Introducing aldehyde functionality to proteins using ligand-directed affinity labeling

Article abstract of DOI:10.1039/d0cc01982h

Aldehyde is a versatile chemical handle for protein modification. Although many methods have been developed to label proteins with aldehyde, target-specific methods amenable to endogenous proteins are limited. Here, we report a simple affinity probe strategy to introduce aldehydes to native proteins. Notably, the probe contains a latent aldehyde functionality that is only exposed upon target binding, thereby enabling a one-pot labeling procedure.

Full text of DOI:10.1039/d0cc01982h

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Introducing Aldehyde Functionality to Proteins Using Ligand-
directed Affinity Labeling
Yinan Song,‡ Feng Xiong,‡ Jianzhao Peng, Yi Man Eva Fung, Yiran Huang, and Xiaoyu Li *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Aldehyde is a versatile chemical handle for protein modification.
Although many methods have been developed to label proteins
with aldehyde, target-specific ones amendable to endogenous
proteins are limited. We report a simple affinity probe strategy to
install aldehyde on native proteins. Notably, the probe contains a
latent aldehyde functionality that is only exposed upon target
binding, thereby enabling a one-pot labeling procedure.
Modified proteins are widely used as powerful tools in many
research fields including chemical biology, biotech, and
medicine.1-3 In the past decades, a plethora of protein labeling
methods have been developed. Among them, site-specific
incorporation of functional groups has been a major focus to
generate homogeneous protein constructs. Aldehyde is a
functional group not typically found in native proteins. Since the
natural amino acid side chains are mostly nucleophilic, the
electrophilicity of aldehyde makes it “bio-orthogonal” and could
Fig. 1: a) Scheme for protein labeling with the epoxy-alcohol-based affinity probe. b) The
precursor EP is coupled with the ligand (L) to form the affinity probe. POI: protein of
interest; Nu: a nucleophilic side chain close to the ligand-binding site.
leading to diverse applications in cell imaging, biosensing, and
inhibitor discovery.1, 22 The Ball group expanded the scope of
modifiable side chains of affinity probes with metal catalysis.23
However, introducing aldehyde to proteins with affinity probes
has been largely underexplored. The studies by the groups of
Hamachi9, 12 and Gothelf8 remain the only examples. Target-
specific labeling of native proteins with aldehyde in complex
biological environment, such as live cells, has not been
reported. Here, we report a simple ligand-directed labeling
strategy to introduce aldehyde to native proteins. As a unique
feature, the aldehyde functionality is latent and is only exposed
after target conjugation, thus enabling a convenient one-pot
labeling procedure. Furthermore, this method also realises the
labeling of membrane proteins with aldehyde on live cells.
Our approach is shown in Fig. 1. The affinity probe has a very
simple design: a ligand and an epoxy alcohol. Epoxide is a
moderate electrophile and its reactivity could be improved by
the proximity effect upon target binding. Previously, epoxides
have been used in affinity probes, activity-based protein
profiling, and covalent inhibitors.24-28 Here, we propose to
exploit the structure of the epoxide opening product. After the
probe binds to the protein of interest (POI), a nearby nucleo-
5
be exploited by various nucleophiles.4, Previously, many
methods have been developed to label protein with aldehyde,5
such as oxidative cleavage,6-9 biomimetic transamination,10, 11
and linker exchange.12, 13 Enzyme-recognizing peptides could be
fused with the target for site-specific aldehyde labeling using
various enzymes.14-18 The powerful unnatural amino acid
mutagenesis and metabolic labeling methods have also been
used to incorporate aldehyde (or ketone) into proteins.19-21
However, chemical methods are mostly limited to purified
proteins, while enzymatic and mutagenesis methods require
genetic manipulation and may not be suitable for native
proteins. Ligand-directed affinity labeling is an effective
approach to achieve target specificity: the ligand guides the
probe to bind the target and the proximity effect facilitates
target-specific labeling. For example, the Hamachi group
pioneered a series of powerful ligand-directed labeling methods
Department of Chemistry and State Key Laboratory of Synthetic Chemistry, The
University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Email: xiaoyuli@hku.hk.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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philic side chain opens the epoxide and forms a vicinal diol,
which can be cleaved using periodate to generate the aldehyde.
The ligand is released from the pocket and the aldehyde could
be further modified with various tags. First, hiding the aldehyde
functionality in the crosslinker simplifies the probe synthesis,
since the probe is only “bi-functional” structurally but has a
latent 3rd functionality. Second, the aldehyde will be exposed
only after protein conjugation, while the free unbound probes
remain “inactive”, thus realizing a one-pot labeling procedure.
The periodate cleavage condition is mild, rapid, and could be
controlled to have minimal impact on protein target.6-8, 29
First, we prepared an epoxy-carboxylic acid precursor EP
through a simple 3-step synthesis (Fig. S1a), which could be
conjugated with an amine-bearing ligand (L) to furnish the
probe (Fig. 1b). To verify the regioselectivity of epoxide
opening, we tested the reaction of an epoxy alcohol with 2-
mercaptoethanol and observed the formation of the desired
vicinal diol product, where the nucleophilic attack took place at
the less hindered carbon (Fig. S2). Next, we tested the method
with a model protein, carbonic anhydrase II (CA-II). EP was
coupled with 4-carboxybenzenesulfonamide (CBS), a known CA-
II inhibitor (Kd: ~1 µM), to form the probe CBS-EP (Fig. 2a). To
examine the stability of the epoxy alcohol towards nucleophile
under physiological condition, CBS-EP was mixed with 5 mM
glutathione in PBS (phosphate-buffered saline; pH 7.4); 4% and
10% epoxide opening were observed after 16 and 24 hours,
respectively (Fig. S3). The probe also showed similar stability at
pH 5.8, which is closer to the physiological pH around cell
surface and in the cytosol of cancer cells (Fig. S4). CBS-EP also
exhibited excellent stability under the typical oxidative cleavage
condition (Fig. S5)30 and showed similar CA-II inhibition activity
as CBS (Fig. S6). Next, CA-II was incubated with CBS-EP and
subjected to diol cleavage. Because of the probe’s “turn-on”
feature, the entire labeling procedure was performed in one
pot; without removing the excess probes, the aldehyde-labeled
CA-II was tagged with a fluorescent and a biotinylated aminooxy
reagent, respectively (FAM-AO and bio-AO; Fig. 2a). As shown
in Fig. 2b, in-gel fluorescence and Western blot clearly showed
the specific labeling of CA-II and the labeling efficiencies were
determined to be 48% (FAM-AO) and 69% (bio-AO),
respectively (Fig. S7). All negative controls (free CBS, no NaIO4,
no CBS-EP) did not give significant labeling. The labeled CA-II
was characterized with mass spectrometry (Fig. 2c), and it
showed a ~50% labeling yield (ii), largely consistent with the gel
analysis. The aldehyde formation was also confirmed in (iii);
notably, the spectra showed a complete oxidative cleavage (ii
to iii), suggesting that the protein-templated epoxide opening
had the same regioselectivity as the intermolecular reaction
(Fig. S2). Furthermore, the labeled CA-II was trypsinized, and
tandem mass spectrometry analysis of the resulting peptides
identified His64 as the modification site (Fig. 2d and S7b). His64
is located near the CBS-binding pocket and is the same
modification site in a previous study also using an epoxide
probe.24 In addition, we tested the method with another
protein: FKBP12 (12-kDa FK506-binding protein). A known
ligand, SLF, was conjugated with EP to form the probe SLF-EP
and used to
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DOI: 10.1039/D0CC01982H
Fig. 2: a) Structure of CBS-EP, FAM-AO, and bio-AO. b) CA-II (10 µM) was labeled with
CBS-EP (20 µM; 16 h, 37 °C), cleaved with NaIO4 (1 mM, 4 °C, 30 min), and tagged with
FAM-AO or bio-AO (40 µM; r.t. 90 min). The reactions were analyzed with SDS-PAGE and
Western blot. c) MALDI-TOF analysis of the labeled CA-II; ○: CA-II (m/z=29025), ∆: CBS-
EP-labelled CA-II (m/z=29475), ●: aldehyde-labelled CA-II (m/z=29091); L: CBS. d) Crystal
structure of CA-II (PDB: 1V9E) with the modification site His64 highlighted.
label FKBP12 (Fig. S8). Western blot and MS results confirmed
that SLF-EP specifically installed an aldehyde on His88 of
FKBP12 (Fig. S8-S10), which is the same as in a previous study
using a different type of affinity probe.31
Next, the labeling method was tested in complex biological
contexts. Albeit being mild, periodate also cleaves the terminal
sialic acids on glycoproteins in eukaryotic cells.32, 33 To suppress
this background, we used deglycosylases to remove the sialic
acids before labeling. First, HeLa cells were lysed and treated
with a deglycosylase mix (Deglyco-Mix kit; New England Biolabs);
then, CA-II and FKBP12 were spiked in the lysates (2% w/w) and
labelled with CBS-EP and SLF-EP, respectively. After diol
cleavage, bio-AO was added to tag the aldehyde. There was no
need to remove free probes and the bio-AO was added to the
lysates directly after diol cleavage. As shown in Fig. 3a, both CA-
II and FKBP12 could be specifically labeled in lysates.
Furthermore, we tested the labeling method on live cells.
Although ligand-directed labeling of membrane proteins has
been realized,34 installing aldehyde specifically on an
endogenous membrane protein has not been reported. CA-12
is a membrane carbonic anhydrase implicated in malignant
cancers. Since CBS is also a ligand for CA-12, CBS-EP was directly
used. First, we verified that CBS-EP could specifically label the
purified CA-12 in buffer (Fig. S11a); next, A549 cells were
cultured in hypoxia to increase CA-12 expression (Fig. S11b) and
then incubated with CBS-EP. After deglycosylation (Fig. S12) and
oxidative cleavage, the cells were tagged with bio-AO and lysed;
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Fig. 4: a) Structure of TPNF. b) Fluorescence spectra of TPNF (100 µM) with CA-II (40
µM); red: labeled with aldehyde; black: unlabeled. Condition: 1x PBS (pH 6.7), 5 mM
aniline, 30 min, r.t., excitation: 430 nm. b) Scheme for the labeling of CA-12 on A549 cells
and the real-time cell images. After installing the aldehyde, the cells were treated with
TPNF (10 µM), aniline (5 mM) in PBS buffer (pH 6.7) at room temperature.
labeling involved buffer exchanges to provide the optimal
conditions for each step (see the SI for details), which may have
washed off some of the non-covalently bound probe. Thus, we
also performed the CA-12 labeling with a completely “no-wash”
procedure and used free CBS to displace the probe after diol
cleavage. The results showed similar cell fluorescence was
obtained and only
a slight signal decrease upon CBS
competition (Fig. S14b), suggesting that the signal was mostly
from the labeled CA-12. Next, the method was further tested
Fig. 3: a) Western blot analysis of CA-II and FKBP12 labeling in HeLa lysates with
CBS-EP and SLF-EP. b) Labeling of the endogenous CA-12 on A549 cells using CBS-
EP. Conditions are the same as in Fig. 2. Loading control: Fig. S13; *: endogenously with folate receptor (FR), another membrane protein and also a
biotinylated protein; IB: immunoblotting. c) CA-12 on A549 cells were labeled
with CBS-EP, tagged with bio-AO, affinity-purified, and analyzed with tandem MS.
y-axis: with CBS-EP; x-axis: without CBS-EP (negative control). d)-e) Labeled CA-12
on A549 cells were tagged with FAM-AO and analyzed with flow cytometry and
prime target for anti-cancer drug delivery on HeLa cells. The
labeling was performed with FA-EP, a probe prepared based on
the known FR ligand folic acid (FA; Fig. S15a). The results
confirmed the specific capture of FR on live HeLa cells (Fig. S15).
In addition, after the probe labeling, oxidative cleavage,
deglycosylation, and bio-AO tagging, we observed little
detrimental effect on cell viability, as well as on the ligand-
binding affinity of CA-2 and FR (Fig. S16).
confocal imaging. CBS-EP: 4 µM, 37 °C, 4 h; NaIO4: 1 mM, 4 °C, 5 min; tagging: bio-
AO/FAM-AO (40 µM), aniline (5 mM), 4 °C, 90 min. In e) A: without CBS-EP; B: with
CBS-EP. See Fig. S14a for flow cytometry histograms.
Western blot analysis showed specific CA-12 labeling (Fig. 3b).
Next, the biotinylated proteins were isolated with streptavidin
beads and characterized with MS, which confirmed the specific
capture of CA-12 from the cell surface (Fig. 3c). In addition, the
CA-12 labeled cells were also tagged with FAM-AO, and both
fluorescence imaging and flow cytometry corroborated the
specific labeling (Fig. 3d-3e). Although CBS is known to bind
multiple CA isoforms, CBS-EP appeared to have only labeled CA-
12. Since NaIO4 is not cell permeable, in principle, only
membrane CAs may be labeled, which includes CA-9 and CA-
12.35 Although both CA-9 and CA-12 are highly expressed on
A549 cells,36 only CA-12 could be efficiently labeled by CBS-EP
in the purified form (Fig. S11c). We reason the lack of CA-9
labeling in Fig 3b and 3c may be due to that CA-9 might bind CBS
weakly on the cell surface or it lacks a properly positioned
nucleophile for probe crosslinking. In addition, the Hamachi
group observed the similar phenomenon with a CBS-based
affinity probe on A549 cells.36
Fluorogenic probes, also known as “turn-on” probes, are
widely used in imaging applications due to the high signal to
noise ratio and the capability of real-time imaging.37 Elegantly
designed fluorogenic probes have been developed to detect
formaldehyde in vivo.38 However, fluorogenic imaging of
proteins via an aldehyde tag has not been reported. To test this,
we used a naphthalimide-based two-photon fluorescent probe
(TPNF; Fig. 4a), which fluoresces upon hydrazone formation.38-
40 To limit the probe to cell membrane, TPNF was modified with
a 5 kDa PEG to prevent it from entering the cell. First, we tested
TPNF with purified CA-II either unlabeled or labeled with an
aldehyde. As shown in Fig. 4b, the labeled CA-II exhibited
significantly higher fluorescence upon TPNF addition,
suggesting the aldehyde was able to activate TPNF. Next, we
labeled CA-12 on A549 cells with an aldehyde using CBS-EP.
After deglycosylation and cleavage, the cells were incubated
with TPNF, and the generation of fluorescence on cell surface
could be observed in real time (Fig. 4c). In addition, we also used
CBS competition to eliminate the signal from the non-covalently
bound probe. The results showed that the cell fluorescence was
Besides the aldehyde on the protein, the diol cleavage
generates another aldehyde on the original probe that may also
be tagged; although there was no extensive washing, the cell
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12. Y. Takaoka, H. Tsutsumi, N. Kasagi, E. Nakata and I. Hamachi,
mostly from the aldehyde label on CA-12 (Fig. S17). Finally, flow
cytometry also confirmed the specific labeling (Fig. S18).
In conclusion, we have developed a simple method to install
aldehyde on proteins. In this method, the latent aldehyde
functionality enables a one-pot labeling procedure; the free
unbound probes were not “activated” and this method is
suitable for real-time imaging of membrane proteins. Like many
affinity labeling approaches, the target specificity/isoform
selectivity of the ligand has important implications for this
method: the labeling outcome is a combination of all the
proteins that can bind the ligand. Thus, additional measures and
controls would be necessary to deconvolute the labeling
outcome and the verification of the labeling specificity is always
important. Unfortunately, the use of oxidative cleavage is a
major limitation due to protein glycosylation. Deglycosylation is
able to suppress the background, but it also alters the native
proteome. In addition, cells contain a range of biomolecules
having the diol moiety but cannot be removed by
deglycosylases, such as the glycosylphosphatidyl- inositol (GTI)-
anchored proteins, the glycosaminoglycan (GAG) chain on
proteoglycans, glycolipids, and the furanose-containing
molecules such as RNA and ATP. These molecules may
complicate the labeling selectivity and careful control
experiments would be required. For in vitro studies, this
approach serves as a simple way to incorporate aldehyde into
proteins; for native proteins, the method may be limited to
certain applications where protein isolation is the main
objective, such as target identification.
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This work was supported by grants from the Research
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Conflicts of interest
There are no conflicts to declare.
Notes and references
‡ These authors contributed equally to this work.
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Table of Content
An affinity probe with a “hidden” aldehyde functionality for protein labeling.