T. Cai et al. / International Journal of Mass Spectrometry 353 (2013) 80–83
81
screen enzyme inhibitors [29,30]. Aimed at getting more compre-
another evaluation system other than AChE activity. Organophos-
phorous (OP) pesticide was used as irreversible inhibitors in this
work, which could take place a chemical reaction with the serine
hydroxyl (OH) in the AChE active site [31]. In the presence of APFO
and PFOA, we evaluated both AChE activities and AChE inhibitions
by OP compounds. The results showed that adding PFOA and APFO
had increased AChE activity. On the other hand, the enzyme inhi-
bition from OP pesticide was also increased. Therefore, APFO and
PFOA might strengthen the influence of OP pesticides and sup-
posed to deepen the hazard of long-time pollutants in humans.
Besides, another AChE inhibitor was introduced to study whether
this increase effect occurred in the situation of reversible mecha-
nism. In contrast to OP pesticide, galantamine belongs to reversible
inhibitors of AChE [32]. With the addition of APFO and PFOA, AChE
inhibitions from galantamine had not shown obvious increase. The
enhancements of AChE inhibitions from irreversible inhibitors are
more obvious than those from reversible inhibitors. In our daily
life, we are confronted with more and more exposure of irreversible
AChE inhibitors such as OP pesticides. Hence, the existence of PFOA
in human blood will strengthen the toxicity of OP pesticides and the
potential long-term effect is serious. The inhibition of AChE activ-
ity in blood leads to the increase of ACh, and causes the a series of
disorder of metabolism.
with adding 10 L of ultrapure water serve as control groups.
Then 10 L of inhibitor (acephate or galantamine) was added and
incubated for 30 min at 37 ◦C with 10 L of ultrapure water as
control group. Finally, the substrate ATCh solution (10 L) par-
ticipated in the enzymatic reaction for 20 min at 37 ◦C. Finally,
the acetonitrile (30 L) was added to the mixture to quench the
reaction.
2.4. MALDI-FTICRMS conditions and analysis
Experiments were conducted with Ionspec 4.7 T HisRes MALDI-
FTICRMS (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 trans-
ferred via a quadruple ion guide to the capacitive coupled closed
cylindrical cell. The intensity of MALDI-laser irradiation was varied
between 40% and 50% 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.
The matrix solution (65 L) was added to each final reaction sys-
tem and mixed completely. Then, the mixture (2 L) was deposited
on the stainless steel target. Samples got dried slowly to produce a
microcrystalline layer and were analyzed by MALDI-FTICRMS.
The abundances of peaks reflected the content of compounds.
The conversion of substrate (C, %) represented the enzymatic activ-
ity, and was calculated as follows:
2. Experimental
2.1. Reagents
ꢀ
ꢁ
Acetylcholinesterase (AChE) from electrophorus electricus
(Type VI-S, EC 3.1.1.7, 426 U/mg), acetylthiocholine (ATCh)
iodide, 2-amino-2-(hydroxymethyl) -1,3-propanediol (Tris) and
2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma
Chemical Co. (St Louis, MO). Acephate (organophosphorus pes-
ticide) was from Shanghai Institute for Food and Drug Control
(Shanghai, China). Ammonium perfluorooctanoate (APFO) and per-
fluorooctanoic acid, PFOA were purchased from TCI Shanghai
(Tokyo Chemical Industry Co., Ltd., Shanghai, China). Galantamine
was bought from Aladdin Reagent Database Inc. (Shanghai, China).
The ultrapure water used in this paper was purified by a Milli-
Q water purification system (Millipore Corp., Bedford, MA, USA).
Methanol and acetonitrile (ACN) were of HPLC-grade quality and
purchased from Merk (Darmstadt, Germany). Polyethylene glycol-
200 (PEG-200), PEG-400, hydrochloric acid (HCl), trifluoroacetic
acid (CF3COOH), glycerol were from Shanghai Chemical Reagent
Corporation (Shanghai, China).
TCh
ATCh + TCh
C =
× 100%
where [TCh] is replaced by the abundance of TCh. [ATCh] is replaced
by the abundance of remaining ATCh as well as the fragment of
ATCh. The C is subsequently used to calculate the enzymatic inhi-
bition (I, %).
The enzymatic inhibition (I, %) was calculated as follows:
ꢀ
ꢁ
C0 − Ci
I =
× 100%
C0
where C0 is the conversion rate of ATCh to TCh with ultrapure
water as control group. Ci is the conversion rate inhibited by AChE
inhibitors.
3. Results and discussion
2.2. Materials preparation
ATCh was used as the substrate of AChE in experiments, and
it would be transformed into TCh by AChE (shown in Scheme 1).
Fig. 1 depicts a typical mass spectrum about this catalysis. In high
resolution MALDI-FTICRMS, the ion signal of m/z 162.1 is from sub-
strate [ATCh]+ and the relative product [TCh]+ is m/z 120.1. The
ion signal of m/z 103.0 is recognized as the fragment of [ATCh]+
(m/z 162.1), which had been discussed in our previous study (28).
The compounds ATCh and TCh contained similar structure and
gave excellent signals in MALDI-FTICRMS. They served as internal
standard for each other in quantitative analysis, so that the change
of abundances of peaks was used to reflect the change of contents
of compounds. Therefore, the abundances of substrate and product
could be used to calculate conversion of substrate (enzyme activ-
ity). With the mix-droplet method and appropriate laser spot, we
could obtain good precision. The data reproducibility of substrate
conversion in MALDI-FTICRMS had been investigated. The percent-
age of relative standard deviation (%R.S.D.) in dot was within 6%,
The substrate ATCh solution (5 mM) was prepared with Tris–HCl
buffer freshly. AChE solutions were prepared by 50 mM Tris–HCl
buffer (pH 8.0) to 5 U/mL and were preserved at −20 ◦C in dark.
Acephate (100 g/L), galantamine (10 mg/L), PFOA (0.2 mM) and
APFO (0.2 mM) solutions were made by fresh ultrapure water and
stored at −20 ◦C. PFOA solution was neutralized with NaOH solu-
tion to pH = 7.4. The matrix solution was prepared daily as follows:
DHB solid (50 mg) was dissolved with methanol (500 L), adding
1% CF3COOH; and then, the DHB solution was mixed with a glyc-
erol/water solution (60/40, vol/vol).
2.3. Measurement of AChE catalyzed reactions
Enzyme reactions were carried out in eppendorf plastic tubes.
AChE solution (10 L) and 10 L of PFOA or APFO were incu-
bated for 30 min at 37 ◦C temperature controlled oven. Systems
Cai, Tingting
