Supra-blot: An accurate and reliable assay for detecting target proteins with a synthetic host molecule-enzyme hybrid

Article abstract of DOI:10.1039/c9cc09699j

In accordance with the rapid increase in demand for selective and spatial chemical tagging, and accurate detection of proteins of interest, we develop a sensitive protein detection method, termed "Supra-blot" capitalizing on high-affinity host-guest interaction between cucurbit[7]uril (CB[7]) and adamantylammonium (AdA). The method can directly detect chemically tagged proteins without false-positive signals caused by endogenous biomolecules. Not only a single specific protein, but also spatially localized proteins in cells were labeled with AdA, and selectively detected by a host molecule-enzyme hybrid, CB[7]-conjugated horseradish peroxidase (CB[7]-HRP) generating amplified chemiluminescence signals. This study shows the great potential of Supra-blot for accurate and reliable detection of proteins of interest in cells.

Full text of DOI:10.1039/c9cc09699j

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DOI: 10.1039/C9CC09699J.
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Supra-blot: an accurate and reliable assay for detecting target
proteins with a synthetic host molecule-enzyme hybrid
Gihyun Sung,b Song-Yi Lee,f Myeong-Gyun Kang,e Kyung Lock Kim,a Jaeyeon An,c Jaehwan Sim,d
Sungwan Kim,c Seungjoon Kim,g Jaewon Ko,g Hyun-Woo Rhee,*f Kyeng Min Park*a and Kimoon
Kim*a,b,c,d
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
In accordance with the rapid increase in demand for selective and and undesired enzymatic degradation of SA under biological
spatial chemical tagging, and accurate detection of proteins of conditions.5, 6 Moreover, the selective binding of SA to biotin-
interest, we develop a sensitive protein detection method, termed tagged proteins suffers interference from endogenously
“Supra-blot” capitalizing on high-affinity host-guest interaction biotinylated proteins, thereby leading to false-positive protein
between cucurbit[7]uril (CB[7]) and adamantylammonium (AdA). detection.7 It is even more serious when used for animal
The method can directly detect chemically tagged proteins without tissues.8-10 Some of these issues have been resolved with bio-
false-positive signals caused by endogenous biomolecules. Not only orthogonal covalent approaches using click coupling
a single specific protein, but also spatially localized proteins in cells reactions.11, 12 However, this covalent coupling system presents
and animal tissues were labeled with AdA, and selectively detected another challenge: the use of a relatively high concentration (a
by a host molecule-enzyme hybrid, CB[7]-conjugated horseradish few 10 μM) of coupling partners.13 When necessary, metal ions
peroxidase (CB[7]-HRP) generating amplified chemiluminescence (e.g., Cu(I)) are utilized for efficient covalent coupling owing to
signals. This study shows the great potential of Supra-blot for slow reaction kinetics, which often causes unwanted
accurate and reliable detection of proteins of interest in cells.
aggregation of proteins.14, 15 Thus, to overcome these barriers,
novel chemical tags that can be selectively conjugated to
proteins using readily accessible chemicals and enzymatic
methods and be rapidly recognized by small, stable and bio-
orthogonal binding partners are required for accurate and
reliable protein detection.
In proteomics, chemical biology has facilitated the selective
modification of proteins of interest with chemical tags that can
be imaged, isolated, and quantified via their non-covalent and
2
covalent binding partners.1, For example, biotin has been
utilized extensively as a chemical tag, since it can be conjugated
to target proteins using various chemical and enzymatic tagging
methods and binds with high selectivity and specificity to its
binding partners, such as streptavidin (SA), for which the
binding affinity (Ka) is ~1013 M-1.3, 4 Although this ligand-protein
binding pair is useful in proteomics, it suffers from intrinsic
drawbacks, including the large size of the components involved,
Recently, adamantyl-(AdA) and ferrocenyl-ammonium (FcA)
derivatives have emerged as new chemical tags for protein
labeling.16-18 Like biotin, they are small and can be selectively
conjugated to any proteins of interest in cells and animals via
chemical and enzymatic methods. In addition, they have an
extremely high binding affinity (Ka >1013 M-1) with selectivity for
their host molecule, cucurbit[7]uril (CB[7]).19-23 Additionally,
this synthetic system possess beneficial features including a
smaller size (~1 kDa) than biotin-SA (~ 53 kDa), negligible
interference of the binding properties by endogenous
biomolecules, no decomposition under biological conditions,
and no enzymatic degradation. However, due to the lack of a
direct sensitive detection method, AdA- and FcA-labelled
proteins have not been directly detected but indirectly through
protein staining using primary and secondary antibodies (Abs)
conjugated with an enzyme for typical western blotting, which
necessitates tedious and laborious antibody treatments and
washing steps.18 In general, horseradish peroxidase (HRP), a
radical-generating enzyme, amplifies chemiluminescent signals
that enable detection of pico- to femto-gram quantities of
proteins of interest by an immunochemical technique.24
a. Center for Self–assembly and Complexity, Institute for Basic Science (IBS), Pohang,
37673, Republic of Korea. E-mail: kmpark@ibs.re.kr, kkim@postech.ac.kr
b. Division of advanced materials science (AMS), Pohang University of Science and
Technology (POSTECH), Pohang, 37673, Republic of Korea.
c. Department of chemistry, Pohang University of Science and Technology
(POSTECH), Pohang, Korea.
d. School of interdisciplinary bioscience and bioengineering (i-bio), Pohang
University of Science and Technology (POSTECH), Pohang, Korea.
e. Department of Chemistry, Ulsan National Institute of Science and Technology
(UNIST), Ulsan, 44919, Republic of Korea.
f. Department of Chemistry, Seoul National University, Seoul, 08826, Republic of
Korea. E-mail: rheehw@snu.ac.kr
g. Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of
Science and Technology (DGIST), Daegu 42988, Republic of Korea.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Combining the ultrahigh binding affinity of CB[7] to AdA and the
signal amplification ability of HRP, we herein demonstrate a
new supramolecular chemistry-based protein detection assay,
termed ‘Supra-blot’, for the accurate, reliable and direct
detection of AdA-labelled proteins using HRP conjugated with
CB[7] (CB[7]-HRP as a host molecule-enzyme hybrid; Fig. 1e).
Specific cellular proteins of interest labelled with AdA on a
genetically designated site, such as AdA-labelled histone H3 in
mammalian cells, was successfully detected by treatment of
CB[7]-HRP with luminol and H2O2 on a blotted nitrocellulose
(NC) membrane. Furthermore, spatially localized proteins
labelled with AdA in cells and animal tissues by peroxidases,
such as an engineered ascorbate enzyme (APEX) and antibody-
conjugated HRP, were successfully detected by Supra-blot as
distinct protein bands on a NC membrane. These demonstrate
the great potential of Supra-blot as a new chemical tool for
accurate and reliable detection of both specific proteins and
proteins spatially localized in regions of interest in cells.
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DOI: 10.1039/C9CC09699J
CB[7]-HRP was prepared by conjugation of monoamine-
functionalized CB[7] (MA-CB[7])25-27 to aldehyde-functionalized
HRP (Act-HRP; EZ-Link Plus Activated Peroxidase, Thermo
Scientific) to form imine between CB[7] and Act-HRP prior to
reductive amination by treatment with NaBH3CN (Fig. S1, See
ESI for the synthetic detail). The conjugation of CB[7] to HRP was
confirmed by SDS-PAGE experiments. Unlike native HRP, the
reacted HRP treated with AdA-conjugated fluorescein
isothiocyanate (AdA-FITC,26 a strong guest of CB[7]) exhibited a
fluorescent band (Fig. S2c) at the same position as the CBB-
stained band matched that of native HRP (Fig. S2b). It suggests
that MA-CB[7] was successfully conjugated to HRP, and the
portal of the conjugated CB[7] was open and thus able to form
a stable complex with AdA-conjugated molecules such as AdA-
FITC. In addition, matrix-assisted laser desorption/ionization
time of flight mass spectrometry of CB[7]-HRP (Fig. S3) revealed
conjugation of ca. two MA-CB[7] to Act-HRP. The enzyme
activity of CB[7]-HRP measured by the O-dianisidine assay28 was
found to be 10.4 ± 0.3 unit mg-1, indicating that HRP retained its
enzymatic activity after conjugation to MA-CB[7].
Fig. 1 Chemical structures a) BG-AdA and b) AdA-phenol and c) MA-CB[7], d) a
hypothetical model of CB[7]-HRP and e) a scheme for detection of AdA-labeled
proteins using Supra-blots.
1a). The protein consists of three different motifs: a target
protein (H3), an expression verification tag (FLAG tag), and a
self-labelling protein tag (SNAP-tag). The FLAG tag epitope
(DYKDDDDK) can be selectively recognized by a corresponding
anti-FLAG primary antibody.29 The SNAP-tag is a mutant of O6-
alkylguanine-DNA alkyltransferase, which selectively labels
chemical tags with BG moiety to itself.30 Expression of the target
protein in cells was confirmed by typical western blotting with
anti-FLAG primary antibody and its corresponding HRP-
conjugated secondary antibody (See ESI), showing
a
chemiluminescence band (~38 kDa) in the membrane from the
cell lysate (Fig. S6). The cell lysate was treated with BG-AdA to
label the H3-SNAP-FLAG with the AdA unit, and Supra-blot was
performed on the membrane. The results showed that cellular
proteins were well-resolved (Fig. 2b), with
a single
Next, we examined the ability of CB[7]-HRP for selective
detection of a specific protein labeled with AdA in a cell lysate.
AdA-conjugated BSA (see ESI) was used as a model protein. A
number of different protein bands from a mixture of AdA-BSA
and cell lysate were well-resolved on the Ponceau S-stained NC
membrane, depending on molecular weight (lanes 3, 4 in Fig.
S5b). After treating the membrane with CB[7]-HRP, luminol, and
H2O2 sequentially, we observed a distinct protein band at ~70
kDa with a chemiluminescence signal (lane 4 in Fig. S5c), the
position of which matched that of AdA-BSA (lane 2 in Fig. S5c)
obtained in the same experiment with phosphate buffered
saline (PBS) instead of cell lysate. This indicates that CB[7]-HRP
selectively detected AdA-BSA in a mixture of cellular proteins.
Encouraged by the blotting results of the model protein, we
tested the Supra-blot method for selective detection of a
specific cellular protein namely, histone H3 (H3) to verify its
applicability. We utilized HEK293T cells expressing the H3-
SNAP-FLAG fusion protein (Fig. 2) for selective labelling of the
target protein with benzylguanine (BG)-conjugated AdA (Fig.
Fig. 2 a) Scheme for AdA labeling of the H3-SNAP-FLAG complex. b) Protein
detection by Ponceau S and c) Supra-blotting
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chemiluminescence band at the position of the target protein of Supra-blot, we compared the Supra-blot results obtained
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DOI: 10.1039/C9CC09699J
(~38 kDa) in the membrane (lane 4 in Fig. 2c). Additionally, no from two types of cells transfected with different APEX-
bands were detected in lysates of non-transfected cells (lanes containing plasmid constructs, both of which targeted the same
1, 2 in Fig. 2c) and cells not treated with BG-AdA (lane 3 in Fig. subcellular compartment (mitochondrial matrix). To validate
2c), demonstrating that H3-SNAP-FLAG was successfully this study, we additionally performed Supra-blot with cells
labelled with AdA, and only the AdA-labelled protein was expressing APEX2 conjugated to mitochondrial ribosomal
detected by Supra-blot with CB[7]-HRP. Not limited to protein L12 (MRPL12-V5-APEX2; MRPL12 is localized to the
mammalian cells, a protein expressed in bacteria (Escherichia mitochondria matrix)33 and compared the results with those
coli) labelled with a polyglycine-conjugaed AdA (GGG-AdA) obtained with cells expressing Matrix-V5-APEX2. As shown in
using Sortase A enzyme was also selectively detected by Supra- lanes 2 and 3 of Fig. 4a, the protein band patterns were similar,
blot (Fig. S7, See ESI for the detail). Interestingly, in the results demonstrating that Supra-blot can reliably detect a unique set
of the model study with AdA-BSA (Fig. S5) and the studies on of localized cellular proteins. Differences and similarities among
cells and bacteria (Fig. 2, S6, S7), bands for the target proteins Supra-blotted protein bands were more recognizable when the
were detected without the false-positives that are unavoidable patterns were converted to a line scan analysis graph with
in typical western blots with streptavidin-conjugated HRP (SA- molecular weight and staining intensity as x and y axes,
31
HRP) when detecting proteins tagged with biotin.7,
This respectively (Fig. 4b). The degree of overlap between graphs
indicates that Supra-blot not only can detect specific proteins, was represented as correlation values (0 to 1 for non-overlap
but is also a more accurate method than typical western and full overlap; Fig. 4c) using correlative functions (See ESI). For
blotting with SA-HRP.
example, Matrix (Matrix-V5-APEX) vs. NES (NES-V5-APEX) and
To explore capabilities beyond detection of a single specific Matrix vs. MRPL12 (MRPL12-V5-APEX) were scored as 0.31 and
protein, Supra-blot was tested on a set of proteins spatially 0.98, respectively, indicating that the Supra-blotted pattern of
localized in a region of interest in a cell. For this investigation, Matrix was more similar to that of MRPL12 than that of NES.
an enzymatic proximity labelling method was employed using Moreover, the correlation value between MRPL12 and Matrix
an engineered ascorbate peroxidase (APEX2) that generates was 0.98, indicating almost full overlap, confirming high
radicals in situ upon H2O2 treatment for tagging AdA-phenol to similarity between the sets of detected proteins when APEX was
neighbouring proteins close to APEX2.3, 4 The enzyme can be located in the same region in cells. This suggests that Supra-blot
targeted to different cellular locations via typical genetic constitutes a reliable technique for detection of spatially
engineering with designed plasmid DNA constructs. In this localized cellular proteins.
study, HEK293T cells were transfected with constructs
A typical western blot of SA-HRP-mediated detection of
harbouring V5 epitope (IPNPLLGLD)-tagged mitochondrial biotinylated proteins yielded unavoidable false-positive signals,
matrix-APEX2 (Matrix-V5-APEX2) or the APEX2 nuclear export
signal (APEX2-V5-NES) to target APEX2 to the mitochondrial
matrix or cytosol, respectively (Fig. 3).32 Successful expression
of Matrix-V5-APEX2 and APEX2-V5-NES was confirmed by
detecting the target proteins via typical western blotting (Fig.
S10) with anti-V5 antibody following the same experimental
procedure used for H3-SNAP-FLAG (See ESI). For APEX proximity
labelling of proteins with AdA-phenol, cells were incubated with
AdA-phenol followed by H2O2 treatment for 1 min (See ESI).
Supra-blot analysis of lysates of the cells clearly revealed
different patterns of protein bands detected with CB[7]-HRP
(Fig. 4a). These results reflected the ability of Supra-blot to
recognize unique sets of proteins localized in subcellular regions
of interest as distinct protein patterns. To verify the reliability
Fig. 4 a) Detection of AdA-phenol-labeled proteins by Supra-blot. b) Line scan
analysis of proteins bands in a). c) Calculated correlation values for protein
bands in b). d) Detection of biotin-phenol-labeled proteins by a western-blot
with SA-HRP. e) Line scan analysis of proteins bands in d). f) Calculated
correlation values for protein bands in e).
Fig. 3 Illustration of APEX2-mediated AdA (or biotin)-phenol labeling of
spatially localized proteins in a cell.
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mostly due to endogenously biotinylated proteins (Fig. 4d-f),5-10
as confirmed by the same experiments with biotin-phenol and
SA-HRP instead of AdA-phenol and CB[7]-HRP. By contrast,
Supra-blot did not suffer from false-positives, as verified using
cells lacking APEX2 (lane 4 in Fig. 4a). Comparison of the
correlation values between Supra-blot with CB[7]-HRP (Fig. 4c)
and western blot with SA-HRP (Fig. 4f) revealed lower
correlation for Supra-blot for different target sites (NES vs. non-
transfected, and NES vs. MRPL12) and higher for the same
target sites (MRPL12 vs. Matrix). This analysis strongly suggests
that Supra-blot provides better accuracy and precision for
detecting spatially localized cellular proteins with negligible
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false-positives. Similar results obtained with
a different
mammalian cell line, HeLa, (Fig. S11) confirmed the reliability of
the Supra-blot for the detection of spatially localized proteins.
In addition, we observed no false-positive protein bands when
Supra-blot was used to analyse Caenorhabditis elegans
(employed as a simple animal model) extracts, unlike the SA-
HRP-based blot (Fig. S12). These results suggest that Supra-blot
may be potentially applicable for the analysis of a wide variety
of biological samples.
In summary, we reported a new protein detecting assay,
termed Supra-blot using a host molecule-enzyme hybrid, CB[7]-
HRP. Specific target proteins in cells and bacteria were labeled
with AdA using SNAP-tag and Sortase A, respectively, and
specifically detected in the form of a protein band on a NC
membrane using CB[7]-based ultrastable and bio-orthogonal
interaction. Furthermore, a set of proteins in regions of interest
in cells and mouse brain tissues were also reliably detected after
proximity labeling of proteins with AdA-phenol mediated by
peroxidases such as APEX2 and antibody-conjugated HRP. Due
to the use of synthetic high affinity host-guest interactions that
are not interfered with biomolecules, Supra-blot provides
accurate detection of target proteins without apparent false-
positives. These findings demonstrate the great potential of
Supra-blot as a reliable, precise and accurate tool for analysis of
specific proteins and spatially localized proteins in cells.
This work was supported by the Institute for Basic Science
[IBS-R007-D1] (to K.K.) and the National Research Foundation of
Korea grant funded by the Korea government (NRF-
2017R1A5A1015366) (to H.-W.R.).
Conflicts of interest
There are no conflicts to declare.
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