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Titel: Biological Nanopore Approach for Single-Molecule Protein

Sequencing

Autoren: Zheng-Li Hu, Ming-Zhu Huo, Yi-Lun Ying, and Yi-Tao Long

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Zitierweise: Angew. Chem. Int. Ed. 10.1002/anie.202013462

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Angewandte Chemie 10.1002/ange.202013462

REVIEW
Biological Nanopore Approach for Single-Molecule Protein
Sequencing
Zheng-Li Hu,[a] Ming-Zhu Huo,[a] Yi-Lun Ying*[ab] and Yi-Tao Long*[a]

Accepted Manuscript

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This article is protected by copyright. All rights reserved.
Angewandte Chemie 10.1002/ange.202013462

REVIEW

Abstract: Proteins are responsible for the occurrence and treatment could be provided, which benefits the unambiguous identification
of many diseases, and therefore protein sequencing will revolutionize of protein variations, such as PTMs.[24–27] Moreover, specific
proteomics and clinical diagnostics. Nanopore electrochemistry has modifications of the nanopore sensing interface could be used to
proved successful for single-molecule DNA sequencing, which optimize nanopore features (e.g. size and charge distribution)
resolves the identities of 4 natural deoxyribonucleotides based on the with an atomic precision, permitting the accurate determination of
current blockages and duration times of their translocations across
the nanopore confinement. However, open challenges still remain for
biological nanopores to sequentially identify each amino acid (AA) of
single proteins due to the inherent complexity of 20 proteinogenic AAs
in charges, volumes, hydrophobicity and structures. Herein, we focus

Accepted Manuscript
on recent exciting advances in biological nanopore approach for
single-molecule protein sequencing (SMPS) from native protein
unfolding, control of peptide translocation, AA identification to
applications in disease detection.

1. Introduction

Function or dysfunction of proteins largely depends on their

primary structures, i.e. the AA sequences. Protein sequencing
would provide important information for disease diagnostics and
treatments. Edman degradation was initially invented to sequence
proteins from N-terminus based on cyclic chemical reactions,
which fails to work without a free α-amino group on the N-terminus
(e.g. N-acetylated proteins) and is difficult to resolve proteins > 50
aa.[1–3] Mass spectrometry (MS) is the current standard method
for protein sequencing but requires enzymatic digestion and/or
fragmentation before detection.[4–6] Protein concentrations vary in
a broad range (~1010) with low copy numbers down to 10-100
molecules in a cell, whereas MS works in a low coverage of
proteome due to a limited dynamic range of mass spectrometers
(~105).[7–9] As such, MS usually requires high-cost equipment and
large amount of samples (e.g. a million copies or an attomole of a
protein) for an accurate detection.[6,10,11] However, enrichment of
proteins based on target-specific biomaterials (e.g. antibody) is
not available for all kinds of proteins.[10,11] Moreover, no methods
exist for protein amplification that analogous to polymerase chain
reaction (PCR) for DNAs. Other drawbacks include insufficient Figure 1. Schematic representation of single-molecule protein sequencing with
sensitivity to identify AAs with identical or similar masses and the a biological nanopore. Top: Illustration of biological nanopore sequencing of a
post-translational modifications (PTMs) of proteins. single translocating protein. A biological nanopore with specific molecular
“brake” site on the nanopore interface is inserted in artificial lipid bilayer.
Recently, nanopores based on electrochemical techniques
Nanopore array is designed to parallel detection of proteins with high-
have developed into a powerful alternative for the realization of throughput. In a typical measurement, (i) a folded protein is unfolded to linear
protein sequencing, because they could provide nano-confined peptide for feasible entry into the nanopore; (ii) then the partially unfolded
space and designable single-molecule sensing interface to protein molecule linearly threads through the nanopore confinement forced by
the electrical field; (iii) eventually the nanopore sensing region identifies single
enable high spatiotemporal resolution and ultimate sensitivity for
AAs residue-by-residue at a controllable pace during the translocation. Bottom:
proteome analysis.[12–15] The interaction of single molecules with Ideal current readouts with distinctive features assigned to each AA
the nanopore sensing interface induces typical ionic current conceptually for calling the linear sequences of single proteins in real time.
modulations, which reflects the composition, charge distribution,
structure and sequence of the translocating molecule.[16–21] A
nanopore, therefore, could directly read the sequences of
individual proteins residue-by-residue, depending on the well- [a] Dr. Zheng-Li Hu, Ms. Ming-Zhu Huo, Prof. Yi-Lun Ying, Prof. Yi-Tao
resolved ionic current alteration (Figure 1). In particular, nanopore Long
State Key Laboratory of Analytical Chemistry for Life Science,
array would contribute to high-throughput parallel detection of
School of Chemistry and Chemical Engineering,
low-abundance proteins, rescuing peptides/proteins from the lack Nanjing University, 163 Xianlin Avenue, Nanjing 210023 (P. R. China)
of in vitro amplification methods.[22,23] This also dictates the bright E-mail: yitaolong@nju.edu.cn, yilunying@nju.edu.cn
prospects to decipher proteins of long-read lengths without the [b] Prof. Yi-Lun Ying
Chemistry and Biomedicine Innovation Center,
needs of labelling and/or enrichment. Besides, a dynamic view of
Nanjing University, 163 Xianlin Avenue, Nanjing 210023 (P. R. China)
protein heterogeneity that is unavailable in an ensemble detection

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Angewandte Chemie
REVIEW
AAs with high spatial and temporal resolution.[28–31]
Different from DNAs, proteins have more complex building
blocks-20 proteinogenic AAs and hundreds of variations including
isomers, enantiomers and PTMs-with very subtle differences in
volumes and structures. These AAs sequentially constitute the
primary structure of a protein, which then folds into higher-order
architectures to display exquisite functionalities. Compared to
recent success of DNA sequencing,[32–35] biological nanopore-
based protein sequencing is still in an early stage and urgently
needs to address multiple challenges:
(i) Structured protein unfolding. The commonly used biological
nanopores, such as α-hemolysin (α-HL),[36] Mycobacterium
smegmatis porin A (MspA),[37] aerolysin (AeL)[38] and
Fragaceatoxin C (FraC) nanopores, [39] have too small internal
constriction (with diameter of 1-2 nm) to accommodate the large
secondary, tertiary and quaternary structures of native proteins.
Thus, a fundamental requirement for nanopore-based protein
sequencing (NPS) is to unfold the 3D topological structures of a
protein to enable its entry and partition into the nanopore
confinement in a stretched profile.
(ii) Peptide translocation control. In general, sufficiently long
residence of single molecules should be provided to allow high
signal-to-noise recording of time-series signals for well-ordering
each AA. As summarised in Figure 2, a peptide traverses across
biological nanopores at high velocities (e.g. majority estimated to
< 1 ms aa-1). However, the time-resolution of currently commercial
nanopore instruments hinders the accurate distinction of different
elements of single peptides.[40,41] Therefore, a timely subject for
NPS is to control the translocation of single peptides within the
nanopore sensing interface.
(iii) Amino acid identification. The 20 proteinogenic AAs dictate
volumes ranging from 0.06 (glycine) to 0.23 (tryptophan) nm3 with
differences as small as 0.001 nm3 (methionine and isoleucine).[42–
44]
They are linked by peptide bonds with an average separation
of ~0.38 nm to compose linear sequences of proteins. [45] Beyond
Zheng-Li Hu received her B.S. in Chemistry
at Shanghai Normal University in 2014 and
PhD in Analytical Chemistry at East China
University of Science and Technology
(ECUST) under the supervision of Prof. Yi-
Tao Long in 2019. She is currently
performing her postdoctoral research at
Nanjing University in the fields of biological
nanopores and single-molecule sensing and
sequencing.
Yi-Lun Ying received her B.S. (2009) and
PhD (2014) in Analytical Chemistry with ((Author Portrait))
Prof. Yi-Tao Long after her studying at
ECUST and University of Birmingham (UK).
Following her postdoctoral research, she
was appointed as an associate professor at
ECUST. Since 2019, she is a professor and
co-PI at the Chemistry and Biomedicine
Innovation Center at Nanjing University. Her
main researches involve electrical-optical
nanopore sensing and advanced artificial
intelligence for nanopore arrays.
((Author Portrait))
This article is protected by copyright. All rights reserved.
10.1002/ange.202013462
this, the prevalence of PTMs including phosphorylation,
acetylation, methylation, glycosylation and ubiquitination, has
made the aim of direct SMPS even more challenging for biological
nanopore techniques.[8,46] Hence, a tremendous challenge is to
improve spatial resolution to provide distinctively featured current
signals for different AAs of the analysed protein.
The first demonstrations have proved the capability of α-HL
nanopores in transporting and identifying peptides (GPP)n with
single, double or collagen-like triple helices.[20,47] A subsequent
work detailed interaction kinetics of theα-HL nanopore with
cationic α-helical peptides (AAKAA)n.[48] A particular study then
suggested that different translocation velocities of an α-helical
peptide Fmoc-DxAyKz through the α-HL and AeL nanopores are
Accepted Manuscript
likely caused by the differences in hydrophobic peptide-nanopore
interactions.[49] Another work elucidated that translocations of
positively charged peptides could be facilitated by engineering
electrostatic binding sites of the nanopore sensing interface. [50]
During the last decade, numerous efforts with biological
nanopores have been invested into peptide and protein sensing,
including conformation/structure characterization, [51–56] variant
recognition,[24,26,27,57–63] interaction investigation,[64–69] and protein
kinase exploration.[70–74] However, despite all the present
achievements in proteomic analysis, there is still a long way
toward SMPS. This minireview focuses on recent progress in
biological nanopore approach for SMPS, and attempts to provide
a comprehensive overview of current experimental efforts and
strategies for overcoming major hurdles of NPS. Finally, we close
it with our insights and perspectives on future developments in the
field of NPS.
2. Unfolding of Structured Proteins
To allow single protein entry into the nanopore confinement, a
handful of methods have been proposed to disrupt the folded
Ming-Zhu Huo received her B.S. in
Chemistry from Shandong University in
2019. She is currently a graduate student
working on peptide/protein single-molecule
detection under the supervision of Prof. Yi-
Tao Long in the School of Chemistry and
Chemical Engineering at Nanjing University.
Yi-Tao Long received his B.S. (1989) from
Shandong University, and MSc (1996) and ((Author Portrait))
PhD (1998) from Nanjing University. After
completing 2-year postdoctoral studies at
Heidelberg University, he worked at the
University of Saskatchewan, University of
Alberta and University of California, Berkeley.
He then was appointed as a professor at
ECUST since 2007 and moved to Nanjing
University in 2019. His main researches
include nanopore electrochemistry,
biointerfaces nano-spectroelectrochemistry
and integrated biosensors for life science.
((Author Portrait))
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Figure 2. Translocation velocities of peptides through commonly used biological
nanopores. Each data point was estimated from the reported longest residences
of peptides. Related references were shown in the right parentheses, where
asterisk indicate the capability for identification of single AA difference.
Compared to the results obtained with wild-type (WT) biological nanopores
(open circles),[25,28,29,44,48,54,66,67,69,73,75–81] translocation velocities of peptides
have been remarkably reduced by designing mutant nanopores (red solid
circles),[30,70,82,83] introducing dipolar-like peptide structures (orange solid
triangles),[57,58,84–86] and optimizing solution conditions, such as temperatures
(blue solid diamonds)[87,88] and pH (violet solid pentagons).[89] Ideal velocities in
suggested region were coloured in light blue.
protein structures. For instance, Congo red (CR) was used as an
inhibitor to prevent β-Amyloid 42 (Aβ 42) aggregation into fibrils
and plaques (Figure 3a).[90] Moreover, an accessible option is to
use the denaturants to open the ready protein folds. Guanidine
hydrochloride (Gdm-HCl) was employed to unfold the WT
maltose-binding protein (MalEwt) and a variant MalE219.[91–93] As
Gdm-HCl increasing to ~1.0 M, the α-HL and AeL nanopores
dictated only long blockades for partially folded proteins, and short
blockades for completely unfolded proteins (Figure 3b).[92]
Previous cases elucidated high-concentration (≥ 2.0 M) urea can
degrade proteins.[94] Despite availability and simplicity, a concern
is protein nanopore denaturation under the extreme denaturing
conditions (e.g. >1.5 M Gdm-HCl or 4 M urea).[93,95,96]
Alternative strategies were proposed. In Figure 3c, MalE219
thermally unfolded when improving temperatures from 20 to 70 ℃,
revealing a same melting temperature of ~45 ℃ using the α-HL
and AeL nanopores.[97] However, high temperatures would
destabilize lipid bilayer and accelerate molecular dynamics. The
fast translocation of peptides hinders the accurate current
recording due to the resolution limit of the existing nanopore
instruments. Recent studies manifested that the hairpin-like
structures of peptides were destabilized by regulating protonation
of AA residues (e.g. histidine) at acidic solution (e.g. pH = 4.5).[53]
Additionally, protein folding states would change by modulating
specific binding between metal ions and AA residues (e.g. Cu2+-
histidine).[66]
To ratchet a protein through the nanopore progressively, early
attempts incorporated a motor enzyme to mediate the unfolding
and translocation of native proteins. For example, a AAA+
unfoldase ClpX, placed in the trans compartment, was employed
to mechanically denature the Smt3 protein perched above the
vestibule of the α-HL nanopore (Figure 3d).[98] Smt3 was modified
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10.1002/ange.202013462
by appending an unstructured polyanion with a ssrA peptide tag
at its C terminal that allows special protein-ClpX binding. ClpX
pulled and linearized Smt3 within the nanopore, resulting in
progressive ionic current decreases followed by a rapid return to
the open pore current. Further study applied unfoldase ClpXP to
modulate sequential unfolding of the structured S2-GT protein
that is composed of one green fluorescent protein (GFP), one titin
fragment (titin I27) and two Smt3.[61] Similarly, incorporation of a
charged polymer tag enables the electrically driven stretching and
traversing of proteins across the nanopore. In recent studies, an
oligonucleotide poly(dC)30 was tagged to support the multistep
unfolding and unidirectional translocation of a model thioredoxin
through the α-HL nanopore (Figure 3e).[99–101]
Accepted Manuscript
3. Controlling of Peptide Translocations
The translocations and trapping times of charged peptides
could be actively or passively controlled by modulating magnitude
and polarity of the applied voltages. As illustated in Figure 4a,
electrophoresis facilated a negatively charged peptide (YEQ) 3,
while hindered a positively charged peptide (YKQ)3 to enter and
migration within the AeL nanopore.[78] In this context, a feasible
idea to slow down peptide journey is to modulate charge
distributions of the molecule itself. [84,86,89,102] In recent works,
oppositely charged tails were introduced to create a dipolar
feature to control peptide sojourn (e.g. CP2a, N’-(E)12-(N)12-(R)12-
C’) inside the α-HL nanopore via alteration of dielectrophoresis
(Figure 4b).[84,89] A dipolar-like peptide can stall within a nanopore
at a fine applied voltage when dielectrophoresis compensates for
electroosmotic flow (EOF).
Unlike the electrophoretic-driven translocations of DNAs, the
combined electrophoresis and EOF accounts for the transports of
peptides/proteins with non-uniform charge distributions.[29,103–106]
Acceptable methods have been proposed to prolong peptide
residence by balancing the electroosmotic and electrophoretic
driving forces. For instance, the concerted action of EOF and
electrophoresis decelerated the movement of peptide CAMA P6
(N’-KWKLFKKIGIGKFLQSAKKF-C’) by carefully tuning the
solution pH.[103,104] By lowering neutral pH below 5, EOF was
augmented due to charge modulation of the α-HL nanopore
induced by protonation of crucial residues (e.g. aspartic acid). The
enhanced EOF played against electrophoresis to prevent the
trans-to-cis oriented motion of CAMA P6, leading to current
oscillations of between B1 and B2 states caused by the back-and-
forth migrations of peptide from pore vestibule to β-barrel domain
(Figure 4c).[104] Similarly, several reports have revealed that
proteins could be trapped inside biological nanopores (e.g. ClyA
nanopore) for several seconds up to tens of minutes via EOF
enhancement.[105–107]
Another potentially powerful approach is to manipulate the
local interactions between peptide and nanopore. In Figure 4d,
the engineered M113F and 2FN (M113F/K147N/T145F) α-HL
nanopores were devised to decrease the translocation velocity of
peptide (Y)6 by introducing an additional aromatic interaction.[82] A
T232K/K238Q mutant AeL nanopore was recently reported to trap
tau peptide (N’-SKIGSTENL-C’) for a longer time than the WT AeL
nanopore, where the T232K site was designed to increase the
electrostatic interaction with charged peptides, and the K238Q
site to enhance the repelling barrier (Figure 4e). [30] Moreover, the
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Accepted Manuscript
Figure 3. Strategies towards the nanopore unfolding of a single protein. (a) Aggregation transitions of β-Amyloid 42 (Aβ42) in the presence of promoter β-cyclodextrin
(β-CD) and inhibitor Congo red, respectively. (b) Denaturing agent-assisted unfolding of a maltose-binding protein variant (MalE219). (c) Thermal unfolding of
MalE219. A same melting temperature (around 45 °C) was obtained by the AeL and α-HL nanopores. (d) Unfoldase ClpX-mediated protein Smt3 unfolding and
translocation. The model protein contains a Smt3 domain (green) at its N-terminus, a negatively charged flexible polyGSD linker (yellow), and a ClpX-targeting ssrA
tag (red) at its C terminus. During the translocation, (i) Empty α-HL nanopore shows open current of ~34 ± 2 pA; (ii) capture of Smt3 leads to an ionic current drop
to ~14 pA; (iii) ClpX-controlled Smt3 unfolding results in a progressive current ramp reaching ~10 pA and (iv) a terminated current reduction to ~3.8 pA; (i') Smt3
migration into the trans solution with an abrupt increase to the open nanopore current. (e) Co-translocational unfolding of thioredoxin (Trx). A model Trx is attached
to a poly(dC)30 DNA leader via a cysteine at its either N- or C-terminus. The representative current trace indicates the four-step unfolding process: step 1→2, DNA
leader threads into the α-HL nanopore; step 2→3, Trx unfolds partially with linearized polypeptide inside the nanopore; step 3→4, Trx unfolds completely with the
remained polypeptide translocating through the nanopore; and step 4→1, unfold Trx leaves the nanopore. Figures reproduced with permission from: a, ref. 90,
Copyright 2011, American Chemical Society; b, ref. 92, Copyright 2012, American Chemical Society; c, ref. 97, Copyright 2012, American ChemicalFigu Society;
d, ref. 98, Copyright 2013, Nature Publishing Group; e, ref. 100, Copyright 2014, Macmillan Publishers Limited.

unphosphorylated and phosphorylated tau peptides were
identified with nearly 100% accuracy. To our best knowledge, this
mutant AeL nanopore gave the slowest translocation velocity of
peptides (about 5-70 ms aa-1) without modulation of solution
environment. Likewise, transport properties of peptides/proteins
could be modulated by manipulating nanopore geometry and
diameter of the nanopore sensing region.[28,31,108]
4. Identification of Amino Acids
Although sequencing 4 natural deoxyribonucleotides has been
achieved, biological nanopore techniques still lack efficient
spatiotemporal sensitivity to identify 20 proteinogenic AAs and
diverse PTMs. Recent years have witnessed many encouraging
developments in nanopore proteomics toward SMPS. For
example, six arginine peptides (5-10 aa in length) differing by a
single AA were resolved independently or in equimolar mixture
using a WT AeL nanopore (Figure 5a). [80] The residence of
cationic peptides were selectively increased by nearly 3 times with
the adoption of an anionic gold cluster Au25(SG)18, accompanying
by a nearly 2-fold reduction in current fluctuations via the
regulation of solution conditions (pH or chaotropic salt).[76]
Another noticeable attempt is the detection of 20 natural AAs
using the AeL nanopore (Figure 5b). [44] In this work, a varied
terminal “X” was chemically attached to an arginine heptapeptide,
which acts as a polycationic carrier to ensure the unidirectional

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Accepted Manuscript
Figure 4. Electrical recording of peptide translocation through a biological nanopore. (a) AeL nanopore detection of the negatively charged (YEQ)3 and positively
charged (YKQ)3 peptides. Left: Electrophoresis-facilitated translocations of (YEQ)3 induce large amounts of current blockades. Right: Electrophoresis-hindered
captures of (YKQ)3 cause only a few transient spikes. (b) Engineered dipolar-like peptide CP2a detected using a single α-HL nanopore. The sequence of CP2a is
N’-(E)12-(N)12-(R)12-C’. In neutral solution, the residence time of CP2a increases due to the balanced electrophoretic forces on the oppositely charged segments at
its N- and C-terminus. (c) EOF-modulated translocation of peptide CAMA P6. At acidic pH values, EOF is augmented to oppose electrophoresis, contributing to a
slow-down peptide moving for capable detection of B1 and B2 sub-states of the positively charged CAMA P6. (d) Study of amyloid-β peptide transport through α-HL
nanopores. Left: Molecular graphics illustration of the WT α-HL nanopore (PDB ID code: 7AHL). Right: Typical current traces of peptide (Y)6 traversing across the
WT, M113F and M113F/147N/T145F (2FN) mutant α-HL nanopores, respectively. Left: The residence of (Y)6 is prolonged due to the additional aromatic interactions
between (Y)6 and engineered phenylalanine (F) sites within the nanopore sensing interface. (e) Mapping phosphorylation sites of tau peptides with the T232K/K238Q
mutant AeL nanopore. Upper left: The T232K/K238Q mutant nanopore was designed to introduce an enhanced electrostatic interaction at T232K site and an
increased repelling barrier at K238Q site. Upper right: Well-separated scatter plots of current and duration for tau peptide (green), pS262-tau peptide (red), pT263-
tau peptide (blue), and pS262/pT263-tau peptide (yellow), respectively. Bottom: The typical current traces reveal longer translocation times of WT, pS262- and
pT263-tau peptides in the T232K/K238Q mutant AeL nanopore than WT AeL nanopore. Figures reproduced with permission from: a, ref. 78, Copyright 2019, Wiley-
VCH; b, ref. 84, Copyright 2015, Nature Publishing Group; c, ref. 104, Copyright 2014, Nature Publishing Group; d, ref. 82, Copyright 2009, American Chemical
Society; e, ref. 30, Copyright 2020, WILEY-VCH.

entry and travel of XR7 peptides. To obtain an exquisite sensitivity,
“X” was electrically confined within the small nanopore sensing
volume for up to tens of milliseconds, allowing AeL identification
of 13 natural AAs (i.e. R, W, F, K, L, N, T, P, D, A, C, S and G)
assigned into small subgroups. Despite satisfied identification of
the electrically charged family (RR7, KR7, HR7, ER7 and DR7) in a
mixed solution, it is still a great difficulty to recognize all the 20
natural AAs in an equimolar mixture.
At the same time, efforts were devoted to direct identification
of single AAs without any attachments. In an initial attempt, single
cysteine (Cys, 0.11 nm3) produced prolonged distinctive current
blockades (e.g. 0.11 ±0.02 ms at 120 mV) when traversing across
a WT AeL nanopore (Figure 5c).[79] While asparagine (Asn, 0.12
nm3) and glutamine (Gln, 0.15 nm3) with larger volumes induced
only undistinguishable current variations, suggesting a relatively
less dominant role of steric hindrance effect for single AA sensing.
Other factors, especially the specific inter-residue interactions,
should be considered to improve nanopore sensitivity.[43,109] As
mentioned before, the T232K/K238Q mutant AeL nanopore has
achieved differentiation of non-, mono- and di-phosphorylated tau
peptides by regulating inter-residue electrostatic interactions.[30]
Up to now, several groups have realized nanopore sensing of
peptide/protein variants.[24–26,30,57–60,63,66] As depicted in Figure 5d,
Cys and homocysteine (Hcy) incorporated to 5’-benzaldehyde
poly(dA)4 (AHA4) produced readable current responses and long
duration (~10 ms) when confined inside the K238Q mutant AeL
nanopore.[60] Another work adopted a dipolar structure consisting
of a negative E10 N-terminus and a positive R10 C-terminus to
orient and slow down peptide passage across the Fragaceatoxin
C (FraC) nanopore, where the balanced EOF and electrophoresis
are responsible for the distinction of unmodified, phosphorylated
and glycosylated serine (S) in an equilibrium position 11 (from the
N-terminus) (Figure 5e).[57]
5. Applications in Disease Detection
On the way to the ultimate goal of NPS, increasing studies
unravel potential opportunities of nanopore proteomics, especially
recently reported applications aiming at disease diagnostics. For
example, structural transformation of α-synuclein (α-syn) was
probed during its early stage fibrillation, which suggests as a

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Angewandte Chemie 10.1002/ange.202013462

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Accepted Manuscript
Figure 5. Identification of single amino acid differences with a biological nanopore. (a) Detection of arginine peptides of different lengths in an equimolar mixture
using the AeL nanopore. Top right: Setup of the AeL nanopore experiments (not to scale). Top left: Illustration of a typical current blockade event. Below: Histogram
of residual current blockages of arginine peptides consisting of 5, 6, 7, 8, 9, and 10 AAs (from right to left). (b) Recognition of terminated proteinogenic amino acids
(X) in peptide XR7. Left: Mean values of experimental Ib/I0 assigned to twenty XR7 obtained by the AeL nanopore. Right: Constructs of peptide X R7, where an AA of
interest (X) is linked to a polycationic carrier R7. (c) Analysis of single amino acids with the AeL nanopore. Left: Schematic of a single cysteine traversing across an
AeL nanopore. Right: Raw current traces of cysteine (Cys), asparagine (Asn) and glutamine (Gln) (from top to bottom), respectively. The red star denotes a typical
blockades event of Cys in the inset. (d) Discrimination of cysteine (Cys) and homocysteine (Hcy) with the K238Q mutant AeL nanopore. Cys/Hcy is labeled by a
specific 5’-benzaldehyde poly(dA)4 (AHA4) probe. Left: Scheme of K238Q AeL nanopore analysis of AHA4, AHA4-C and AHA4-H. Right: Representative blockade
events of single AHA4-H, AHA4-C and AHA4 (from left to right), respectively. (e) Phosphorylation and glycosylation detection with the FraC nanopore. Left:
Schematic representation of unmodified and modified peptides measured by a FraC nanopore. Right: Histogram of the relative blockade of the unmodified,
phosphorylated and glycosylated peptides, respectively. Peptide sequence is N’-(E)10-XGSGSGSKGS-(R)10-C’, where X represents serine (S) or its variants. Figures
reproduced with permission from: a, ref. 80, Copyright 2018, Nature Publishing Group; b, ref. 44, Copyright 2020, Nature Publishing Group; c, ref. 79, Copyright
2020, The Royal Society of Chemistry; d, ref. 60, Copyright 2019, The Royal Society of Chemistry; e, ref. 57, Copyright 2019, American Chemical Society.

pathological hallmark of Parkinson’s disease (Figure 6a). [110] If
captured inside an α-HL nanopore, the natively unfolded α-syn
monomer either transiently translocated through the nanopore
lumen, or transformed into partially folded intermediate to exit
from the nanopore vestibule. Similarly, the α-HL nanopore was
employed to investigate Alzheimer’s disease (AD)-associated Aβ
aggregation, revealing that the amyloidosis process of Aβ is
affected by its initial structure features and could be inhibited by
β-CD.[54,90] The WT and mutated FraC nanopores were adopted
to detect peptide/protein biomarkers.[28,29] An engineered W116S
FraC nanopore discriminated angiotensin I (Ang I), II (Ang II), III
(Ang III) and IV (Ang IV) peptides simultaneously in a mixture,
which are key players in regulating blood pressure and the fluid
balance (Figure 6b).[28]
Nanopore sensing of protein kinases is another burgeoning
area, which is potential therapeutic targets for many diseases
including cancers and leukemias. As shown in Figure 6c, the AeL
nanopore system was utilized to directly monitor the catalysis of
protein kinase A (PKA), which resulted in serine phosphorylation
of the substrate peptide (S-peptide, N’-LRRASLG-C’).[67] In this
work, PKA activity was quantified by analysing the relative event
frequency f/f1 μM of the phosphorylated peptide (P-peptide, N’-
LRRApSLG-C’), where f1 μM corresponds to the enhanced event
frequency resulting from the interior label of 1 μM P-peptide.
Further nanopore assay manifested that H-89 inhibited PKA
activity with a half-maximal inhibitory concentration (IC50) of 104
nM, while the combination of FsK and IBMX triggered PKA
activation in MCF-7 cell lysates.
Synergistic binding of Pim-1 kinase to MgATP and a peptide
sensor attached to an engineered α-HL nanopore was measured,
which yielded pseudo-first-order association rate constants k’+2 of
60 ± 10 μM for the Pim-1-sensor binary complex and k’+3 of 35 ±
5 μM for the Pim-1-MgATP-sensor ternary complex (Figure 6d).[72]
The ATP-competitive Pim kinase inhibitor 1 was titrated to inhibit
Pim-1 against the Pim-1-sensor complex formation. An inhibition
constant of 130 ± 30 nM was obtained for inhibitor 1 in the

7
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Angewandte Chemie 10.1002/ange.202013462

REVIEW

Accepted Manuscript
Figure 6. Applications in disease diagnostics using biological nanopores. (a) Characterization of early fibrillation from natively unfolded α-syn monomer to partially
folded α-syn intermediate via an α-HL nanopore. Transient states between block current level Lb and capture blockade current level Lc might be produced by the
elongation/ordering of α-syn intermediate inside nanopore. (b) Simultaneous measurement of angiotensin (Ang) peptide biomarkers with the engineered W116S
FraC nanopores. Ang I, II, III and IV peptides could be discriminated directly by the magnitudes of ionic current blockades. (c) Evaluation of PKA activity through
the aerolysin nanopore. Top: nanopore sensing schematic and current traces of S-peptide and PKA-catalyzed P-peptide. Below: quantitative analysis of PKA
inhibition by the kinase inhibitor H-89, and PKA activation in MCF-7 cells stimulated with Fsk/IBMX. Here, f/f1 μM suggests the relative event frequency of P-peptide
to reduce the frequency errors among different experiments, where f indicates P-peptide event frequency in an analyzed sample, f1 μM represents the increased
event frequency generated by the interior label of 1 μM P-peptide. (d) Investigation of Pim-1 kinase kinetics with an engineered α-HL nanopore sensor. Above:
illustration of the novel heptameric α-HL nanopore containing a single functionalized subunit, which bears a serine/glycine linker, a pseudosubstrate peptide sensor
and a TEV protease recognition site (“TEV RS”). Middle: kinetic model for synergistic binding of Pim-1 and MgATP to the peptide sensor. Bottom: determination of
Pim-1 inhibition caused by an ATP-competitive kinase inhibitor 1 against Pim-1-sensor interaction. Figures reproduced with permission from: a, ref. 110, Copyright
2013, American Chemical Society; b, ref. 28, Copyright 2019, Nature Publishing Group; c, ref. 67, Copyright 2019, American Chemical Society; d, ref. 72, Copyright
2015, Wiley-VCH.

presence of 162 nM Pim-1, 100 μM ATP and 10 mM MgCl2. A
more recent work investigated the reversible phosphorylation/
dephosphorylation of the sensor peptide induced by Pim-1 and a
protein phosphatase.[74] In addition, an engineered phi29
nanopore was incorporated with an Epithelial Cell Adhesion
Molecule (EpCAM) peptide probe to specifically detect EpCAM
antibody (Ab) in the serum, demonstrating the feasibility and
versatility of biological nanopores for disease diagnostics and
medical utilization.[64]
6. Summary and Outlook
This review has summarised the undergoing methodologies to
promote the developments of NPS, including modification of
target peptides/proteins, engineering of biological nanopores, and
alteration of the experimental conditions (e.g. electrical field, pH
and temperature). The translocation velocities of peptides have
been reduced by ~1-4 orders of magnitude with mutant biological
nanopores (Figure 2). A direct consequence is the discrimination
of peptides differing by a single AA. However, it is still difficult to
develop a protein sequencer, because the nanopore sensing
volume is too large for AAs (e.g. glycine of 0.06 nm3), such as the
AeL nanopore with a sensing region of ~2.4 nm and inner
diameter of ~ 1 nm.[44,111,112] Moreover, the interaction difference
between the nanopore and single AAs is not sufficient to produce
the corresponding current identities. In this aspect, site-directed
manipulation of the nanopore sensing interface has appeared to
improve spatiotemporal sensitivity. Advances in electrochemical
instruments with lower noise and higher bandwidth would also
benefit protein profiling with a higher signal-to-noise ratio.
Compared to biological nanopores, solid-state nanopores

8
This article is protected by copyright. All rights reserved.
Angewandte Chemie
REVIEW
could provide complementary options for peptide/protein sensing Keywords: protein sequencing • biological nanopore • single-
in hash conditions because they have evolved broad molecule sensing interface • nanopore electrochemistry •
environmental tolerances including voltage, temperature, pH and disease diagnostics
denaturants.[113–116] Hence, integrated nanopore arrays could be
developed for high-throughput parallel protein detection by [1]
combining biological nanopore sequencer with scalable solid- [2]
state nanopores.[117,118] Moreover, a deep dialogue between
nanopore and other on-going methods, such as fluorescent [3]
fingerprinting and tunneling currents, will expand the opportunities
for SMPS.[119–121] A feasible corporation may lead to a nanopore
[4]
coupled multi-compartment device to simultaneously monitor the
[5]
transversal tunneling current and longitudinal ionic blocked
current for highly accurate SMPS.[122,123]
[6]
Concerted efforts in interdisciplinary areas will open up new [7]
possibility for NPS. Recently, researches in the fields of protein [8]
engineering and computational enzyme design have collaborated
to create an enzyme termed as “Edmanase”, which is capable of
step-wise cleaving off N-terminal AAs under biocompatible [9]
conditions.[124] This creation provides experimental evidence for
[10]
the feasibility of exopeptidase-based NPS, where the removed
AAs are identified one-by-one by the nanopore at a controlled [11]
pace. Considering the complexity of large amounts of nanopore
data, machine learning algorithms should be included in the future [12]
repertoire to efficiently decode current readouts for reconstruction [13]
of the original protein sequences.
Avenues towards SMPS with biological nanopores are full of [14]
[15]
challenges and opportunities. Next decade will highlight the cost-
effective and highly parallelized NPS. Theoretical, methodological
[16]
and technical innovations are on the horizon to improve sensitivity,
selectivity and reliability of NPS, and therefore would facilitate [17]
label-free, multiplexing and high-throughput profiling of proteome [18]
in individual cells. If realized, NPS will undoubtedly bring a great [19]
revolution to single-cell proteomics in understanding phenotypic [20]
identities of cells, unbiased proteome imaging of cellular [21]
heterogeneity, studying dynamic cellular development and etc.
[22]
The innovative technique of nanopore electrochemistry could also
[23]
be extended to clinical applications, such as discrimination of
PTMs and protein biomarkers, offering new insights in protein [24]
function/dysfunction at a single AA level. If combined NPS with
single-molecule DNA/RNA sequencing, an unprecedented view [25]
will be provided for molecular diagnostics and personalized
medicine. [26]
[27]
[28]
Acknowledgements [29]
This work is supported by the National Natural Science [30]
Foundation of China (21834001 and 21922405), the China
[31]
Postdoctoral Science Foundation (2020M680066), the Excellent
Research Program of Nanjing University (ZYJH004) and the [32]
Fundamental Research Funds for the Central Universities
(14380239). Yi-Lun Ying is sponsored by National Ten Thousand [33]
Talent Program for young top-notch talent. We specially thank the
kind help on the figure drawing from Kai-Li Xin, Chao-Nan Yang,
[34]
Drs. Qiang Zhu and Shao-Chuang Liu.
[35]
[36]
Conflict of interest
[37]
[38]
The authors declare no conflict of interest.
9
This article is protected by copyright. All rights reserved.
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Angewandte Chemie 10.1002/ange.202013462

REVIEW
Entry for the Table of Contents

Accepted Manuscript
Biological nanopore approach is promising for single-molecule protein sequencing (SMPS) by offering single-molecule confined
sensing interface to enable a high spatiotemporal resolution and an ultimate sensitivity. Here, we focus on recent advances in
biological nanopores from protein unfolding, peptide translocation, amino acid identification to diagnostic application, and close with
an outlook of interdisciplinary efforts for SMPS realization.

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This article is protected by copyright. All rights reserved.