Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins

Article abstract of DOI:10.1039/c4ob00622d

A traceable linker that is potentially applicable to identification of a target protein of bioactive compounds was developed. It enabled not only thiol-induced cleavage of the linker for enrichment of the target protein but also selective labelling to pick out the target from contaminated non-target proteins for facile identification. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00622d

Organic &
Biomolecular Chemistry
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Development of a traceable linker containing a
thiol-responsive amino acid for the enrichment
and selective labelling of target proteins†
Jun Yamamoto,a Masaya Denda,a Nami Maeda,a Miku Kita,a Chiaki Komiya,a
Tomohiro Tanaka,b Wataru Nomura,b Hirokazu Tamamura,b Youichi Sato,a
Aiko Yamauchi,a Akira Shigenaga*a,c and Akira Otaka*a
Cite this: Org. Biomol. Chem., 2014,
12, 3821
Received 24th March 2014,
Accepted 17th April 2014
DOI: 10.1039/c4ob00622d
www.rsc.org/obc
A traceable linker that is potentially applicable to identification of target can be connected to a biotinylated linker molecule that
a target protein of bioactive compounds was developed. It enabled allows it to be purified over streptavidin beads using the
not only thiol-induced cleavage of the linker for enrichment of the highly specific biotin–streptavidin interaction.1,3 The immobi-
target protein but also selective labelling to pick out the target lized target can then be released from the streptavidin beads
from contaminated non-target proteins for facile identification.
for subsequent sequence analysis by attenuating the biotin–
streptavidin interaction. The high affinity of this interaction
(Kd = 10−15 M),4 however, sometimes hampers the liberation of
the target from the beads, and several alternatives have been
developed for the liberation of the target, including the use of
a cleavable linker between the bait and biotin.5 The use of
such a linker allows for the efficient elution of the target
protein from the beads via linker cleavage, but this approach
can be limited by the contamination of the target protein with
non-target proteins, which can prevent the identification of
the target.6 The development of a method that allows for the
cleavage of the linker moiety to be conducted under mild con-
ditions with generation of an orthogonal functional group that
is not seen in proteins is therefore strongly desired. The use of
an orthogonal functional group in this context should allow
for the introduction of an isotopic or fluorescent tag that
would facilitate identification of the tagged target by mass
spectrometry (isotopic tag) or sodium dodecyl sulfate (SDS)-
PAGE (fluorescent tag).
A wide variety of biologically active organic molecules, includ-
ing natural products, peptides, and synthetic small molecules,
exert their biological activity through specific interactions with
a target biological macromolecule. Among the macromolecules
present in living systems, proteins represent one of the most
important target classes, with enzymes, receptors, and ion
channels being of particular interest. In the fields of chemical
biology and drug discovery, the identification of novel protein
targets with which organic molecules such as biologically
active ligands can interact is critical to understanding complex
biological signalling pathways and developing novel thera-
peutic agents, although studies of this type can be time-con-
suming and laborious. The identification of a protein target
involves a sequence of specific processes, including (1) effec-
tively ‘fishing’ for a target protein using a biologically active
ligand as a bait; (2) enrichment of the hooked target; and (3)
sequence analysis of the target by the Edman degradation or
mass spectrometry.1 For the first step, photo-affinity label-
ling1a,b,2 and activity-based probe technology,1c–e which allow
the covalent attachment of a ligand to a target of interest by
photo-irradiation or chemical reaction respectively, have
shown good potential in terms of their application to low
affinity binding pairs. Using these approaches, the hooked
We recently developed a trimethyl lock7-based stimulus-
responsive amino acid that we applied to a nuclear-cytoplasmic
shuttle peptide and a hypoxia-responsive fluorophore.8 The
molecular basis of this amino acid-induced amide cleavage is
shown in Fig. 1. Peptide 1, bearing an O-protected stimulus-
responsive amino acid, was converted to phenol 2 by exposure
to an appropriate stimulus (e.g., PG = o-nitrobenzyl; stimulus =
UV irradiation). Subsequent lactonization of the phenol led to
the cleavage of peptide 2 to give fragments 3 and 4.
It was envisioned that the incorporation of this amide scis-
sion system into a cleavable linker would allow for the enrich-
ment and identification of target proteins, although two
additional requirements would need to be considered, includ-
ing (1) the cleavage of the linker should be mediated by a
reagent that does not react irreversibly with the target mole-
aInstitute of Health Biosciences and Graduate School of Pharmaceutical Sciences,
The University of Tokushima, Shomachi, Tokushima 770-8505, Japan.
E-mail: shigenaga.akira@tokushima-u.ac.jp, aotaka@tokushima-u.ac.jp
bInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,
Chiyoda-ku, Tokyo 101-0062, Japan
cJST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
†Electronic supplementary information (ESI) available: Supplementary scheme
and figures, experimental section, 1H NMR and MS spectra. See DOI: 10.1039/
c4ob00622d
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cule; and (2) it should be possible for the cleavage products to
react chemoselectively during the post-cleavage labelling
process to allow for the target protein to be discriminated
from the contaminated non-target proteins. With these
requirements in mind, we designed a new linker molecule, as
shown in Fig. 2. A thiol compatible with biomolecules was
used to trigger the cleavage of the linker.9 An aminooxy moiety
was used for the chemoselective labelling of the eluted target,
because this functionality would react selectively with the alde-
hyde group on the labelling reagent.10 An azide group was
placed at one end of the linker and biotin was placed at the
other to allow for the introduction of the linker into the target
protein–alkyne conjugate and the enrichment of the hooked
target with streptavidin beads, respectively. A general scheme
depicting different functional aspects of the linker is shown in
Fig. 2. The linker is initially introduced into the target protein
bearing an alkynylated bait using click chemistry.11 Following
enrichment of the hooked target on streptavidin beads, the
target would be eluted through a thiol-induced linker cleavage.
This cleavage process would generate an aminooxy group on
the target that could be chemoselectively labelled with an alde-
hyde derivative. Given that these linker molecules would allow
for the target proteins to be traced, we named them traceable
linkers. The traceable linkers would be more stable and easier
to be cleaved on demand than equilibrium-based hydrazone
type linkers12 which were also designed for purification and
labelling of the targets. In this study, we prepared the flexible
miniPEG derivatives 5 and 6, as well as a rigid proline-rod
derivative 7.13
Fig. 1 Stimulus-responsive amide bond cleavage induced by a stimu-
lus-responsive amino acid (PG: a protective group removable by an
appropriate stimulus).
We initially investigated the use of an enantioselective syn-
thesis for the construction of the traceable linkers composed
of a thiol-responsive amino acid. We previously reported the
preparation of a racemic thiol-responsive amino acid bearing a
thiol-removable p-nitrobenzenesulfonyl (pNs) group as the
Fig. 2 Structure of traceable linkers (pNs: p-nitrobenzenesulfonyl).
Scheme 1 Click chemistry of traceable linker 5 with model peptide 8 followed by thiol-responsive cleavage and selective labelling. Reagents and
conditions: (a) 8, CuSO4, Na ascorbate, TBTA, PBS, DMSO, tert-BuOH; (b) 2-mercaptoethanol, NP40, Na phosphate buffer (pH 7.8), DMSO, 37 °C; (c)
o-bromobenzaldehyde, aniline. (R: biotin–NH(CH2)5CO-miniPEG-; F: phenylalanine; G: glycine; R: arginine; Y: tyrosine).
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phenolic protective group.14 The enantioselective synthesis
was therefore achieved starting from the chiral intermediate S1
(Scheme S1 in ESI†).15 The thiol-responsive amino acid was
then incorporated into the traceable linkers using Fmoc-based
solid phase peptide synthesis (Fmoc SPPS) using a 1-(4,4-
dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl
(ivDde)
group as an ε-amino protective group.
A model reaction was conducted prior to the enrichment
and selective labelling of the alkynylated protein using alkyny-
lated peptide 8 instead of the protein (Scheme 1). Click chem-
istry was performed between the traceable linker 5 and the
model peptide 8 in the presence of tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine (TBTA)16 in phosphate buffered
saline (PBS) with organic co-solvents. After 1 h of the reaction,
the traceable linker–peptide conjugate 9 was generated in high
purity (Fig. S1 in ESI†). We then proceeded to investigate the
thiol-induced cleavage and labelling of the model peptide con-
jugate in a one-pot manner. The conjugate 9 was treated with
2-mercaptoethanol at 37 °C in a mixture of sodium phosphate
buffer–DMSO with NP40.17 The cleavage of the linker reached
completion within 24 h to yield the model peptide 10 bearing
an aminooxy group and the biotin derivative 11. o-Bromobenz-
aldehyde was then added to the reaction mixture as a bromine-
based labelling reagent6b,18 followed by aniline, which was
used to accelerate the formation of the oxime.19 Following a
reaction time of 1 h, the selective labelling of the model
peptide was complete to yield the labelled peptide 12 and
intact 11.
Because the traceable linker 5 has been shown to be respon-
sive to the thiol-induced cleavage and selective labelling pro-
cesses, we proceeded to evaluate the applicability of this
traceable linker strategy to the enrichment and selective label-
ling of an alkynylated protein. A general scheme is shown in
Fig. 3A. In this study, an alkynylated enolase was used as the
target protein covalently modified with the alkyne bearing sub-
strate. The thiol-intact linker 15 was synthesized as a negative
control for the thiol-induced release of the protein from the
streptavidin beads (Fig. 3B). The click chemistry of linkers 5–7
and 15 was initially examined. A mixture of the alkynylated
enolase and an intact one prepared according to the litera-
ture12b was treated with the linker in the presence of CuSO4,
sodium ascorbate and TBTA. SDS was also added for solubili-
sation of the enolase. Following a reaction time of 1 h and sub-
sequent purification by SDS-PAGE, the biotinylated proteins
and all proteins were visualized by western blot analysis using
a streptavidin-horseradish peroxidase conjugate (SAv-HRP) and
Lumitein staining, respectively. As shown in Fig. 4A, the
linkers were successfully introduced into the enolase in all
cases. It was also observed that the efficiency of the click
chemistry depended on the structure of the R moiety and the
amino acid on the aminooxy group. Although reason for the
difference of the reactivity is not clear at present, the result
suggests that optimization of not only the reaction conditions
but also the structure of the linker is essential for efficient
introduction of the linker onto a target protein. After the click
chemistry, the proteins were treated with streptavidin beads
Fig. 3 (A) Schematic representation of enrichment and labelling of
alkynylated enolase by traceable linker. (B) Structure of thiol-intact
negative control linker 15.
for 24 h. Following a period of washing, the beads were
reacted with 2-mercaptoethanol in sodium phosphate buffer
(pH 7.8) containing NP40 at 37 °C for 24 h (Fig. 3A and 4B).
The resulting product was centrifuged and the supernatant
was treated with the fluorophore 1420 in the presence of
aniline for 24 h. When the traceable linker 5, 6, or 7 was
employed, the thiol-induced elution and labelling of the
enolase proceeded successfully, whereas small amounts of pro-
teins remained on the beads after the thiol treatment (elution
efficiency calculated based on Lumitein staining in Fig. 4B:
68% for 5, 66% for 6, and 63% for 7).21 In the case of the thiol-
inert negative control 15, although the enolase was success-
fully adsorbed onto the bead, it was not released by the thiol
treatment. These observations suggested that the traceable
linkers enabled the thiol-responsive release of the target
protein in a similar manner to conventional cleavable linkers
to allow for subsequent labelling of the aminooxy group of the
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2-mercaptoethanol followed by the fluorophore 14 in a similar
manner to that shown in the footnote of Fig. 4. Following puri-
fication by SDS-PAGE, the labelled enolase was detected using
its fluorescence tag. As shown in Fig. S3 in ESI,† labelling was
only observed when the traceable linker was used. This result
demonstrated that the cleaved traceable linker was suitable for
the selective labelling of the target protein in the presence of
other proteins.
Pull-down experiments were finally conducted for the
enrichment and selective labelling of the alkynylated enolase
in a protein mixture, and the results are depicted in Fig. 5. A
mixture consisting of the alkynylated enolase, bovine serum
albumin (BSA) and ovalbumin (1/1/1 (w/w)) was subjected to
enrichment (the click chemistry with the traceable linker 5,
adsorption on streptavidin beads, and thiol treatment) and
selective labelling with the fluorophore 14. A procedure similar
to that described for the alkynylated enolase shown in Fig. 4
was used in this particular case and the experimental details
are provided in the ESI.† After enrichment with streptavidin
beads, ovalbumin was excluded from the protein mixture as
shown in Fig. 5 [Lumitein staining: proteins adsorbed on the
beads before the thiol-treatment (SDS release) and proteins
eluted from the beads after the thiol-treatment (cleavage + lab-
elling)]. In this experiment, the eluent was found to be con-
taminated with BSA, in the same way as conventional cleavable
linker systems can be contaminated with non-target proteins.6
The eluent containing the enolase and the BSA contaminant
was then treated with the fluorophore 14 in the presence of
Fig. 4 Monitoring of the reactions shown in Fig. 3A using SDS-PAGE.
(A) After click chemistry. Linker 5–7 or 15 (0.10 mM) was introduced into
the alkynylated enolase (0.50 g L−1) using a mixture of CuSO4 (1.0 mM),
Na ascorbate (0.50 mM), TBTA (0.10 mM), SDS (1% (w/v)), PBS and co-
solvents over 1 h. (B) Adsorption on streptavidin beads followed by thiol-
induced elution and labelling. Proteins were treated with streptavidin
beads for 24 h following the click chemistry. After washing, the beads
were reacted with 2-mercaptoethanol (100 mM) and NP40 (1% (v/v)) in
Na phosphate buffer (10 mM, pH 7.8) at 37 °C for 24 h. The product was
centrifuged and the supernatant was treated with fluorophore 14
(0.10 mM) and aniline (100 mM) for labelling. The reaction mixture was
stirred for 24 h. [a] Biotinylated proteins were detected by western blot-
ting analysis using a SAv-HRP. [b] All proteins were visualized by Lumi-
tein staining. [c] Fluorescein-labelled proteins were detected at λex
=
488 nm and λem = 530 nm without staining. [d] Proteins after thiol treat-
ment followed by the labelling reaction. [e] Proteins remaining on strep-
tavidin beads after the thiol treatment. The beads after centrifugation
followed by removal of the supernatant was suspended in SDS-PAGE
sample loading buffer, and the resulting mixture was heated at 100 °C
for 5 min. After centrifugation, the supernatant was analysed.
Fig. 5 Pull-down experiment for the alkynylated enolase in a protein
mixture consisting of the alkynylated enolase, BSA, and ovalbumin.
Enrichment (click chemistry with traceable linker 5, adsorption on strep-
tavidin beads, and thiol treatment) and selective labelling with fluoro-
phore 14 were performed in a similar manner to that described in the
footnote of Fig. 4. Following purification by SDS-PAGE, the labelled pro-
ducts or all proteins were visualized by fluorimetry without staining
(FTC: λex = 488 nm, λem = 530 nm) or Lumitein staining (Lumitein),
respectively. [a] Mixture of the alkynylated enolase, BSA, and ovalbumin
(1/1/1 (w/w)). [b] The beads were suspended in SDS-PAGE sample
loading buffer prior to the thiol-treatment, and the mixture was heated
at 100 °C for 5 min. The mixture was then centrifugated, and the super-
natant was analysed. [c] Samples after enrichment and selective labelling
of the alkynylated enolase.
cleavage products. Following the enrichment and labelling
processes, the traceable linker 5 gave the brightest band of all
of the traceable linkers for fluorescein-labelled enolase, and
this linker was therefore used in all of the subsequent
experiments.
We then proceeded to investigate the orthogonal nature of
the aminooxy group generated by the cleavage of the linker.
The enolase–linker conjugate derived from the click chemistry
with the traceable linker 5 or control 15 was treated with
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aniline for 24 h, which resulted in the selective labelling of the
enolase and allowed for the target enolase to be distinguished
from the non-target contaminants (Fig. 5; FTC detection: clea-
vage + labelling). These results demonstrate that the traceable
linker is applicable to the enrichment and selective labelling
of the target protein for facile identification, even when the
target protein has been contaminated with non-target proteins
in the eluent obtained from the streptavidin beads.
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9 Photo-cleavage reaction is also compatible with bio-
molecules. In this study, however, it was not used because
the photo-cleavage usually generates carbonyl functional-
ities (e.g. an aldehyde from an o-nitrobenzyl group) that
would hamper the following chemoselective labelling by
reacting with an aminooxy group.
Conclusions
In summary, we developed the traceable linker as an advanced
cleavable linker. This new linker not only enabled the thiol-
induced cleavage of the linker for the enrichment of the target
protein in a manner similar to that of conventional cleavable
linkers, but also allowed for the selective labelling of the target
so that it could be distinguished from contaminated non-
target proteins. Depending on an experimental design, in prin-
ciple, a stimulus for cleavage of the traceable linker can be
altered simply by changing a phenolic protection of the stimu-
lus-responsive amino acid.22 Combination of this traceable
linker-based technique with the target selective introduction
of an alkyne unit by the use of photo-affinity labelling or
activity-based probe technology1,2 therefore represents a new
methodology for the facile clarification of the targets of the
biologically active compounds, including drug candidates. The
application of this traceable linker technique to the identifi-
cation of the target proteins of naturally occurring bioactive
compounds is currently underway in our laboratory.
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Acknowledgements
This research was supported in part by PRESTO, Japan Science
and Technology Agency (JST) and a Grant-in-Aid for Scientific
Research from the Japanese Society for the Promotion of
Science (JSPS) and the Ministry of Education, Culture, Sports,
Science and Technology, Japan (MEXT). Astellas Foundation
for Research on Metabolic Disorders and Takeda Pharma-
ceutical Company are also acknowledged. JY and MD are grate-
ful for JSPS fellowship. A propargyl glycine used in this study
was a gift from Nagase & Co., Ltd.
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requires that the linker cleavage and selective labelling
should be performed in the presence of detergents. In this
model reaction, therefore, NP-40 that is widely used as a
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non-ionic detergent for solubilization of proteins was
employed.
the ESI†). The concentration of intracellular glutathione
that is the most abundant thiol in cells is 0.2 to 10 mM,
therefore, our traceable linker is supposed to be compatible
with proteomes with endogenous glutathione. Moreover,
the glutathione and other small thiols can be removed by
dialysis or ultrafiltration before the reaction with the trace-
able linker. For the concentration of endogenous gluta-
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21 When the concentration of 2-mercaptoethanol was reduced 22 A fluoride-responsive traceable linker has already been
to 10 mM, the linker cleavage was not observed (Fig. S2 in
developed and it will be published in due course.
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