Rapid and Selective Labeling of Endogenous Transmembrane Proteins in Living Cells with a Difluorophenyl Ester Affinity-Based Probe

Article abstract of DOI:10.1002/asia.202001049

The long-term stability of affinity-based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho-difluorophenyl ester reactive module exhibit long-term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well-suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia-induced transmembrane carbonic anhydrases were revealed by live cell imaging and in-gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in-depth studies of their distribution and functions in biological processes.

Full text of DOI:10.1002/asia.202001049

DOI: 10.1002/asia.202001049
Communication
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Rapid and Selective Labeling of Endogenous Transmembrane
Proteins in Living Cells with a Difluorophenyl Ester Affinity-Based
Probe
Hsin-Ju Chan,[a] Xin-Hui Lin,[a] Syuan-Yun Fan,[a] Jih Ru Hwu,[a, b] and Kui-Thong Tan*[a, b, c]
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with the fluorescent protein fusion technology, protein labeling
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with small molecule fluorophores enables the incorporation of
a wide range of fluorophores with superior photophysical
properties to the protein of interest (POI), coupled with the
precise control of the location and timing of labeling by the
appropriate regulation of probe delivery. Furthermore, protein
labeling also exhibits a strong advantage over fluorescent
protein fusion technology for the imaging of membrane
proteins where significant fluorescence from the fluorescent
proteins can be observed in intracellular secretory pathways.[3]
Currently, most protein labeling strategies rely on protein/
peptide tags and bioorthogonal chemistry-based methods.
Although these genetic approaches are robust and versatile,
the major drawback is that they are applicable only to
genetically engineered proteins, thus cannot be directly
employed in the study of endogenous proteins in live cells.
As a complementary strategy to the labeling tags, affinity-
based protein labeling is a tag-free approach that is applicable
to study endogenous membrane protein of live cells.[4] In a
typical design, the small molecule probe binds to the POI by a
specific protein–ligand interaction, inducing a chemical reaction
between the reactive group and an amino acid near the active
pocket of the protein via proximity effect. To date, a variety of
reactive modules have been developed and used in affinity-
based protein labeling, including activated esters, sulfonyl
chlorides, epoxides, maleimides, α-halocarbonyls, tosylate, and
acyl imidazole.[5] Although these reactive moieties have been
applied for proteomics analysis, protein imaging, biosensor
construction and irreversible inhibition of protein activity, the
reactive nature of the electrophiles on the probe is also the
cause of many selectivity and stability problems.[4c,6]
Ideally, biocompatible electrophile exhibiting large reaction
rate constants is highly desirable for efficient labeling of
endogenous proteins. However, high reactivity may also result
in elevated levels of nonspecific reactions. For example,
derivatives of N-hydroxysuccinimide (NHS, pKa =6.0) and penta-
fluorophenol (pKa =5.5) activated esters exhibit fast reaction
with proteins, but they are not stable in an aqueous buffer and
decompose slowly in DMSO, a popular organic solvent used for
preparing stock solution.[7] The main reason for the low stability
and high reactivity of these two ester groups is due to the low
pKa values of the electrophile. Therefore, it is important to
modulate the pKa value of electrophile in order to create an
affinity-based protein labeling probe with optimal reactivity and
stability.
Abstract: The long-term stability of affinity-based protein
labeling probes is crucial to obtain reproducible protein
labeling results. However, highly stable probes generally
suffer from low protein labeling efficiency and pose
significant challenges when labeling low abundance native
proteins in living cells. In this paper, we report that protein
labeling probes based on an ortho-difluorophenyl ester
reactive module exhibit long-term stability in DMSO stock
solution and aqueous buffer, yet they can undergo rapid
and selective labeling of native proteins. This novel electro-
phile can be customized with a wide range of different
protein ligands and is particularly well-suited for the
labeling and imaging of transmembrane proteins. With this
probe design, the identity and relative levels of basal and
hypoxia-induced transmembrane carbonic anhydrases were
revealed by live cell imaging and in-gel fluorescence
analysis. We believe that the extension of this difluorophen-
yl ester reactive module would allow for the specific
labeling of various endogenous membrane proteins, facili-
tating in-depth studies of their distribution and functions in
biological processes.
Membrane proteins are involved in a variety of vital biological
processes including selective transport of molecules, cell
adhesion, intracellular signal transduction and cell-cell commu-
nication. Due to their requisite roles in the survival of
organisms, many membrane proteins are central targets for
therapeutics and medical diagnosis.[1] Therefore, the analysis of
the structure, function and expression level of membrane
proteins is highly significant in medical and pharmaceutical
applications as well as for fundamental research.
Among the modern methods to study membrane proteins
in live cells, several methods based on the selective protein
labeling are widely popular and highly effective.[2] As compared
[a] H.-J. Chan, X.-H. Lin, S.-Y. Fan, J. Ru Hwu, Prof. Dr. K.-T. Tan
Department of Chemistry, National Tsing Hua University, 101 Section 2,
Kuang Fu Road, Hsinchu 30013, Taiwan (Republic of China)
E-mail: kttan@mx.nthu.edu.tw
[b] J. Ru Hwu, Prof. Dr. K.-T. Tan
Frontier Research Center on Fundamental and Applied Sciences of Matters,
National Tsing Hua University, 101 Section 2, Kuang Fu Road, Hsinchu
30013, Taiwan (Republic of China)
[c] Prof. Dr. K.-T. Tan
Department of Medicinal and Applied Chemistry, Kaohsiung Medical
University, Kaohsiung 807, Taiwan (Republic of China)
In this paper, we report that an ortho-difluorophenyl ester,
which shows high stability in DMSO stock solution and aqueous
buffer, can be incorporated in affinity-based protein labeling
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/asia.202001049
Chem Asian J. 2020, 15, 1–6
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probes for the efficient and selective labeling of target protein
in complex biological environments (Figure 1a). The reported
experimental pKa value for ortho-difluorophenol is 7.51.[8] As
compared with the previous ortho-dibromophenol electrophile
which has two bulky bromo atoms and a pKa value of 5.59,
protein labeling probes based on ortho-difluorophenol should
display faster labeling rates and higher stability.[9] Therefore we
believe that this difluorophenyl ester reactive module can
overcome the shortcomings of many previously reported
protein labeling probes. To generate the affinity-based labeling
probes for selective, sensitive and rapid protein labeling, a cell-
impermeable Cy5 dye was incorporated with 2,6-difluorophenol
and the corresponding protein ligand (Figure 1b). Based on this
modular design, probe 1 and 2 were synthesized which consist
of a binding ligand, arylsulfonamide and biotin, for the specific
labeling of human carbonic anhydrase II (hCAII) and streptavi-
din proteins.[10]
difluorophenyl ester linked with a Cy5 dye. No obvious
fluorescent band was observed by treating Cy5-DF with hCAII
protein (Figure S1). Furthermore, we also studied the labeling
sensitivity of 1 and found that the probe can label as low as
50 nM of hCAII in just 15 minutes (Figure S2).
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Similar to the results from 1, the labeling of streptavidin
(MW ~53 KDa, tetramer) with 2 in E.coli lysates displayed a
single band which was not observed in the presence of 100 μM
biotin (Figure 2b). The labeling selectivity of 1 and 2 were also
tested with different non-target proteins (BSA, lactoferrin, RNase
A, HSA, and lysozyme) and only in the presence of hCAII and
streptavidin showed one distinctive band (Figure S3). For 2, we
also performed the labeling reaction with streptavidin in fetal
bovine serum (FBS) which has about 56 mg/mL of total proteins
as determined by BCA assay (Figure S4). The results showed
that as little as 5 ng of streptavidin can be labeled in FBS. By
using MALDI-TOF, we also identified the labeling sites for the
recombinant hCAII to be at Lys190 and Lys192, respectively
(Figure S5). Overall, these results indicate that our labeling
probes based on the difluorophenyl ester electrophile can
modify the target protein selectively and rapidly in complex
environments containing various non-target biomolecules.
The stability of labeling probes in stock solutions upon
long-term storage and freeze/thaw cycles is a fundamental
concern for the reproducibility of the results. It is known that
many chemical compounds decompose over time when stored
in DMSO, which is the most common solvent employed for
preparing stock solutions.[11] To examine the chemical integrity
of difluorophenyl ester protein labeling probes, we conducted
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We first examined the selectivity of protein labeling by
incubating 1 and recombinant hCAII (0.5 μM each) for 15
°
minutes in E.coli lysate at 37 C and analyzed by using in-gel
fluorescent imaging (Figure 2a). The result showed that a clear
single fluorescent band corresponding to hCAII (MW ~32 KDa)
appeared when the cell lysate was incubated with 1. On the
other hand, no detectable fluorescence was observed in the
presence of 100 μM hCAII inhibitor, ethoxzolamide (EZA),
indicating the specific ligand controlled labeling of the
difluorophenyl ester probe in complex biological medium. To
further prove that the protein labeling occurs solely based on
the protein-ligand affinity binding, we also synthesized a
controlled reagent Cy5-DF which consists of only the reactive
°
HPLC analysis of 1 stored at À 80 C in DMSO stock solution
(Figure 2c and Figure S7). The results from the HPLC chromato-
gram showed that 97% of 1 remained after 8 months of
storage. In comparison, Cy5 derivatized with NHS ester
degraded to 76% purity after 1-month storage in the same
condition (Figure S8). Furthermore, we also studied the stability
of 1 in pH 7.4 PBS buffer (Figure S9). Approximately 80% of 1
remained after 24 hours of incubation. Evidently, the half-life of
1 in aqueous buffer is much longer as compared with the
highly reactive NHS and pentafluorophenyl esters reported in
the literature.[7b] We believe that the long term stability of the
probe in the DMSO stock solution and aqueous buffer can be
attributed to the moderate pKa value of difluorophenol.
Next, we analyzed the labeling kinetics of hCAII with 1 by
monitoring the increase in fluorescence intensity over time at
SDS-PAGE gel (Figure 2d and Figure S10). Full labeling can be
achieved after 1 hour of incubation and the second-order rate
constant (k2) was calculated to be about 404 MÀ 1 sÀ 1. With the
given k2 values, the reaction rate of difluorophenyl ester is
comparable to the thiol-maleimide Michael addition
labeling.[5b,11] Although the two electrophiles share similar
reaction rates, protein labeling using difluorophenyl ester has
many advantages over the maleimide electrophile; there are
numerous lysine residues on the protein surface to facilitate
reactions with the phenyl ester, the formation of a highly stable
peptide bond in contrast to the thio-succinimide adduct which
often undergo a site-dependent retro-Michael reaction leading
to deconjugation, and there is less interference from the
Figure 1. (a) Schematic illustration of an affinity-based protein labeling
probe design based on the difluorophenyl ester as a reactive electrophile.
(b) Chemical structures of difluorophenyl ester protein labeling probes. 1
and 2 are for human carbonic anhydrase (hCA) and streptavidin proteins,
respectively.
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Figure 2. SDS-PAGE in-gel analysis of the labeling of (a) 1 with recombinant hCAII and (b) 2 with streptavidin for 15 minutes in PBS buffer and 6 mg/mL E. coli
°
lysates at 37 C. The concentrations of the probes and proteins are 0.5 μM. The gel was analyzed by in-gel fluorescence imaging (FL) and stained with
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Coomassie Brilliant Blue (CBB). (c) HPLC trace of probe 1 on day 1 and after storage in DMSO at À 80 C for 8 months. X denotes decomposed byproduct of
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probe 1. (d) Time course of fluorescence increase for the reaction of hCAII with 1 at 37 C in pH 7.4 PBS buffer. The inset shows the kinetic plot of the apparent
reaction rate constant kobs (sÀ 1) versus various concentrations of 1 (R2 =0.99).
biological medium which often contains high level of thio
derivatives. Moreover, the preparation of protein conjugates
using maleimide chemistry can be a cumbersome process. The
reducing agents such as dithiothreitol (DTT) or tris(2-carbox-
yethyl)phosphine (TCEP) used to activate cysteines, can also
inactivate maleimide and so must be removed before con-
jugation. Thus, by introducing the ortho-difluorophenol moiety
with moderate electrophilicity as a leaving group, we have
successfully created a general affinity-based protein labeling
probe approach with rapid protein labeling rate without
compromising the stability of the probe.
To demonstrate the application of our protein labeling
probes for live cell imaging, we first applied 1 to label HeLa
cells with overexpressed recombinant CFP-hCAII-PDGFR protein
on the extracellular surface. The transfected HeLa cells were
incubated with 0.5 μM 1 for 15 minutes and fluorescence
images were taken after removing the excess probe. The
fluorescence from the Cy5 channel overlaid very well with the
CFP-hCAII-PDFGR protein expressed on the cell surface (Fig-
ure 3a and Figure S11). In contrast, addition of EZA reduced the
Cy5 fluorescence while the emission from the CFP channel was
not affected (Figure 3a, ii). These results indicate that 1 can
specifically label and image extracellular hCAII with high
specificity.
Carbonic anhydrases (CAs) form a family of enzymes that
catalyze the inter-conversion between carbon dioxide and
carbonic acid. It has been reported that transmembrane-type
human carbonic anhydrases hCAIX (MW ~50 KDa) and hCAXII
(MW ~44 KDa) are substantially expressed under the condition
of hypoxia in many tumor cell lines which makes hCAs a
valuable biomarker for preclinical and diagnostic imaging.[13] To
show that our difluorophenyl ester probes are sensitive and
selective enough to detect native proteins, 1 was further
applied for the imaging and identification of basal trans-
membrane CA isozyme of MCF7 under normoxia condition
(Figure 3a, iii and iv). The imaging results showed that clear Cy5
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Figure 3. Live cell labeling of overexpressed and endogenous transmem-
brane carbonic anhydrases with 1. (a) Live-cell imaging of HeLa cells
transfected with CFP-hCAII-PDGFR plasmid and MCF7 cells expressing native
transmembrane carbonic anhydrase. CFP and PDGFR encode for cyan
fluorescent protein and platelet-derived growth factor receptor, respectively.
(i) and (iii) were treated with 1 only, while (ii) and (iv) were with 1 and
100 μM EZA. Images were taken after incubating the cells with 0.5 μM 1 for
°
15 minutes at 37 C in DMEM medium. Scale bar: 20 μm. (b) SDS-PAGE in-gel
fluorescence analyses of the labeling of endogenous hCAIX with 0.5 μM 1 in
Figure 4. Live cell labeling of normoxia and hypoxia-induced carbonic
anhydrase isozymes of A549 cells with 1. (a) Images of normoxia and
hypoxia-mimetic A549 cells labeled with 0.5 μM 1 for 15 minutes in DMEM
medium. (i) and (iii) were treated with 1 only, while (ii) and (iv) were with 1
and 100 μM EZA. Scale bar: 20 μm. (b) In-gel fluorescence analyses of the
labeling of living A549 cells with 0.5 μM 1 for 15 minutes in the absence or
presence of EZA inhibitor. The cells were cultured under normoxia and
hypoxia-mimetic conditions, respectively. M=Molecular weight marker.
living MCF7 cells in the absence or presence of 100 μM EZA.
fluorescence along the plasma membrane were observed for
the MCF7 cells treated with 1 and were not detected in the
presence of EZA inhibitor (Figure S12). To characterize the
extracellular CA isozymes, the MCF7 cells labeled with 1 were
lysed and identified by in-gel fluorescence and Western blot
analysis. The fluorescent gel showed that distinct fluorescent
bands at around 50 KDa can be observed clearly and was not
visible in the presence of EZA (Figure 3b). It is important to
note that hCAIX contains a unique N-linked glycosylation site
that bears high mannose-type glycan structures. As a result,
multiple fluorescent bands can be observed with in-gel
fluorescent analysis. In line with the in-gel fluorescence results,
Western-blot bands were observed for MCF7 cells with anti-
hCAIX antibody and not with anti-hCAXII antibody (Figure S13).
These results are consistent with many previous reports which
indicate that the major extracellular CA isozyme for MCF7 cells
is hCAIX.[13b,14]
It has been reported that A549 cells express higher level of
hCAXII proteins when cultured under hypoxia conditions.[13a,15]
Since the sulfonamide ligand has similar binding affinities to
hCAIX and hCAXII, the relative abundance of hCAXII under
normoxia and hypoxia conditions can be determined by
measuring the fluorescence intensity of the 1-labeled hCAXII
bands. Thus, we applied 1 to label A549 cells cultured under
normoxia and hypoxia-mimetic conditions to determine the
relative level of hCAXII. To induce the overexpression of hCAXII,
A549 cells were cultured in the presence of 200 μM CoCl2 for
24 hours to mimic the hypoxia condition.[16] As shown in
Figure 4a, stronger fluorescence signals were observed on the
cell membrane of the hypoxia-mimetic A549 cells (Figure S14).
To estimate the relative levels of hCAXII, the 1-labeled cells
were lysed and analyzed by in-gel fluorescence imaging and
quantified using ImageJ software (Figure 4b). In contrast to
MCF7 cells in which hCAIX is the major isozyme, a fluorescent
band at around 44 KDa was observed which corresponds to the
molecular weight of hCAXII. Under the hypoxia conditions, the
expression level of hCAXII was increased by about 35%. For
comparison, western-blot analysis showed that hypoxia-A549
cells expressed about 30% higher level of hCAXII (Figure S15).
These results are in accordance with many previous findings
which indicate that higher level of hCAXII was found in A549
cells under hypoxia condition.[13d,14,17] Thus, cell-impermeable 1
can be harnessed as a new sensitive and selective protein
labeling probe to study the expression of different trans-
membrane hCAs as demonstrated by the quantitative analysis
of the hCAXII level under basal and stimulated conditions.
In summary, we have developed a stable yet reactive
affinity-based protein labeling probe approach based on the
ortho-difluorophenyl ester for the selective and rapid labeling
of target proteins in the complex biological environments. The
moderate pKa value of difluorophenol imparts long-term
stability of the probe in DMSO stock solution and aqueous
buffer, while maintaining high labeling reaction efficiency with
its target protein. Owing to the modular design and the
versatile synthetic scheme, different ligands can be customized
easily on the probes to label various natural (or engineered)
proteins with nucleophilic amino acids located near the ligand
binding site, as shown in the cases of streptavidin and hCAs.
With the cell-impermeable sulfonamide probes, we have also
successfully shown that the major CA isozymes expressed on
the cell surface of MCF7 and A549 cells to be hCAIX and hCAXII
respectively. Furthermore, the sensitivity of proteins labeling
was comparable to the standard protein detection methods,
such as the Western blot technique. We envision that the
extension of this difluorophenyl ester reactive module should
allow for multi-color imaging of various endogenous membrane
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Acknowledgements
We are grateful to the Ministry of Science and Technology (Grant
No.: 107-2113-M-007-003), Ministry of Education (“Aim for the Top
University Plan”; Grant No.: 107QR001I5) and National Tsing Hua
University (Grant No.: 108QI009E1), for financial support. We also
thank the Instrumentation Center of National Tsing Hua University
for HRMS measurements.
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The authors declare no conflict of interest.
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Keywords: Protein labeling · Membrane proteins · Hypoxia ·
Fluorescent Imaging · Difluorophenyl ester
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Manuscript received: September 3, 2020
Accepted manuscript online: September 15, 2020
Version of record online: September 29, 2020
Chem Asian J. 2020, 15, 1–6
www.chemasianj.org
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Communication
COMMUNICATION
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Endogenous protein labeling:
Affinity-based protein labeling
probes based on an ortho-difluoro-
phenyl ester reactive module exhibit
long term stability in stock solution
and aqueous buffer, yet they can
undergo rapid and selective modifi-
cation of native proteins in living
cells.
H.-J. Chan, X.-H. Lin, S.-Y. Fan, J.
Ru Hwu, Prof. Dr. K.-T. Tan*
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Rapid and Selective Labeling of En-
dogenous Transmembrane Proteins
in Living Cells with a Difluorophenyl
Ester Affinity-Based Probe
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