Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry

Article abstract of DOI:10.1021/acs.analchem.6b03975

Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis-Menten constants K_m for these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine K_m and turnover number k_cat for trypsin digestion of angiotensin II (K_m and k_cat are determined to be 6.4 mM and 1.3 s^-1, respectively).

Full text of DOI:10.1021/acs.analchem.6b03975

Article
pubs.acs.org/ac
Online Monitoring of Enzymatic Reactions Using Time-Resolved
Desorption Electrospray Ionization Mass Spectrometry
Si Cheng,† Qiuhua Wu,*,†,‡ He Xiao,† and Hao Chen*,†
†Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio
University, Athens, Ohio 45701, United States,
‡Department of Chemistry, College of Science, Agricultural University of Hebei, Baoding 071001, China
S
* Supporting Information
ABSTRACT: Electrospray ionization mass spectrometry (ESI-MS) is power-
ful for determining enzymatic reaction kinetics because of its soft ionization
nature. However, it is limited to use ESI-favored solvents containing volatile
buffers (e.g., ammonium acetate). In addition, lack of a quenching step for
online ESI-MS reaction monitoring might introduce inaccuracy, due to the
possible acceleration of reaction in the sprayed microdroplets. To overcome
these issues, this study presents a new approach for online measuring
enzymatic reaction kinetics using desorption electrospray ionization mass
spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in
a buffered aqueous media (e.g., a solution containing Tris buffer or high
concentration of inorganic salts) could be directly detected. Furthermore, by
adjusting the pH and solvent composition of the DESI spray, reaction can be
online quenched to avoid the postionization reaction event, leading to fast and
accurate measurement of kinetic constants. Reaction time control can be
obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this
study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by
acetylcholinesterase. Derived Michaelis−Menten constants Km for these two reactions were determined to be 214 μM and 172
μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-
MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine Km and turnover number kcat for trypsin
digestion of angiotensin II (Km and kcat are determined to be 6.4 mM and 1.3 s−1, respectively).
ammonium acetate for both enzymatic reaction and for
inetic studies play an essential role in the elucidation of
1
Kchemical and biochemical reaction mechanisms. Typi- ionization. Another challenge is the necessity of online reaction
quenching, which is critical for obtaining accurate kinetics
measurement. Recent studies showed that the reaction could be
accelerated in the charged droplets generated from the
spray.23−26 It was reported that reaction rate in microdroplets
could be significantly accelerated by a factor of a thousand
compared to the one in bulk solution.27 Therefore, if the
reaction were not quenched prior to MS analysis, it could be
accelerated during the spray process of ionization, which would
make it difficult to obtain accurate kinetics measurements.
However, online quenching of reaction prior to ESI-MS
analysis is inconvenient and was not reported before. To tackle
these issues, we proposed a new approach of using desorption
electrospray ionization mass spectrometry (DESI) for online
measurement of enzymatic reaction kinetics.
cally, kinetic measurements are carried out using optical
methods, such as fluorescence and absorption spectroscopies.
Nowadays mass spectrometry (MS) has become a powerful
tool for the study of reaction kinetics.2−15 The advantage of
using MS over spectroscopic techniques is that MS has a high
chemical specificity and does not require chromophoric
substrates. It can also provide the molecular weight and
structure information for reaction products and intermediates.
With the advances in the field of MS, various elegant MS-based
approaches, particularly those employing the soft electrospray
ionization (ESI) technique,12,16−22 have been reported for
online reaction kinetic studies. However, the reports about
online measurement of enzymatic reaction kinetics using ESI-
MS are still limited.10,12,22 In general, there are conflicts of ESI
ionization condition with enzymatic reaction media. For
instance, enzymatic reactions occur in buffered aqueous
environments with certain pH and ionic strength (containing
inorganic salts or nonvolatile buffers), while ESI usually needs
the addition of acid and organic additives into samples for
efficient ionization. These additives would denature enzymes.
One compromised solution is to use volatile buffers, such as
DESI, a representative spray-based ambient ionization
technique, was introduced in 2004 by Cooks and co-workers
as a milestone in the field of MS.28,29 Numerous of applications
Received: October 9, 2016
Accepted: January 26, 2017
Published: January 26, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.analchem.6b03975
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
of DESI-MS have been reported, ranging from drug detection
to tissue imaging.28−31 Traditionally DESI addresses solid
samples deposited onto a surface. In our and other’s
laboratories, DESI was extended for the analysis of liquid
samples.32−36 Analyte ionization by liquid sample DESI occurs
via interaction of the liquid sample with charged droplets
generated by the DESI spray and the resulting ions are
collected and analyzed by a nearby mass spectrometer. It is
useful for directly analyzing samples including large proteins/
protein complexes from their native environments.33,37−42
Furthermore, it is also possible to use liquid sample DESI-
MS for capturing transient organometallic or electrochemical
reaction intermediates.43−46 On the basis of the capability of
DESI for ionizing a high-speed liquid jet, generated from fast
mixing of reactants, at different positions, kinetics measurement
of fast reaction with a high time resolution (300 μs) was
achieved.47
reaction (e.g., a solution containing Tris buffer or high
concentration of inorganic salts). Also, different from our
previous kinetics experiment,47 the formation of a liquid jet is
not required in this case. Therefore, the consumption of
substrate sample could be greatly reduced. In this study, three
hydrolysis reactions catalyzed by β-galactosidase, acetylcholi-
nesterase and trypsin were chosen as enzymatic reaction
models to validate the proposed DESI-MS method.
EXPERIMENTAL SECTION
■
Chemicals. 2-Nitrophenyl-β-D-galactopyranoside (ONPG),
bovine serum albumin (BSA), acetylcholine (Ach), acetylcho-
linesterase (AchE), trypsin, magnesium chloride (MgCl2),
potassium chloride (KCl), trizma, and choline were purchased
from Sigma-Aldrich (St louis, MO, USA). β-Galactosidase from
Escherichia coli and acetonitrile (ACN) were purchased from
EMD Millipore (Billerica, MA, USA). Angiotensin II and
angiotensin II (3−8) were bought from Genway (San Diego,
CA, USA). Angiotensin III antipeptide was purchased from
CPC Scientific (Sunnyvale, CA, USA). Ammonia (NH3·H2O),
methanol (MeOH), and acetic acid (HOAc) were purchased
from Fisher Scientific (Hampton, NH, USA). 2-Nitrophenol-
3,4,5,6-d4 (ONP-d4) and choline-d4 were purchased from CDN
isotope (Pointe-Claire, Quebec, Canada). Silica capillary was
purchased from IDEX (Lake forest, IL, USA). P890 micro-Tee
mixer was purchased from Upchurch Company (Oak Harbor,
WA, USA).
Scheme 1 illustrates the principle underlying the DESI-MS
method for online measurement of enzymatic reaction kinetics,
Scheme 1. Schematic Showing the Apparatus of Time-
Resolved DESI-MS
Method. Experiments were performed using either a DECA
MAX LCQ ion trap mass spectrometer (Thermo Finigan) or
an LTQ Orbitrap mass spectrometer (Thermo Scientific)
equipped with a homemade liquid sample DESI source.
Scheme 1 shows the configuration of the online time-resolved
DESI-MS apparatus. The nebulization nitrogen gas pressure
was set at 160 psi. The high voltage applied to the DESI sprayer
was +5 kV for the positive ion mode and −5 kV for the negative
ion mode. The enzyme and the substrate were introduced by
two syringes separately (driven by a single syringe pump with
two channels) and mixed in a micro-Tee mixer that is
connected with a piece of silica capillary (ID 100 μm, 3.5 cm
long) to serve as a microreactor for reaction.
The reaction buffer for hydrolysis of 2-nitrophenyl-β-D-
galactopyranoside by β-galactosidase consisted of 2.0 mM KCl,
0.1 mM MgCl2, and 0.1% BSA (w/w) and its pH was adjusted
to 7.8 with aqueous ammonia. The enzyme was dissolved in the
reaction buffer to prepare a 9 nM enzyme solution. The
substrate was dissolved in the same reaction buffer at different
concentrations (91, 182, 373, 455, 545 μM) with addition of 10
μM ONP-d4 as an internal standard (IS) for the product
quantification purpose. The enzyme solution and the substrate
solution injection flow rates were kept the same and were
varied from 10 to 1 μL/min (corresponding to the reaction
time from 0.99 to 9.96 s). The spray solvent for hydrolysis of 2-
nitropenyl-β-D-galactopyranoside by β-galactosidase was NH3·
H2O/ACN/H2O (0.1:50:50 by volume, ammonia concen-
tration is 15 mM) for enhancing the product ionization
efficiency in the negative ion mode and also for quenching the
reaction (see the quenching test result in the discussion below
and in Figure S4).
in which enzyme and substrate solutions undergo mixing in a
micro-Tee mixer that is connected with a short piece of silica
capillary to serve as a microreactor for reaction. After exiting
the microreactor capillary, the reaction mixture is directly
ionized by DESI for MS detection. The reaction time can be
varied by changing injection flow rates of enzyme and substrate
solutions. There are several advantages of using DESI-MS for
enzymatic reaction monitoring. First, unlike ESI which requires
organic solvent/acid additives to be added into the sample for
obtaining optimal sensitivity, DESI allows the direct ionization
of sample from aqueous solution with good sensitivity.38 This is
important for enzymatic reaction analysis because the enzyme
could be deactivated with these additives, as mentioned above.
This feature of DESI is ascribed to the freedom to adjust its
spray solvent composition, for example, adding acid, base or
acetonitrile into the spray solvent. Meanwhile, addition of these
additives into the spray solvent could be used for online
reaction quenching during the ionization step. Thus, the
potential postionization reaction in the spray droplets could be
avoided. Furthermore, DESI has high salt tolerance47−49 and is
suitable for directly detecting enzymatic reaction product or
monitoring the consumption of the substrate in the buffered
solution which is typically required media for enzymatic
Acetylcholinesterase was dissolved in 10 mM Tris buffer with
the final enzyme concentration of 1.4 μM. Acetylcholine was
dissolved in the same buffer at different concentrations (0.14,
0.35, 0.56, 0.7, 1.4 mM) with addition of 70 μM choline-d4 as
an internal standard. The same sample introduction system
B
DOI: 10.1021/acs.analchem.6b03975
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
(Scheme 1) was used for studying the hydrolysis of
acetylcholine catalyzed by acetylcholinesterase. ACN was used
as the spray solvent for quenching the reaction as it is an
organic solvent.14
ase produces choline and acetic acid (eq 2, Scheme 2). The
choline can be detected at m/z 104 by DESI-MS in the positive
ion mode. Hydrolysis of angiotensin II (sequence DRVYIHPF)
by trypsin produces angiotensin II (3−8) (sequence VYIHPF,
eq 3, Scheme 2), which can be detected at m/z 775 by DESI-
MS in the positive ion mode.
The enzyme β-galactosidase dissolved in a reaction buffer
consisting of 2.0 mM KCl, 0.1 mM MgCl2, and 0.1% BSA (w/
w, pH 7.8). Monovalent potassium ions (K+), as well as
divalent magnesium ions (Mg2+), are required for obtaining the
enzyme’s optimal activity55 and the optimal working pH of β-
galactosidase is 6−8.56 Both enzyme and ONPG dissolved in
the same buffer were infused (at the same flow rates) into the
micro-Tee mixer for reaction followed by direct DESI-MS
detection. By changing the injection flow rates from 10 to 1
μL/min, different reaction times varying from 0.99 to 9.96 s
were resulted which affected the yield of the reaction product
ONP. Figure 1 displays the acquired DESI-MS spectra showing
For the study of trypsin digestion kinetics, angiotensin II was
chose as the reaction substrate. Different concentrations of
angiotensin II (1.5, 2, 2.5, 3, 3.5 mM) and 0.06 mM trypsin
were prepared in 25 mM ammonium bicarbonate. 40 μM
angiotensin III antipeptide was added to the substrate solution
as an internal standard. The same sample introduction system
(Scheme 1) was used for examining the hydrolysis of
angiotensin II by trypsin. The DESI spray solvent for this
reaction was MeOH/H2O/HOAc (50:50:3, by volume) for
enhancing sample ionization efficiency and for quenching the
reaction (see the quenching test result in the discussion below
and in Figure S6).
RESULTS AND DISCUSSION
■
Enzymes plays very important role in many disciplines, such as
biochemistry, medicine, and pharmacology. One key issue in
enzyme-based assays is the accurate characterization of the
enzymatic reaction constants such as Michaelis−Menten
constant Km and turnover number kcat.50,51 The Michaelis−
Menten kinetics is one of the most commonly used models to
describe the bulk reaction of enzymes,52,53 and Km and kcat are
two of the important constants in enzymatic reactions. Km
quantifies the affinity between enzyme and substrate and kcat
reveals the turnover rate of the reaction. A typical strategy used
to calculate Km and kcat is the Lineweaver−Burk plot (L−B
plot),54 which is based on relationship between two reciprocals
of both the initial reaction rate (v0) and the substrate
concentration (C0).
Figure 1. Time-resolved DESI-MS spectra showing the hydrolysis
reaction of ONPG (545 μM) catalyzed by β-galactosidase (9 nM, pH
at 7.8) sampled by DESI-MS at (a) 0.99, (b) 1.25, (c) 2.49, and (d)
9.96 s, respectively.
Scheme 2 shows three enzymatic reactions that were
examined in this study. Hydrolysis of ONPG by β-galactosidase
produces 2-nitrophenol (ONP) and galactose (eq 1, Scheme
2). In the negative ion mode, the product ONP can be
deprotonated to form the ONP anion and detected at m/z 138
by DESI-MS. Hydrolysis of acetylcholine by acetylcholinester-
the enzymatic reaction of 545 μM substrate and 9 nM enzyme
sampled at different reaction times. In this experiment, for
quantification of the product ONP, ONP-d4 was added into the
substrate solution to serve as an internal standard. Our test
results showed that ONP and ONP-d4 have the same DESI
ionization efficiency, even at different sample injection flow
rates, becaue of the similarity of their structures (Figure S1).
Therefore, the concentration of ONP from the enzymatic
reaction can be calculated based on the relative intensity of the
ONP peak (m/z 138) relative to the IS peak (m/z 142) and the
known concentration of ONP-d4. To confirm whether the
addition of internal standard has influence on the reaction
kinetics, experiments using different concentrations of internal
standard were carried out. The result showed that the product
concentration was the same when the concentration of internal
standard ONP-d4 changed from 10 to 5 μM (Figure S2),
indicating that the added internal standard has no effect on
reaction kinetics.
Scheme 2. Equations Showing Three Enzymatic Reactions
That Were Studied: Hydrolysis of 2-Nitrophenyl-β-D-
galactopyranoside by β-Galactosidase (Eq 1), Hydrolysis of
Acetylcholine by Acetylcholinesterase (Eq 2), and Digestion
of Angiotensin II by Trypsin (Eq 3)
As shown in the acquired DESI-MS spectra (Figure 1), the
intensity of m/z 138 relative to m/z 142 increased with the
increased reaction time, showing the progress of the β-
galactosidase-catalyzed hydrolysis with time. In this case,
although the reaction sample contains inorganic salts such as
KCl and MgCl2, the product ONP can be well ionized by
DESI-MS (Figures 1 and S3-a). In comparison, when ESI was
used for the ionization of the same sample, weaker signal (by 4-
fold) of m/z 138 was observed and the background of the ESI-
C
DOI: 10.1021/acs.analchem.6b03975
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
MS spectrum was quite high (Figure S3-b), probably because of
the salt suppression effect. In Figure 1, a peak at m/z 135 was
observed, which was from the DESI spray background.
varied at 91, 182, 373, 455, and 545 μM. Each concentration
was sampled at 5 different reaction times in triplicate
measurements. The reaction time was calculated by using the
total dead volume of Tee mixer and the microreactor capillary
(0.332 μL) divided by the total flow rate of substrate and
enzyme solutions. Once the reaction commences, the intensity
ratio of the product ion to the internal standard ion increases
linearly in accordance with the classical Michaelis−Menten
kinetics (this initial part of the enzyme reaction has a linear
trend and the slope of the linear fit is the initial reaction velocity
v0). Using the obtained values of v0 for different substrate
concentrations (from Figure 2), the reaction constant Km can
be obtained, using the L−B plot. The L−B plot is described by
eq 4 where 1/v0 has a linear relationship with 1/C0, in which
the slope is Km/Vmax and the y-intercept is 1/Vmax. (where Vmax
is the maximum reaction rate and C0 is the initial substrate
concentration)
The spray solvent used in this case was NH3·H2O/ACN/
H2O (0.1:50:50 by volume), which was chosen for two reasons.
First, because of the acidity of ONP, ammonia was added into
the spray solvent to facilitate the deprotonation of ONP into an
anion. Second, such a basic solvent would be able to quench the
enzymatic reaction, which is important and necessary for
preventing the possible postionization reaction process. To test
this hypothesis, DESI-MS spectra were recorded for the
ionization of the reaction mixture under the condition that
the distance between the microreactor capillary outlet tip and
the inlet of mass spectrometer (d, Scheme 1) was changed. Our
data showed that the intensity ratios of m/z 138 to m/z 142
was nearly kept the same (8.6−8.7%, Figure S4-a−c) with
changing distances, indicating the reaction was well quenched
when the basic spray solvent was used for ionization. In
contrast, when the spray solvent was changed to ACN/H2O
(50:50 by volume) without ammonia, the ratio increased from
14% to 21% when the distance d increased from 0.2 to 1 cm
(Figure S4-d−f). It is very likely that the reaction had not been
completely quenched upon DESI ionization with the spray
solvent of ACN/H2O. The results above tell that simultaneous
reaction quenching and efficient ionization could be achieved
by DESI-MS with the use of an appropriate spray solvent. By
contrast, carrying out online reaction quenching is inconvenient
for ESI-MS, since the quenching solution needs to be infused
through a second Tee-mixer, which would increase the
complexity of the apparatus and delay the MS detection of
the reaction product (therefore not suitable for fast reaction
monitoring). In addition, we carried out an additional
experiment to show that our DESI quenching could be more
effective and faster than the traditional offline quenching. When
the reaction solution in the capillary reactor was directed into a
10 μL quenching solution of NH3·H2O/ACN/H2O (0.1:50:50
by volume), we found that the reaction product yield was
slightly higher than that from online DESI quenching, based on
the subsequent MS measurement. It is likely that the mixing of
reaction sample with the DESI sprayed droplets in a “confined
volume” is faster than mixing with the bulk quenching solution,
leading to faster quenching. Similar phenomenon was reported
in a recent study reporting that ultrafast mixing of reactants in
microsecond time scale can be achieved using microdroplets
instead of using bulk solutions.57
Km
Vmax C0
1
v0
1
1
Vmax
=
+
(4)
(5)
Vmax = kcat[E]
The reciprocal of v0 (i.e., the slope of the curve shown in
Figure 2) was plotted against the reciprocal of C0 (Figure 3). A
Figure 3. Lineweaver−Burk plot for characterizing Km.
least-squares linear fit provides the slope of the fitting line (Km/
V
max) and the y-intercept (1/Vmax), which determines Km to be
214 μM. This result of Km measurement is in good agreement
with the value of 300 μM reported before using spectroscopy.58
Furthermore, based on the measured value of Vmax from Figure
3 and eq 5 where [E] is the concentration of the enzyme, the
value of kcat was calculated to be 97 s−1. Compared to
spectroscopic methods, the method based on MS measurement
has advantage of no need for chromophoric substrates. The
success in measuring the key kinetics constants of this high-
speed enzymatic reaction is ascribed to the capability of DESI-
MS for both efficient ionization of high salt-containing samples
and online quenching of reaction. To the best of our
knowledge, no literature was found for using MS to study
this reaction kinetics in the past.
Figure 2 displays the change of the reaction product
concentration vs the reaction time. The substrate concentration
To further verify the applicability of this DESI-MS method,
the hydrolysis of acetylcholine by acetylcholinesterase was also
studied as another test example. Ach is a neurotransmitter in
peripheral and central nervous systems.59 AchE could reduce
the Ach level in neurosystem by hydrolyzing Ach to choline and
acetic acid. In the DESI-MS experiment, both AchE and Ach
were dissolved in 10 mM Tris buffer and then underwent
mixing for reaction in the microreactor, following by direct
DESI-MS analysis. The DESI spray solvent used was ACN for
aiding the ionization and quenching the reaction. Figure 4
Figure 2. Plot showing the change of the reaction product
concentration with the reaction time. The substrate ONPG
concentration varied from 91 to 545 μM. The β-galactosidase
concentration of enzyme was set as 9 nM.
D
DOI: 10.1021/acs.analchem.6b03975
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
Figure 5. Time-resolved DESI-MS spectra showing the reaction
between 3.5 mM angiotensin II and 0.06 mM trypsin sampled by
DESI-MS at (a) 1.17, (b) 1.76, (c) 3.51, and (d) 7.02 s, respectively.
Figure 4. Time-resolved DESI-MS spectra showing the reaction
between 1.4 mM Ach and 1.4 μM AchE sampled by DESI-MS at (a)
0.33, (b) 0.49, (c) 0.99, and (d) 3.32 s, respectively.
[VYIHPF + H]+ as a result of the c-terminal cleavage of the
substrate’s arginine residue. The internal standard ion
[GVYVHPV + H]+ appears at m/z 770. A separate experiment
showed the ionization efficiency of the product vs the internal
stand is 0.77 (data not shown), which was used for
measurement of the product concentrations at different
reaction times. As also shown in Figure 5, with increasing
reaction time, the intensity of the product ion increased. In this
experiment, MeOH/H2O/HOAc (50:50:3 by volume) was
chosen as DESI spray solvent, for enhancing the ionization
efficiency and quenching the reaction. As displayed in Figure 5,
the product ion is clearly seen in the positive ion mode with
high intensity. Furthermore, another separate experiment
showed that the reaction was well quenched with this chosen
solvent (Figure S6). DESI-MS spectra were recorded for the
ionization of the reaction mixture under the condition that the
distance between the microreactor capillary outlet tip and the
inlet of mass spectrometer was changed. Our data showed that
the intensity ratio of m/z 775 to m/z 770 remained constant
(220%, Figure S6-a−c) when distance increased from 0.5 to 1
cm. In contrast, when the spray solvent was changed to
MeOH/H2O (50:50 by volume), the ratio increased signifi-
cantly from 310% to 500% with changing distances (Figure S6-
d−f).
displays DESI-MS spectra showing the enzymatic reaction of
1.4 mM substrate and 1.4 μM enzyme in 10 mM Tris buffer
sampled at different times. The choline-d4 was added as the
internal standard (seen at m/z 108) for assisting quantification
of the reaction product choline (seen at m/z 104). According
to the time-dependent DESI-MS spectra, m/z 104 (relative to
m/z 108) increased with increasing reaction time. At the same
time, the peak of the substrate (m/z 146) decreased with time.
As displayed in the spectrum (Figure 4), the ion signal for m/z
104 is good, even in the presence of 10 mM Tris buffer. Again,
this shows the high tolerance of DESI to matrices.33,48 Figure
S5-a and S5-b shows the change of the reaction product with
the reaction time for five different substrate concentration
measurements (0.14, 0.35, 0.56, 0.7, and 1.4 mM) and the L−B
plot, respectively. The Km was found to be 172 μM. Again, this
result is in line with the value reported in the previous ESI-MS
study (230 μM),14 in which the reaction was offline quenched
by ACN solution. Compared to the ESI-MS approach, our
method has features with simpler apparatus and faster
detection. Also, based on eq 5, kcat was found to be 234 s−1.
Trypsin is an important protease for protein digestion in
typical MS-based shotgun proteomics experiments because of
its high specificity, widespread availability, and ease of use.60
This pancreatic serine protease cleaves peptides on the C-
terminal side of lysine and arginine residues. Surprisingly,
although trypsin is widely used for MS-based research, the
reaction kinetics study for the digestion of proteins/peptides by
trypsin using MS is rare.12 Because of the success of the
measurements of kinetics parameters of the aforementioned
two hydrolysis reactions, we believe that our DESI-MS can
serve for this purpose. In this experiment, peptide angiotensin
II (sequence DRVYIHPF) was chosen as the substrate.
Angiotensin II solutions at different concentrations containing
40 μM internal standard of angiotensin III antipeptide
(sequence GVYVHPV, chosen because of its similar structure
to the trypsin digestion product VYIHPF) was prepared in 25
mM ammonium bicarbonate and infused into the micro-Tee
mixer via one inlet. 0.06 mM trypsin dissolved in 25 mM
ammonium bicarbonate was infused into the micro-Tee mixer
via the other inlet. Figure 5 shows the DESI-MS spectra of the
enzymatic reaction of 3.5 mM substrate and 0.06 mM enzyme
sampled at different reaction times. In Figure 5, the peak at m/z
775 corresponding to the protonated digestion product
Figure S7-a and S7-b displays the change of the reaction
product concentration with the reaction time for different
substrate concentration measurements (1.5, 2, 2.5, 3, and 3.5
mM) and the L−B plot, respectively. By the L−B plot, Km was
measured to be 6.4 mM, indicating the weak affinity of trypsin
toward this peptide. Also, kcat was calculated to be 1.3 s−1.
Under the conditions used in this experiment, this trypsin
digestion turnover rate is the slowest among the three
hydrolysis reactions measured in this study.
CONCLUSIONS
■
In summary, we present a new online DESI-MS approach for
measuring enzymatic reaction kinetic constants. Two striking
features of DESI-MS method in comparison to the traditional
ESI-MS approaches are revealed. First, by choosing a proper
spray solvent, the reaction product could be well ionized from
aqueous solution containing high salt content or nonvolatile
buffer which are required for optimal activity of enzymes.
Second, the online reaction quenching could be realized, again
through the judicious selection of the spray solvent. Three
E
DOI: 10.1021/acs.analchem.6b03975
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
(16) Konermann, L.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1998,
9, 1248−1254.
enzymatic reactions with different turnover rates were explored
and demonstrated the capability of DESI-MS for online
enzymatic reaction monitoring. We believe that DESI-MS
would be of great value for measuring many other enzymatic
reaction kinetics, in particular for fast reactions as DESI
tolerates high sample injection rates.61
(17) Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408−6414.
(18) Lee, E. D.; Mueck, W.; Henion, J. D.; Covey, T. R. J. Am. Chem.
Soc. 1989, 111, 4600−4604.
(19) Li, Z.; Sau, A. K.; Shen, S.; Whitehouse, C.; Baasov, T.;
Anderson, K. S. J. Am. Chem. Soc. 2003, 125, 9938−9939.
(20) Li, Z.; Sau, A. K.; Furdui, C. M.; Anderson, K. S. Anal. Biochem.
2005, 343, 35−47.
(21) Sowole, M. A.; Vuong, S.; Konermann, L. Anal. Chem. 2015, 87,
9538−9545.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.6b03975.
(22) Fu, Q.; Tang, J.; Cui, M.; Zheng, Z.; Liu, Z.; Liu, S. J.
Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2015, 990, 169−173.
(23) Badu-Tawiah, A. K.; Campbell, D. I.; Cooks, R. G. J. Am. Soc.
Mass Spectrom. 2012, 23, 1077−1084.
MS spectra and kinetics data (PDF)
(24) Girod, M.; Moyano, E.; Campbell, D. I.; Cooks, R. G. Chem. Sci.
2011, 2, 501−510.
AUTHOR INFORMATION
■
(25) Bain, R. M.; Pulliam, C. J.; Thery, F.; Cooks, R. G. Angew.
Chem., Int. Ed. 2016, 55, 10478−10482.
Corresponding Authors
*E-mail: qiuhuawu@126.com.
*E-mail: chenh2@ohio.edu.
(26) Yan, X.; Bain, R. M.; Cooks, R. G. Angew. Chem., Int. Ed. 2016,
55, 12960−12972.
(27) Lee, J. K.; Kim, S.; Nam, H. G.; Zare, R. N. Proc. Natl. Acad. Sci.
U. S. A. 2015, 112, 3898−3903.
ORCID
Hao Chen: 0000-0001-8090-8593
Notes
The authors declare no competing financial interest.
́
(28) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science
2004, 306, 471−473.
(29) Wiseman, J. M.; Ifa, D. R.; Song, Q.; Cooks, R. G. Angew. Chem.,
Int. Ed. 2006, 45, 7188.
(30) Jiang, J.; Zhang, H.; Li, M.; Dulay, M. T.; Ingram, A. J.; Li, N.;
You, H.; Zare, R. N. Anal. Chem. 2015, 87, 8057−8062.
(31) Perry, R. H.; Splendore, M.; Chien, A.; Davis, N. K.; Zare, R. N.
Angew. Chem., Int. Ed. 2011, 50, 250−254.
ACKNOWLEDGMENTS
■
Financial supports from NSF Career Award (CHE-1149367),
NSF IDBR (CHE-1455554), NSF MRI Instrument Grant
(CHE-1428787), Program of Study Abroad for Young
Teachers by Agricultural University of Hebei, the National
Natural Science Foundation of China (No. 31471643 and
31671930), and the Natural Science Foundation of Hebei
Province (B2016204136 and B2017204025) are gratefully
acknowledged.
(32) Miao, Z.; Chen, H.. Proceedings of 56th ASMS Conference on
Mass and Allied Topics, 2008.
(33) Miao, Z.; Chen, H. J. Am. Soc. Mass Spectrom. 2009, 20, 10−19.
(34) Ma, X.; Zhao, M.; Lin, Z.; Zhang, S.; Yang, C.; Zhang, X. Anal.
Chem. 2008, 80, 6131.
(35) Chipuk, J. E.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2008, 19,
1612−1620.
(36) Moore, B. N.; Hamdy, O.; Julian, R. R. Int. J. Mass Spectrom.
2012, 330−332, 220−225.
REFERENCES
■
(37) Zheng, Q.; Chen, H. Annu. Rev. Anal. Chem. 2016, 9 (1), 411−
448.
(1) Callender, R.; Dyer, R. B. Chem. Rev. 2006, 106, 3031−3042.
(2) Liesener, A.; Karst, U. Anal. Bioanal. Chem. 2005, 382, 1451−
1464.
(38) Miao, Z.; Wu, S.; Chen, H. J. Am. Soc. Mass Spectrom. 2010, 21,
1730−1736.
(3) Houston, C. T.; Taylor, W. P.; Widlanski, T. S.; Reilly, J. P. Anal.
Chem. 2000, 72, 3311−3319.
(39) Pan, N.; Liu, P.; Cui, W.; Tang, B.; Shi, J.; Chen, H. Analyst
2013, 138, 1321−1324.
(4) Konermann, L.; Simmons, D. A. Mass Spectrom. Rev. 2003, 22,
1−26.
(40) Ferguson, C. N.; Benchaar, S. A.; Miao, Z.; Loo, J. A.; Chen, H.
Anal. Chem. 2011, 83, 6468−6473.
(5) Lento, C.; Audette, G. F.; Wilson, D. J. Can. J. Chem. 2015, 93,
7−12.
(41) Liu, P.; Zhang, J.; Ferguson, C. N.; Chen, H.; Loo, J. A. Anal.
Chem. 2013, 85, 11966−11972.
(6) Bothner, B.; Chavez, R.; Wei, J.; Strupp, C.; Phung, Q.;
Schneemann, A.; Siuzdak, G. J. Biol. Chem. 2000, 275, 13455−13459.
(7) Pi, N.; Armstrong, J. I.; Bertozzi, C. R.; Leary, J. A. Biochemistry
2002, 41, 13283−13288.
(42) Yao, Y.; Shams-Ud-Doha, K.; Daneshfar, R.; Kitova, E.; Klassen,
J. J. Am. Soc. Mass Spectrom. 2015, 26, 98−106.
(43) Zheng, Q.; Liu, Y.; Chen, Q.; Hu, M.; Helmy, R.; Sherer, E. C.;
Welch, C. J.; Chen, H. J. Am. Chem. Soc. 2015, 137, 14035−14038.
(44) Brown, T. A.; Chen, H.; Zare, R. N. J. Am. Chem. Soc. 2015, 137,
7274−7277.
(8) Ge, X.; Sirich, T. L.; Beyer, M. K.; Desaire, H.; Leary, J. A. Anal.
Chem. 2001, 73, 5078−5082.
(9) Gao, H.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2003, 14, 173−
(45) Brown, T. A.; Chen, H.; Zare, R. N. Angew. Chem., Int. Ed. 2015,
54, 11183−11185.
181.
(10) Attwood, P. V.; Geeves, M. A. Anal. Biochem. 2004, 334, 382−
389.
(11) Dennhart, N.; Letzel, T. Anal. Bioanal. Chem. 2006, 386, 689−
(46) Brown, T. A.; Hosseini-Nassab, N.; Chen, H.; Zare, R. N. Chem.
Sci. 2016, 7, 329−332.
(47) Miao, Z.; Chen, H.; Liu, P.; Liu, Y. Anal. Chem. 2011, 83, 3994−
3997.
698.
(12) Robbins, M. D.; Yoon, O. K.; Barbula, G. K.; Zare, R. N. Anal.
Chem. 2010, 82, 8650−8657.
(48) Cheng, S.; Wu, Q.; Dewald, H. D.; Chen, H. J. Am. Soc. Mass
Spectrom. 2016, 1−8.
(13) Xu, Z.; Yao, S.; Wei, Y.; Zhou, J.; Zhang, L.; Wang, C.; Guo, Y. J.
Am. Soc. Mass Spectrom. 2008, 19, 1849−1855.
(14) Hu, L.; Jiang, G.; Xu, S.; Pan, C.; Zou, H. J. Am. Soc. Mass
Spectrom. 2006, 17, 1616−1619.
(49) Zhang, Y.; Chen, H. Int. J. Mass Spectrom. 2010, 289, 98−107.
(50) Copeland, R. A. In Enzymes; John Wiley & Sons, Inc., 2002; pp
350−366.
(51) Copeland, R. A. In Enzymes; John Wiley & Sons, Inc., 2002; pp
109−145.
(15) Northrop, D. B.; Simpson, F. B. Bioorg. Med. Chem. 1997, 5,
641−644.
F
DOI: 10.1021/acs.analchem.6b03975
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
(52) Johnson, K. A.; Goody, R. S. Biochemistry 2011, 50, 8264−8269.
(53) Butterworth, P. J. Cell Biochem. Funct. 2005, 23, 293−294.
(54) Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658−666.
(55) Juers, D. H.; Matthews, B. W.; Huber, R. E. Protein Sci. 2012, 21,
1792−1807.
(56) Tenu, J.-P.; Viratelle, O. M.; Garnier, J.; Yon, J. Eur. J. Biochem.
1971, 20, 363−370.
(57) Lee, J. K.; Kim, S.; Nam, H. G.; Zare, R. N. Proc. Natl. Acad. Sci.
U. S. A. 2015, 112, 3898−3903.
(58) Yoon, J. H.; McKenzie, D. Enzyme Microb. Technol. 2005, 36,
439−446.
(59) Wessler, I.; Kirkpatrick, C. J.; Racke,
77, 59−79.
́
K. Pharmacol. Ther. 1998,
(60) Giansanti, P.; Tsiatsiani, L.; Low, T. Y.; Heck, A. J. R. Nat.
Protoc. 2016, 11, 993−1006.
(61) Zhang, Y.; Yuan, Z.; Dewald, H. D.; Chen, H. Chem. Commun.
2011, 47, 4171−4173.
G
DOI: 10.1021/acs.analchem.6b03975
Anal. Chem. XXXX, XXX, XXX−XXX