Article

Cite This: Anal. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/ac

A Wild-Type Nanopore Sensor for Protein Kinase Activity

Fu-Na Meng,§,† Yi-Lun Ying,*,†,‡ Jie Yang,† and Yi-Tao Long*,†,‡
†
School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China
‡
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University,
Nanjing 210023, P. R. China
*
S Supporting Information
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ABSTRACT: Protein kinases play a critical role in regulating virtually all cellular processes. Here, we developed a novel one-
step method based on a wild-type aerolysin nanopore, which enables kinase activity detection without labeling/modification,
immobilization, cooperative enzymes and complicated procedures. By virtual of the positively charged confinement of the
aerolysin nanopore, the kinase-induced phosphopeptides are specially captured while the positively charged substrate peptides
might move away from the pore by the electric field. Combining with internal standard method, the event frequency of the
phosphopeptides exhibited a dose-dependent response with kinases. The detection limit of 0.005 U/μL has been achieved with
protein kinase A as a model target. This method also allowed kinase inhibitor screening, kinase activity sensing in cell lysates
and the real-time monitoring of kinase-catalyzed phosphorylation at singe molecule level, which could further benefit
fundamental biochemical research, clinical diagnosis and kinase-targeted drug discovery. Moreover, this nanopore sensor shows
strong capacity for the other enzymes that altered substrate charge (e.g., sulfonation, carboxylation, or amidation).

P rotein kinases are a critical family of enzymes that regulate

virtually all cellular processes, including differentiation,
proliferation, motility, and apoptosis, and thus cell function.1−3
Nanopore sensor provides a single-molecule platform for
sensitive detection.14,15 When a certain voltage is applied
across a nanoscale pore, target molecules will be driven into
Modulation of cell function by kinases is the result of the the pore and interact with it, resulting in the typical blockages
phosphorylation of target protein substrates that are involved of the pore’s ionic current. Both the target identity and
in intracellular signaling pathways.4,5 The phosphorylation of quantity can be determined by analyzing the current
protein substrates is evidenced to activate or deactivate target signatures. The nanopore technique has been extensively
proteins at the molecular level. The resulting activation or employed to detect DNA/RNA,16−20 peptides,21,22 pro-
deactivation of target proteins can in turn lead to aberrant teins,23,24 enzymes25−28 and host−guest molecules.29,30 Aero-
signal transduction if levels of kinase activity are altered, as is lysin,31−33 a promising protein sensor, has been applied to
the case in many disease states.6,7 Therefore, monitoring investigate the peptides with different charges,21,34,35 discrim-
protein kinase activity is significant for fundamental bio- inate very short oligonucleotides with single base resolution36
chemical research, clinical diagnosis, and kinase-targeted drug and monitor enzymatic degradation kinetics,37 et al. Although
discovery. the protein kinase inhibitors and the binding constants
A variety of methods have been developed to assess protein between protein kinases and the substrate could be evaluated
kinase activities, including radiometric assays,8 fluorescence,9,10 by nanopore via genetic engineering and chemical modify-
electrochemistry,11 surface plasmon resonance12 and mass ing,38,39 the direct detection of kinase activity has not been
spectrometry.13 These methods mentioned above have their achieved by nanopore up to now. Besides, the chemical
own unique advantages, such as high sensitivity and high modification of nanopores complicated the analysis procedures
specificity, etc. However, most of them suffer from certain and increased the cost. Here we utilize the positively charged
drawbacks such as harmful radioactive labels, sophisticated and confinement of the wild-type aerolysin nanopore to achieve
costly fluorescence-labeling peptide, multistep detection one-step method for the kinase activity sensing including
procedures, inadequate contact in reactants and expensive
recognition proteins. Thus, it is still challenging but highly Received: March 29, 2019
desirable to develop new principle for simple, convenient, Accepted: June 26, 2019
label-free detection for protein kinase. Published: June 26, 2019

© XXXX American Chemical Society A DOI: 10.1021/acs.analchem.9b01570

Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry Article

Figure 1. (a) Schematic representation of PKA activity detection via a wild-type aerolysin nanopore. Positive-charged S-peptide (LRRASLG)
hardly induced any blockage events while the product P-peptide produced large amounts of current blockage events after catalyzed by PKA. The
red circle on the P-peptide represents the phosphate group with two negative charges. The raw current traces were recorded with the reaction
solution containing 100 μM S-peptide before and after catalyzed by PKA (0.1 U/μL). The detailed information on this PKA catalysis experiment is
shown in the Experimental Section. The detection buffer was composed of 1.0 M KCl, 20 mM MgCl2 and 50 mM Tris (pH 7.5). The potential was
set at +100 mV, which was applied from stem side and the cap side was connected to the ground. (b) Correlation curve between the relative event
frequency of P-peptide and [PKA]. Based on the curve, the PKA can be quantified via the nanopore. (c) The phosphorylation evolution of S-
peptide in the course of the enzyme catalysis reaction. The aerolysin nanopore achieved to real-time monitor the kinase catalysis at single molecule
level.

ultrasensitive kinase activity detection in cell lysate, the kinase (Shanghai, China). cAMP-dependnet protein kinase (PKA)
inhibitor screening and the real-time monitoring of the kinase catalytic subunit was purchased from New England Biol-
kinetics. A previous research utilized the α-hemolysin nano- abs (Beverly, MA, USA). Forskolin (Fsk) was purchased from
pore to detect the protein phosphorylation by tagging the GL Biochem Ltd. (Shanghai, China). 3-Isobutyl-1-methylxan-
protein on the C terminus with a 30-mer oligodeoxycytidine.40 thine (IBMX), H-89 and decane were obtained from Sigma-
In this paper, the peptide phosphorylation without any labeling Aldrich (St Louis, MO, USA). ATP and ADP were purchased
and chemical modifications can be determined via the from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai,
aerolysin nanopore, aiming to obtain the target kinase activity. China). MCF-7 cells were from MoXi Biotech. Co. Ltd.
The principle that underlies the sensing platform is shown in (Shanghai, China). All other reagents were of analytical grade
Figure 1. Here, we selected protein kinase A (PKA) as a model and were used as received without further purification.
target and its recognition consensus sequence LRRASLG41 as Nanopore Formation and Data Collection. The details
the substrate peptide (S-peptide) to examine the kinase of the nanopore formation are described in our previous
activity. Under the catalysis of PKA, the serine of S-peptide will research.29 The two chambers beside the nanopore were filled
be phosphorylated, resulting in phosphorylated peptide (P-
with the buffer solution (1 M KCl, 20 mM MgCl2 and 50 mM
peptide) (Supporting Information (SI) Figure S1). The P-
Tris at pH 7.5). The experiments were conducted using eONE
peptide induced the evident current blockages while the
amplifier (Elements SRL, Cesena, Italy). The sampling rate
positively charged molecule, S-peptide, might move away from
the pore and hardly caused any blockages under the bias was set to 100 kHz and the final bandwidth 5 kHz. The data
voltage. Combining with internal standard method, the were acquired by EDR 3 software (Elements SRL, Cesena,
frequencies of the blockage events induced by P-peptide can Italy). Data analysis was performed using MOSAIC software44
be used to quantify the kinase activity. This one-step method and Origin 8.0 (OriginLab Corporation, Northampton, MA).
realizes facile quantitative analysis of kinase without peptide The event frequencies were obtained by plotting the relation
labeling/modification and other cooperative enzymes, repre- curves between cumulative event counts versus recording time.
senting significant advantages over traditional kinase assays.9,42 PKA Activity Detection and Inhibition Study. In the
This strategy is also successfully applied to evaluate the kinase presence of ATP and Mg2+, PKA can transfer γ-phosphate
inhibitor and monitor drug-triggered PKA activation in cell group of ATP to the serine residue of S-peptide at pH 7.5.
lysates. Moreover, this nanopore system can realize a real-time Then, the assays for assessing kinase activity were performed as
monitoring of the kinase-catalyzed phosphorylation, which follows. Typically, the PKA-catalyzed phosphorylation reaction
provides an approach to obtain the kinase kinetics at single was carried out in a 100 μL volume of reaction solution (1 M
molecule level. KCl, 20 mM MgCl2, 50 mM Tris, pH 7.5) containing 100 μM

■ EXPERIMENTAL SECTION
Materials. All aqueous solutions for analytical studies were
S-peptide, 100 μM ATP and a certain concentration of PKA at
30 °C for 12 h. Afterward, the reaction system was
immediately put into water bath with the constant temperature
prepared with ultrapure water (reaching a resistivity of 18.2 65 °C for 20 min to denature the enzyme. Then, the reaction
MΩ·cm at 25 °C) from the Milli-Q System (EMD Millipore, system was added into the cap side chamber of the aerolysin
Billerica, MA, USA). We obtained the 1,2-Diphytanoly sn- nanopore, which contained 900 μL buffer solution, after it
glycero-3-phosphocholine from Avanti Polar Lipids Inc., restored to the room temperature (27 ± 1 °C). Then, the
Alabaster, AL,USA. Proaerolysin was prepared according to current traces of aerolysin nanopore were recorded for
our previous work and activated by digestion with trypsin for 4 analyzing the reaction system. The experimental procedures
h at room temperature.43 S-peptide (LRRASLG) and P- for the inhibition assay were similar to those stated above for
peptide (LRRApSLG) were synthesized by GL Biochem Ltd. detection PKA activity, except for the preincubation of a fixed
B DOI: 10.1021/acs.analchem.9b01570
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry Article

PKA concentration 0.5 U/μL with varying concentrations of

inhibitors in the reaction system.
■ RESULTS AND DISCUSSION
Measurement of PKA Activity via One-Step Method.
MCF-7 Cell Culture and Cell Lysate Preparation. As shown in Figure 1, the reaction solution containing S-
MCF-7 cells (1 × 105 cells) were cultured in RPMI-1640 peptide without PKA could not induce any current blockages
medium (GIBCO) supplemented with 10% fetal bovine serum of aerolysin nanopore while the blockage events emerged
(FBS, Sigma), streptomycin (100 μg/mL) and penicillin (100 evidently in the presence of 0.1 U/μL PKA. The enlargement
μg/mL) in a humid atmosphere with 5% CO2 at 37 °C. Then of the typical blockage events was shown in Figure 2a. From
the cultured medium was replaced by serum-free RPMI-1640
(1 mL) and the cells were incubated for 4 h before drug
stimulation. Subsequently, the cultured cells were treated with
different concentrations of Fsk and IBMX in dimethyl sulfoxide
(DMSO) to activate intracellular PKA. DMSO (equal volume)
without Fsk/IBMX solution was added to the medium for no
drug unstimulated sample as the control. After 45 min of
stimulation, the cultured cells were removed by scraping and
lysed in Dulbecco’s phosphate-buffered saline (DPBS) by
sonication (200 W) for 5 s × 6 times at an interval of 5 s for
each time. The cell lysate were clarified by centrifugation at Figure 2. (a) The typical blockage events induced by the product P-
14 000 rpm for 30 min at 4 °C. To eliminate the interference peptide in the PKA-catalyzed reaction system. (b) The corresponding
of other small molecules in the clarified lysates, including short 2D scatter plot induced by the catalysis product P-peptide. The plot
DNA/RNA, sugars, peptides and small proteins etc., the lysates was obtained via collecting the events for 15 min. The data were
were continuously centrifuged by Amicon 10 K Ultra recorded after S-peptide was catalyzed by PKA (0.1 U/μL). The
Centrifugal Filter Device (Merck KGaA, Darmstadt, Ger- recording potential was +100 mV. The detailed information on this
many). Therefore, the molecules whose molecular weight less PKA catalysis experiment is shown in the Experimental Section.
than 10 kDa were almost excluded from the lysate. Then the
lysates were ready for phosphorylation reactions. the 2D contour plot (Figure 2b), we can find the blockage
The total protein concentration of cell lysate was assessed by events mainly distributed in the region with duration time (t)
BCA protein assay kit with BSA as the standard. Briefly, ranging from 0.3 to 10 ms and I/I0 falling between 0.4 and 0.7.
standard solutions with different concentrations of BSA (25− Here, I is the residue blockage current and I0 is the open pore
2000 μg/mL) were incubated with BCA reagent for 30 min at current. Since S-peptide, ATP, PKA and the catalysis
37 °C. The absorbance of the resulting solutions was recorded byproduct ADP could not induce any blockages (Figure 1
at 562 nm using a UV−vis spectrophotometer and the and SI Figure S2), the observable current blockages were
calibration curve of the standard concentration versus the derived from the catalyzed product P-peptide. Its identity
absorbance was obtained by using a linear-regression program. signature was further confirmed by direct measurement of the
The correlation coefficient of the absorbance with respect to pure sample of P-peptide via aerolysin nanopore. As shown in
Figure 3 and SI Figure S3, P-peptide resulted in the current
the concentration was >0.99. Finally, an aliquot of the cell
blockages clearly, which were also located in the same region
extract was mixed with BCA reagent and detected as described
with duration time ranging from 0.3 to 10 ms and I/I0 lying
above. Its total protein concentration was then calculated by
between 0.4 and 0.7. The event frequency grew as the
reference to the calibration curve.
concentration of P-peptide increased (Figure 3d and SI Figure
PKA Assay in MCF-7 Cell Lysates. The experimental
S4). It was found that the relative event frequency compared to
procedures were similar to those stated above for detecting 1 μM P-peptide (f/f1 μM) increased linearly as [P-peptide]
PKA activity, except that the incubation of the cell lysates grew (Figure 3e). The relative frequency will help reduce the
instead of a certain concentration of PKA. The total protein frequency errors among different experiments. Thus, the
concentrations of the cell lysate in the reaction system were blockage events of the reaction solution were truly ascribed
kept at 12 μg/mL. to the catalyzed product P-peptide.
In Situ Monitoring of the Kinase-Catalyzed Phos- To probe the relationship between the PKA concentrations
phorylation. In an attempt to perform a real-time reaction and its catalysis induced event frequencies, different concen-
monitoring, the enzymatic catalysis was directly carried out in trations of PKA ranging from 0.005 to 0.5 U/μL were
the cap side compartment. Five U/μL PKA, 100 μM ATP, and investigated by the aerolysin nanopore (Figure 4 and SI Figure
10 μM S-peptide were added into the cap side compartment S5−S7). Then, the resulting event frequencies were analyzed.
filled with the detection buffer composed of 1.0 M KCl, 20 Since the relative frequency could help reduce the frequency
mM MgCl2 and 50 mM Tris (pH 7.5). After a certain errors among different experiments, we utilized the relative
incubation time, the number of blockage events was counted frequency to quantify the kinase-induced P-peptide in the
for a time-interval of 1 min and the events were recorded for 9 reaction solution. As the event frequencies of standard P-
min. The slopes of “recording time-cumulative number” fitting peptide increased linearly with its concentration ranging from
lines represent the event frequencies. We considered the 0 to 11 μM (Figure 3e), we likewise select 1 μM P-peptide as
average event frequency within the 9 min as the true frequency interior label for detecting [P-peptide] produced in the
after a certain incubation time. With the same procedure, we reaction solution which ranged from 0 to 10 μM. The interior
statistics the event frequencies after different incubation times label, 1 μM P-peptide, was added into the cap side chamber
and obtained the phosphorylation evolution curve of S-peptide after recording for the reaction solution (the frequency named
in the course of the enzyme reaction. The temperature was as f) and the enhanced frequency resulting from 1 μM P-
controlled at 27 ± 1 °C. peptide was regarded as f1 μM (Figure 4 and SI Figure S5−S7).
C DOI: 10.1021/acs.analchem.9b01570
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Analytical Chemistry Article

Figure 4. (a−c) The raw current traces of the reaction solution

without (left) and with (right) adding 1 μM P-peptide as interior
label. The reaction solution contained 100 μM S-peptide, which was
catalyzed by varying concentrations of PKA at 0.01 U/μL (a), 0.05
U/μL (b) and 0.2 U/μL (c), respectively. The detailed information is
described in the Experimental Section. The width of the light yellow
band in the current trace was calculated according to the current
Gaussian peak width of the reaction solution (SI Figure S8), which
was almost the same with that from pure sample experiments of P-
peptide (SI Figure S4). (d) The cumulative blockage number versus
recording time (min) for the reaction solution catalyzed by 0.01 U/
μL PKA without (black) and with (red) adding 1 μM P-peptide.
Results for more reaction solution catalyzed by different [PKA] was
Figure 3. (a−c) The raw current traces and the 2D contour plots of shown in SI Figure S7. The slopes of fitted lines represent the event
different concentrations of P-peptide: 1 μM (a), 5 μM (b), 10 μM frequencies. (e) Correlation between the f/f1 μM of blockage events
(c). The width of the light yellow band in the current trace was and [PKA] in the reaction solution. All the experiments were
calculated according to the current Gaussian peak width for standard performed at +100 mV.
P-peptide (SI Figure S4). All the 2D plots were based on the blockage
events within 10 min. (d) The cumulative blockage number versus
recording time (min) for different concentrations of P-peptide: 0.25, preincubation of a fixed PKA concentration 0.5 U/μL with
1, 3, 5, 7, 10, 11 μM. The slopes of fitted lines represent the event
varying concentrations of H-89 in the reaction system. The
frequencies (f). The frequencies for more concentrations of P-peptide
were shown in SI Figure S4. (e) The linear relationship between the inhibitor H-89 could not induce any interfere (SI Figure S2).
relative event frequency and the concentrations of P-peptide. The As the concentration of H-89 increased from 0.1 to 105 nM,
different concentrations were achieved via continuously adding P- the f/f1 μM gradually decreased from 3.69 ± 0.32 to 0.29 ±
peptide into the cap side chamber during the experiment. All the 0.07, which is attributed to PKA activity decreasing (Figure 5a,
experiments were performed at +100 mV. SI Figure S9 and Table S2). Notable, the half-maximal
inhibitory concentration (IC50) value of H-89 was determined
Then, we utilized the relative frequency f/f1 μM to quantify to be 104 nM, which is in agreement with previous literature
PKA activity. When the concentration of PKA increased from values.47
0.005 to 0.5 U/μL, the f/f1 μM increased from 0.05 ± 0.01 to Evaluation of PKA activity in cell lysates. Protein kinase
4.14 ± 0.47 (SI Table S1). We identified a positive correlation plays crucial role in intracellular signaling pathways and their
between PKA concentration and its corresponding f/f1 μM, activities are highly regulated in cells, so a practical kinase assay
which could be linearly fitted on a log−log plot (Figure 4e). should be applicable for kinase detection in real biological
The detection limit for PKA in the nanopore system was 0.005 samples. Thus, we testify whether the nanopore-based PKA
U/μL. Such a detection limit is comparable with those of other assay could work in MCF-7 cell lysates. It is well-known that
various sensitive PKA detection methods10,45 in spite of our stimulation of MCF-7 cells by the combination of Fsk and
extremely simple detection strategy. IBMX can greatly increase the intracellular level of cAMP,
Screening of PKA Inhibitor. To demonstrate the ability leading to the activation of cAMP-dependent PKA.48 There-
of the nanopore sensor to screen kinase inhibitor, the fore, in this study, MCF-7 cells were stimulated with different
inhibition of the small molecule kinase inhibitor H-89 to doses of Fsk/IBMX for 45 min and then the cell lysates were
PKA activity was measured (Figure 5a). The inhibitor H-89 extracted. The PKA activities in these cell lysates were explored
competitively inhibits PKA by binding to the ATP binding by the nanopore assay. The experimental protocols were
cleft.46 The experimental procedures were similar to those similar to those stated above for detecting PKA activity, except
stated above for PKA activity detection, except for the for the incubation of the cell lysates instead of PKA. The total
D DOI: 10.1021/acs.analchem.9b01570
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry Article

time recording of the PKA-catalyzed phosphorylation, which

serves as a method for monitoring phosphorylation kinetics at
the single molecule level.

■ CONCLUSIONS
In conclusion, the wild-type aerolysin nanopore provided a
novel one-step method for PKA activity without exogenous
labels, immobilization and cooperative enzymes. The detection
was based on the special response of aerolysin nanopore to the
phosphorylated peptide in the reaction system. This nanopore
method can be used as a sensing platform to identify kinase
inhibitor as well as assaying kinase activity in cell lysate. More
importantly, the aerolysin nanopore provides a label-free
approach for real-time monitoring the kinase kinetics. The
nanopore strategy may be used to evaluate the activity and
kinetics of other enzymes that catalyze post-translational
modifications that alter substrate charge (e.g., sulfonation,
carboxylation, or amidation), thus providing a platform to
Figure 5. (a) Relationship between the relative event frequency (f/ screen a broad spectrum of protein or biomolecule
f1 μM) and [H-89] added in the reaction solution. The IC50 was modifications.

■
obtained from this figure. (b) The f/f1 μM of the reaction solution with
the unstimulated cell lysate, 10 μM Fsk/20 μM IBMX-stimulated cell
ASSOCIATED CONTENT
lysate and 50 μM Fsk/100 μM IBMX-stimulated cell lysate. All the
experiments were performed at +100 mV. (c) The cumulative * Supporting Information
S
blockage number versus recording time (min) for the phosphorylation The Supporting Information is available free of charge on the
process after different incubation times. The slopes of fitted lines ACS Publications website at DOI: 10.1021/acs.anal-
represent the event frequencies after different incubation times. (d) chem.9b01570.
The phosphorylation evolution of S-peptide in the course of the
enzyme reaction. The reaction system included 10 μM S-peptide, 100
Additional nanopore data as noted in text PDF)
μM ATP, 20 mM MgCl2, and 5 U/μL PKA. The data were recorded
at +100 mV. The reaction temperature was 27 ± 1 °C. ■ AUTHOR INFORMATION
Corresponding Authors
protein concentrations of the cell lysates incubated in the *(Y.L.Y.) E-mail: [email protected].
reaction solution were kept at 12 μg/mL. The nanopore results *(Y.T.L.) E-mail: [email protected].
demonstrated that the Fsk/IBMX-stimulated cell lysates ORCID
aroused obvious blockage events with 0.3 ms < t < 10 ms Yi-Lun Ying: 0000-0001-6217-256X
and 0.4 < I/I0 < 0.7 (Figure S10 and S11). The event Yi-Tao Long: 0000-0003-2571-7457
frequency ascended with increasing concentration of stimu- Present Address
lants (Figure 5b and SI Figure S12). Thus, this clearly §
The author now works in Heze University, Heze City 274015,
manifested that the cell lysate-actuated nanopore response Shandong Province, P. R. China.
truly resulted from the successful activation of PKA by drug
Notes
stimulation. These results conclusively show that the proposed
The authors declare no competing financial interest.

■
nanopore strategy can measure changes in cellular kinase
activities in response to drug stimuli. ACKNOWLEDGMENTS
Real-Time Monitoring of the Kinase-Catalyzed
Phosphorylation. In the real-time monitoring of enzymatic This research was supported by the National Natural Science
catalysis, any blockage events were hardly observed before the Foundation of China (21834001 and 61871183), and
addition of PKA to the cap side chamber containing 100 μM Excellent Research Program of Nanjing University
ATP, 10 μM S-peptide and 20 mM MgCl2 (SI Figure S13, t = (ZYJH004). Dr. Yi-Lun Ying is sponsored by National Ten
0 h). After addition of 5 U/μL PKA, the blockage events were Thousand Talent Program for Young Top-Notch Talent,
clearly observed and the event frequency increased as Shanghai Rising-Star Program (19QA1402300) and “Chen-
incubation time grew (SI Figure S13). Via statistical analysis, Guang” Project supported by Shanghai Municipal Education
the blockage event also distributed in the target region (SI Commission and Shanghai Education Development Founda-
tion (17CG27).

■
Figure S14). Since the active PKA could not induce any
interfere (SI Figure S2), the variation of the event frequency
here resulted from the increase of the catalyzed product P-
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F DOI: 10.1021/acs.analchem.9b01570
Anal. Chem. XXXX, XXX, XXX−XXX