890 gas-chromatograph (Agilent Inc.
USA). The analytical column was an HP-5MS fused
silica capillary column (30 m×0.25 mm ×0.25 μm)
(Varian Inc. USA).
Figure 1 The MALDI-FTMS spectrum obtained from AChE
Samples were analyzed in the electron impact ioni-
zation (EI) mode with the ionization energy of 70 eV.
The compounds were separated on an HP-5MS and
eluted with helium gas (99.999%) at a constant flow rate
catalyzed ATCh to TCh system.
Relation between AChE inhibition and inhibitor con-
centration
－
1
of 1.0 mL•min , and employed split injection mode at
a split ratio of 1∶20. The initial oven temperature was
maintained at 100 ℃ for 1 min, and then progressively
We investigated the relation between AChE inhibi-
tions and inhibtior concentrations, in order to examine
the reliability of enzyme-inhibited method to screen
AChE inhibitors based on MALDI-FTMS. Three OP
pesticides were used as AChE inhibitors, and they were
diluted to different concentration range based on their
different toxicity.[31] As shown in Figure 2, it illumi-
nated that AChE inhibitions caused by dichlorovos
－
1
heated up to 280 ℃ at a ratio of 20 ℃•min , finally
kept for 15 min. The injection port temperature and in-
terface temperature were both 260 ℃. Ionization source
temperature, quadrupole temperature and transfer line
temperature were kept at 230, 150 and 280 ℃ respec-
tively. Full scan mass spectra were acquired in the mass
range within 50—500 amu. Data acquisition was per-
formed in full scan mode and screened by selected ion
monitor (SIM).
(DDVP) in the range between 2 and 500 µg•L－ in-
1
creased along with the incremental DDVP concentration.
Similarly, Figures 3 and 4 displayed the corresponding
inhibition curves of malathion (2—100 µg•L ) and
－
1
Results and Discussion
MALDI-FTMS performance for low weight mole-
cule
MALDI-FTMS has superiority in determining com-
pounds. Figure 1 depicts a typical mass spectrum from
AChE catalyzed ATCh to TCh system based on high
resolution MALDI-FTMS. The signals of substrate ace-
tylthiocholine [ATCh]＋ (m/z 162.1) and the product
thiocholine [TCh]＋ (m/z 120.1) are recognized clearly.
The ion signal of m/z 103.0 is from the fragment of
acetylthiocholine (m/z 162.1), which had been discussed
in our previous study.[28] Previously, it was reported that
the relative change of abundances of peaks could reflect
the change of contents of compounds in our laboratory.
Therefore, the abundances of substrate and product
Figure 2 AChE inhibition caused by DDVP (2—500 µg•L－1).
Chin. J. Chem. 2012, XX, 1—5
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3

Cai et al.
FULL PAPER
used for detecting AChE activity and served as control
group. These decedents were characterized as poisoning
symptoms, which had been caused by unknown com-
pounds. There were so many candidate toxicants that it
had difficulties in looking for the criminal poison. In
this case, whole blood samples from these decedents
were extracted and prepared for detecting AChE activity
in order to judge whether these persons died for irre-
versible AChE inhibitors. All of the whole bloods were
diluted by 10 times since AChE in healthy persons with
high catalytic efficiency was not easily operational and
practicable. When enzymatic reactions in the whole
bloods were quenched by ACN, lots of proteins in
whole bloods would deposit. And the following cen-
trifugation would remove the deposited proteins and
exclude the disturbance from macromolecules in matrix.
Meanwhile, the concentration of enzyme substrate
ATCh used here for AChE activity was much higher
than low molecular weight compounds in blood samples
and would exclude the disturbance of these compounds.
Plus, ATCh used in our work was not a native substrate
of AChE and other substrates of AChE such as ACh in
matrix would not disturb the targeted compound analy-
sis.
－
Figure 3 AChE inhibition caused by malathion (2—100 µg•L ).
1
As shown in Table 1, in comparison with AChE ac-
tivity of healthy people, three decedents (marked as
1390, 1477 and 1462) had obvious lower enzyme activ-
ity, while another decedent (marked as 1420) had nor-
mal even higher enzyme activity. These results indi-
cated that it was irreversible AChE inhibitors that
caused those three decedents’ death probably. Mean-
while, the decedent with a 1420 symbol probably died
for some other toxicants. Although numerous AChE
inhibitors existed in our life, taking enzyme activity as
the single criterion for responding the enzyme inhibitor
was convenient for fast screening, and helpful for ex-
cluding non-AChE inhibitors poisoning.
Figure 4 AChE inhibition caused by rogor (10—1000 µg•L－1).
－
1
rogor (10 and 1000 µg•L ) respectively. It demon-
strated that MALDI-FTMS was a reliable technique to
detect AChE activity and inhibition, which laid the
foundation for studying AChE inhibitors.
Besides, the limits of detection about these AChE
inhibitors had been studied by detecting AChE activity.
When the percentage of inhibition was more than 20%,
the sample was designated of causing significant inhib-
iting action. OP pesticides solutions were diluted con-
tinuously to obtain the reasonable LODs. The detection
limits of malathion obtained from our method was as
Table 1 AChE activity of five whole bloods from decedents
and healthy person
Whole blood
#1390
Conversion/%
0
0
#1477
－
1
low as 0.035 µg•L , which was much lower than the
#1462
0
－
1
result from the literature (1 µg•L ),[13] which showed
advantages for assaying AChE inhibitors in real samples
and demonstrated the good sensitivity of our proposed
method.
#1420
100
70.8
Blank
Detection of AChE inhibitor via GC-MS/MS
Detection of AChE activity of five whole blood sam-
ples
In order to testify whether there had AChE inhibitors
in those whole bloods with inactive AChE and find out
what these inhibitors were, we introduced GC-MS/MS
to detect the whole blood samples directly. The results
were as follows: high concentration dichlorphos and
dipterex were detected in sample marked as 1390; low
concentration dichlorphos was detected in sample
marked as 1477; rogor and omethoate were detected in
Finally, we applied this mass spectrometry based
method to detect AChE activity of whole blood of four
decedents with unknown death reasons and healthy
persons. The whole blood samples of healthy persons
were from persons who all died in car accidents. The
whole bloods from these persons were collected and
mixed to obtain the final healthy samples which was
4
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Chin. J. Chem. 2012, XX, 1—5

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay
912.
sample marked as 1462; sumicidin was detected in
[4] Guo, J. X.; Wu, J. J. Q.; Wright, J. B.; Lushington, G. H. Chem. Res.
sample marked as 1420. Among these compounds, di-
chlorphos, dipterex, rogor and omethoate were all OP
pesticides, which were definite AChE inhibitors.
Meanwhile, sumicidin was not designed as AChE in-
hibitor, thus, the whole blood containing sumicidin had
equal AChE activity with whole blood from healthy
person. Therefore, through detecting AChE activity, it
was convenient and fast to identify the existence of
AChE inhibitors. Coupling with MALDI-FTMS, high
throughput detection had been realized easily, and this
method was suitable for detecting real samples.
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Conclusions
A simple and convenient approach about screening
irreversible AChE inhibitors by MALDI-FTMS is de-
veloped in this work. It is helpful for excluding
non-AChE inhibitor toxicant quickly and judging the
existence of AChE inhibitors via detecting AChE activ-
ity. In comparison with other detection method, mass
spectrometry provides accurate analysis results about
enzyme activity. Furthermore, MALDI-FTMS shows
advantages to analyze real samples, due to its salt toler-
ance and high-throughput. The method used in this pa-
per identified that three of decedents had died for irre-
versible AChE inhibitors, whose whole bloods were
figure out containing OP pesticides in deem by the fol-
lowing GC-MS/MS analysis. The application of
MALDI-FTMS detecting AChE activity and screening
irreversible AChE inhibitors, offers an alternative
choice for rapid screening enzyme inhibitors.
Acknowledgments
This work was supported by the Science and Tech-
nology Commission of Shanghai Municipality project
(No. 10595812600), the Key Laboratory of Evidence at
the Scene Open Subject (No. 2011xcwzk07), the Na-
tional Natural Science Foundation of China (Nos.
20902104, 21072115) and the Innovation Method fund
of China (No. 2010IM030900).
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