Cleavable trifunctional biotin reagents for protein labelling, capture and release

Article abstract of DOI:10.1039/c3cc42076k

Trifunctional biotin reagents incorporating cleavable linkers are evaluated for their usage in protein enrichment. A linker based on the Dde protecting group leads to efficient release of protein targets under mild conditions. It additionally contains a masked trypsin cleavage site, which eliminates the majority of the tag during tryptic digestion.

Full text of DOI:10.1039/c3cc42076k

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Cleavable trifunctional biotin reagents for protein
labelling, capture and release†
Cite this: Chem. Commun., 2013,
49, 5366
Yinliang Yang and Steven H. L. Verhelst*
Received 21st March 2013,
Accepted 22nd April 2013
DOI: 10.1039/c3cc42076k
www.rsc.org/chemcomm
Trifunctional biotin reagents incorporating cleavable linkers are
evaluated for their usage in protein enrichment. A linker based on
the Dde protecting group leads to efficient release of protein
targets under mild conditions. It additionally contains a masked
trypsin cleavage site, which eliminates the majority of the tag
during tryptic digestion.
Site-specific modification of proteins by small molecule
chemical probes is one of the main techniques in chemical
proteomics.1 Examples include lipid and carbohydrate deriva-
tives for metabolic labelling of post-translationally modified
proteins2 and activity-based probes (ABPs) for the tagging of
active enzymes.3 Ideally, the labelling of protein targets is carried
out in models that resemble physiological conditions as well as
possible, such as living cells or animals. Bioorthogonal ligation
methods have provided opportunities to study proteins in situ
and in vivo. To this end, probes containing alkyne or azide
moieties are functionalized after protein modification by
Cu(I)-catalyzed azide–alkyne cycloaddition, Staudinger ligation or
Cu-free cycloaddition.4 Trifunctional tags, incorporating a ligation
handle, a biotin and a fluorophore, have become popular, since
they can be applied for both visualization and affinity purifica-
tion.5–7 However, due to the strong interaction between biotin and
streptavidin harsh conditions are necessary for the elution of
enriched proteins. This usually leads to contamination of the
sample with non-specifically bound proteins and endogenously
biotinlytated proteins, which complicates target identification.
Cleavable linkers8 have been employed to solve this problem.
In this study, we design trifunctional reagents with cleavable
linkers in order to facilitate mild release of target proteins during
enrichment from proteomes (Fig. 1). We compare five linkers
that cleave under different conditions, evaluate their compat-
ibility with bioorthogonal ligation, and determine the efficiency
of the release. We also report on a new linker based on the
Fig. 1 Cleavable trifunctional tags in chemical proteomics serve the purpose of
sensitive detection, enrichment of targets and selective elution.
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protecting
group,9 which has – to our knowledge – not yet been used in
chemical proteomics applications. We show here that this linker
leads to efficient protein elution under very mild conditions.
Additionally, in the trifunctional reagent with the Dde linker, a
masked trypsin cleavable bond eliminates the fluorescent tag
upon tryptic digestion, reducing the molecular weight of the final
modification when compared to other trifunctional tags.
The cleavable trifunctional tags were designed as follows
(Fig. 2). All tags incorporate an azide group to enable click
chemistry-mediated derivatization of alkyne-labelled proteins. A
PEG spacer is introduced to increase the solubility. A carboxy-
tetramethylrhodamine (Rh) fluorophore, which remains attached
to the protein after cleavage of the linker, serves the purpose of
sensitive detection. Finally, the biotin enables efficient protein
enrichment. As cleavable linkers (Fig. 2) we use:
1 A vicinal diol synthesized from tartrate,10,11 which can be
cleaved under oxidative conditions.
2 A diazobenzene derivative,12–14 which is cleavable under
reductive conditions (sodium dithionite).
3 A bisaryl hydrazone. This biocompatible linker can be
cleaved by hydroxylamine.15–17 It has not yet been used in
combination with bioorthogonal ligations.
¨
¨
¨
Lehrstuhl fur Chemie der Biopolymere, Technische Universitat Munchen,
Weihenstephaner Berg 3, 85354 Freising, Germany. E-mail: verhelst@wzw.tum.de;
Fax: +49 8161 714404; Tel: +49 8161 713505
4 A disulfide linker, which is commercially available and can
be cleaved under reductive conditions.18
† Electronic supplementary information (ESI) available: Detailed experimental pro-
cedures, supplementary figures, schemes and tables. See DOI: 10.1039/c3cc42076k
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Fig. 2 The structures of cleavable trifunctional tags 1–5. All contain an azide, a Rh fluorophore, a cleavable linker (boxed) and a biotin.
5 Besides the four above-mentioned cleavable linkers, we
designed a linker that is novel in chemical proteomics. It is
based on the Dde group, which is used as a protecting group for
the e-amino group of lysine in solid phase peptide synthesis
(SPPS). The conventional way of deprotecting the Dde group
consists of using 2% hydrazine.9
All reagents were synthesized by a combination of SPPS and
solution phase synthesis (Schemes S1–S5, ESI†). Having the
cleavable tags 1–5 in hand (Fig. 2), we first tested whether they
can be used for the detection of alkyne modified proteins in a
complex proteome. To this end, we treated a lysate of the
macrophage cell line RAW264.1 with alkyne-E-64 (Fig. 4), an
activity-based probe for cathepsin proteases.19 We then
attached the cleavable tags to the alkyne-E-64 labelled proteins
Fig. 3 (a) Labelling of alkyne-E-64 labelled cathepsins in RAW 264.7 cell lysate
with a tetramethylrhodamine fluorophore and with trifunctional tags. (b) Pull
down of labelled cathepsins by streptavidin beads. Left lanes: labelling after click
chemistry with carboxy-tetramethylrhodamine fluorophore or trifunctional tags.
Middle lanes: supernatant (sup) after capture with streptavidin beads. Right lane:
by Cu(I)-catalyzed click chemistry, and the lysate was resolved
on a gel. As determined before by mass spectrometry, cathepsin
Z and B are the two main targets of E-64 in the lysate.10,19 All
tags display similar labelling compared with a fluorophore-
azide derivative, illustrating the compatibility of the cleavable
tags with the bioorthogonal labelling strategy (Fig. 3a). They
also showed efficient depletion of the labelled cathepsins from
a proteome by using immobilized streptavidin (Fig 3b, middle
lanes and Table S1, ESI†), while boiling released the targets
again (right lanes). The pull down efficiency was slightly lower
release of immobilized proteins by boiling with SDS sample buffer (D).
A
carboxytetramethylrhodamine coupled 3-aminopropylazide (Rh–N3) was used
as a control to show that no non-specific binding of labelled cathepsins to
streptavidin beads occurs.
for the disulfide and the Dde linker, possibly due to premature For cleavage of the diol linker, we used 1 and 10 mM NaIO4
cleavage or steric hindrance around the biotin.
(3 Â 20 min; directly followed by quenching of the periodate
At this point, we investigated the release of the tagged with sample buffer). Under both conditions, the target proteins
proteins. For each individual linker, the specific elution was were efficiently released (Fig. 4 and Fig. S1, ESI†). The 1 mM
optimized. In all experiments, we determined the efficiency of NaIO4 treatment gave rise to 74% cleavage efficiency. For the
the elution using fluorescent gel band densitometry by com- diazobenzene linker, which was treated with 200 mM Na2S2O4,
parison of the proteins selectively eluted and residually bound the cleavage efficiency was 58%. The bisaryl hydrazone linker
to the immobilized streptavidin (Table S1, ESI†). We also tested could not be cleaved by 100 mM hydroxylamine (Fig. S3, ESI†),
whether elution conditions were specific (i.e. if they resulted in but the addition of aniline, a reported catalyst in the cleavage of
cleavage of the biotin from the tagged proteins; ESI,† Fig. S1–S5). these linkers, led to a release of 47% of the proteins (Fig. 4).
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azidolysine residue of the trifunctional tag will be left behind
on a target protein. Modifications that have a large size and
undergo fragmentation in tandem MS experiments are unde-
sired for the identification of probe-labelled tryptic peptides.
Hence, this secret trypsin site may aid in future identification
of the modification sites of probes and post-translational
modifications.
In conclusion, we have synthesized and analyzed five differ-
ent cleavable trifunctional biotin tags for tandem labelling of
proteins and subsequent detection or enrichment. Overall, the
trifunctional tags containing a diol or Dde linker were the most
efficient in the chemical release experiments. The Dde group
represents to our knowledge a novel cleavable linker in protein
enrichment strategies. This linker can be easily synthesized and
cleaved under mild conditions. Moreover, after tryptic diges-
tion, only a small residual modification results. We expect this
linker to find application in proteomics experiments aimed at
the identification of modification sites. Further studies along
these lines will be reported in due course.
Fig. 4 Evaluation of the elution of alkyne-E-64 targets from streptavidin beads
with the cleavable trifunctional tags. Left lane: direct release by boiling with SDS
sample buffer (D). Middle lane: chemical cleavage (cc). (1) 1 mM NaIO4, 100 mM
phosphate, pH 7.4, 3 Â 20 min. (2) 200 mM Na2S2O4, 100 mM phosphate, pH 7.4,
3 Â 20 min. (3) 100 mM NH2OH and 100 mM aniline 100 mM phosphate, pH 4.6,
4 h. (4) 0.5 M DTT in H2O, 1 h. (5) 0.05% SDS, 200 mM Tris, pH 8.5, 2 h. Right lane:
(D after cc), boiling the streptavidin beads with SDS sample buffer after chemical
release to elute unreleased proteins.
Funding was provided by the DFG (Emmy Noether program
to SV), the Chinese Scholarship Council (fellowship to YY), the
Center for Integrated Protein Science Munich and the Graduate
Unfortunately,
a further increase in the concentration of
¨
Centre Weihenstephan of the Technische Universitat Mu¨nchen.
hydroxylamine did not lead to full release (Fig. S3, ESI†). This is
in accordance with recent experiments by Claessen et al., who
reported poor elution of targets enriched from a whole proteome
using a similar bisaryl hydrazone linker.17 The disulfide linker is a
widely used and commercially available linker, which can be
cleaved by reducing agents. However, one problem of a disulfide
linker is the potential disulfide exchange. This has been reported
before in the labelling of biomolecules by clickable disulfide
cleavable linkers.20 We indeed observed a high degree of disulfide
exchange when analysing samples in non-reducing gels (Fig. S4,
ESI†). The cleavage efficiency was only moderately effective with
60% after treatment with 0.5 M DTT (Fig. 4).
Besides the four above-mentioned cleavable linkers, which
have previously been applied in the capture and release of
proteins, we designed a linker based on the Dde protecting
group (5, Fig. 2). The conventional way of deprotecting the Dde
group in peptide synthesis consists of using 2% hydrazine. This
turned out to be not compatible with fluorescent scanning
(Fig. S5a, ESI†). However, the protein targets were released and
visualized almost quantitatively (92%) with a solution of 0.05%
SDS and 200 mM Tris at pH 8.5 (Fig. 4). Both reagents are
necessary for efficient cleavage. Other mild conditions did not
lead to the release of target proteins (Fig. S5b, ESI†), whereas
other linkers are unaffected by the Tris-SDS treatment (Fig S5c,
ESI†), indicating that the release is dependent on the presence
of the Dde linker. LC-MS analysis showed that linker 5 treated
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