Development of trifunctional probes for glycoproteomic analysis

Article abstract of DOI:10.1039/c0cc00345j

A new trifunctional probe, assembled using a cleavable linker, is useful for efficient enrichment and detection of alkynyl sugar-tagged biomolecules. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0cc00345j

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Development of trifunctional probes for glycoproteomic analysisw
Charng-Sheng Tsai,a Po-Yu Liu,ab Hsin-Yung Yen,ab Tsui-Ling Hsua and Chi-Huey Wong*ac
Received 11th March 2010, Accepted 16th April 2010
First published as an Advance Article on the web 13th May 2010
DOI: 10.1039/c0cc00345j
A new trifunctional probe, assembled using a cleavable linker,
is useful for efficient enrichment and detection of alkynyl sugar-
tagged biomolecules.
Glycosylation is an important co-/post-translational modifi-
cation that has profound effects on protein structure and
function.1 However, the complex and non-templated nature
of glycans has been a barrier for studying many of their
basic features, especially on a proteome-wide scale. In order
to understand the role of glycans in numerous physio-
logical and pathological processes, metabolic oligosaccharide
engineering (MOE) has been employed to insert sugar analogues
appended with bioorthogonal alkynyl or azido groups in place
of the native sugars via glycan synthesis pathways in cells.2
Fig. 1 Application of trifunctional probes in glycoproteomics and the
structure of trifunctional probes I and II.
Following further labeling with proper probes via Cu(I)-catalyzed
Azide–Alkyne [3+2] Cycloaddition (CuAAC),3 the tagged
glycoproteins can be visualized and profiled on a proteome-
scale. Click-activated fluorogenic probes have widely emerged
as indispensable tools for labeling biomolecules,2c,d,4 as these
probes give lower background signals that are commonly
observed in many fluorochromes.5 Fluorogenic probes bearing
azido or alkynyl groups can form a 1,2,3-triazole ring under
CuAAC to trigger fluorescence.2c,d,5b,c Our previous studies
have demonstrated that peracetylated alkynyl sugar derivatives
of fucose (Fucyne) and N-acetylmannosamine (ManNAcyne)
can be incorporated into fucosylated and sialylated proteins,
respectively, where they are selectively labeled by fluorogenic
azido-1,8-naphthalimide or coumarin probes using CuAAC.2c,d
These click-activated probes can be used for visualizing and
quantitatively studying cellular glycoconjugates.
In this paper, we report the synthesis of trifunctional probes,6
which combines the features of labeling alkynyl group-tagged
biomolecules, generating fluorescence upon CuAAC, and
enabling affinity enrichment of labeled biomolecules, for testing
their applications in glycoproteomic analysis. These trifunc-
tional probes consist of three major components: an azido
group and click-activated fluorescent and biotin moieties.
Here we examine the applications of two trifunctional
probes with different linkers for their abilities in both target
detection via 3-azido-7-aminocoumarin and enrichment via
biotin, when reacting with alkynyl sugars (Fig. 1). Probe I
carries an azido group for both conjugation and fluorescent
triggering. When the 1,2,3-triazole ring was formed by
CuAAC, it activated the fluorescent property of 3-azido-7-
aminocoumarin to provide a high fluorescent signal for rapid
and sensitive target detection, while the biotin group in probe I
offered the advantage of glycoprotein enrichment. In order to
increase the elution yield of labeled targets that bind to
streptavidin matrices, we further introduced a cleavable linker
(cystamine) between 3-azido-7-aminocoumarin and biotin in
probe II. Thus after binding to streptavidin matrices, the
labeled targets can be recovered by treatment with reducing
agents such as dithiothreitol (DTT) or tris-(2-carboxyethyl)
phosphine (TCEP)7 to break the disulfide bond and give
fluorescent labeled biomolecules.
As shown in Scheme 1, the synthesis of probes I and II
started with two alkylations and formylation of 3-aminophenol
to give 1, which was further condensed with ethyl nitroacetate
under the Knoevenagel condensation reaction8 to yield 2. The
nitro group was subsequently reduced in presence of Lindlar
catalyst to give 3. The azido group was introduced to the C-3
position of 3 using tert-butyl nitrite (t-BuONO) and azidotri-
methylsilane (TMSN3) to yield 4,9 followed by treatment with
trimethylsilyl triflate (TMSOTf) and 2,6-lutidine to give 5.
Probes I and II were constructed by amide bond formation
between 5 and the TFA salt of the dioxaoctanediamine
derivative of biotin or the TFA salt of the cystamine derivative
of biotin in the presence of EDCI.
a Genomics Research Center, Academia Sinica, 128 Academia Road
Section 2, Nankang, Taipei 115, Taiwan.
E-mail: chwong@gate.sinica.edu.tw; Fax: +886-2-2785-3852;
Tel: +886-2-2789-9400
b Institute of Biochemical Science, National Taiwan University,
1 Roosevelt Road Section 4, Taipei 106, Taiwan
c Department of Chemistry, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
E-mail: wong@scripps.edu
The reactivity and click-activated fluorogenic property of
probes I and II were tested on ManNAcyne. As expected, the
ligation reaction produced a significant increase in fluores-
cence intensity (Fig. 2). The wavelength of maximum emission
w Electronic supplementary information (ESI) available: Synthetic
procedures for probes I and II, 2D-gel and table of identified proteins.
See DOI: 10.1039/c0cc00345j
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Fig. 3 Detection of ManNAcyne-labeled glycoproteins in PC3 cell
extracts. Lanes 1–4: Coomassie blue stain for visualizing total proteins
of each sample. Lanes 5–8: protein gel analyzed by fluorescence
imaging (Ex: 419 nm; Em: 490 Æ 25 nm).
Scheme 1 Synthesis of probes I and II. Reagents and conditions:
(a) CH3I, K2CO3, DMF, 100 1C, 60%; (b) tert-butyl bromoacetate,
2,6-lutidine, DMF, rt, 60%; (c) POCl3, DMF, rt, 95%; (d) ethyl
nitroacetate, piperidine, AcOH, n-butanol, reflux, 96%; (e) Lindlar
catalyst, H2, MeOH, rt; (f) tBuONO, TMSN3, CH3CN, rt, 65% in two
steps; (g) TMSOTf, 2,6-lutidine, CH2Cl2, rt; (h) the TFA salt of
dioxaoctanediamine derivative of biotin, EDCI, HOBT, TEA,
DMF, rt, 55% in two steps; (i) the TFA salt of cystamine derivative
of biotin, EDCI, HOBT, TEA, DMF, rt, 58% in two steps.
intensity generated from probe I was stronger than that from
probe II, which was similar to their corresponding fluorescent
moieties alone.
We also tested whether these trifunctional probes were able to
enrich sialylated proteins. Probe-labeled ManNAcyne-treated cell
extracts were incubated with streptavidin beads. After removing
unbound proteins, probe I- and probe II-labeled proteins were
eluted with 1 mM biotin and 50 mM TCEP, respectively. Our
results showed that the elution yield of probe I-labeled lysate was
less than 1% and the elution yield of probe II-labeled lysate was
more than 90%. We observed specific enrichment and efficient
elution of sialylated proteins labeled by probe II (Supporting
Information Fig. S1w). In summary, probe II is more suitable for
the enrichment of sialylated proteins.
We further analyzed the enriched glycoproteins by 2D
electrophoresis. Labeled glycoproteins from the cells fed with
ManNAcyne showed specific fluorescent signals compared to
the cells fed with control ManNAc (Fig. 4). The results showed
detectable protein spots in the ManNAcyne-, but not the
ManNAc-treated sample, indicating that the enrichment by
probe II is specific. Comparing the signals given by probe II
and protein stain, the spots can be classified as either high or
low sialic acid/protein ratios. According to this, we picked up
five spots (two and three with high or lower sialic acid/protein
ratio, respectively) for protein identification by LC-MS/MS
(Supporting Information Table S1w). Among these five spots,
two were predicted to have potential glycosylation sites (spots
2 and 4) and three have been proved to be glycoproteins (spots
1, 3, and 5),10 validating that probe II enabled the enrichment
of glycoproteins appended with sugar analog(s). Here, the
results demonstrated that our trifunctional probing system is
useful for glycoproteomic study.
Fig. 2 The fluorescence spectra of probe I and probe II and their click
products TA-probe I and TA-probe II.
for both probes I and II was 495 nm when excited at 430 nm,
whereas the parent compounds of probes I and II gave very
low fluorescence under this setting (Fig. 2a and b). TA-probe I
and TA-probe II (reaction products with triazole rings) displayed
46-fold and 18-fold fluorescence enhancement compared to
probe I and probe II, respectively. The spectral properties of
probes I and II, and TA-probes I and II were similar to those
of the corresponding 3-azido-7-diethylaminocoumarin and
their CuAAC adducts (the compounds without biotin moiety),
respectively.5b
We next examined whether these probes could be applied
to analyze sialylated glycoproteomes. After feeding with
ManNAcyne or control ManNAc, prostate cancer PC3 cells
were lysed for harvesting their protein extracts. The soluble
fractions of lysates were next labeled with probe I or probe II
and then separated by gel electrophoresis. The fluorescence
imaging of protein gels showed specific signals in the lysates
derived from PC3 cells fed with ManNAcyne (Fig. 3, lanes
6 and 8) but not ManNAc (Fig. 3, lanes 5 and 7). The signal
In conclusion, we have developed and synthesized two
trifunctional probes. Notably, probe II is useful for the
efficient enrichment and detection of alkynyl sugar-tagged
biomolecules (e.g., sialylated proteins). Therefore, this trifunc-
tional probe can be a powerful tool for use in profiling
and comparing low-abundant glyco-biomolecules at different
physiological/pathological stages for identification of glycan-
related biomarkers and targets, for example, in cancer cells.
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Fig. 4 After reaction of probe II with PC3 cell lysate was resolved in
2D electrophoresis (A) fluorescence image (left) and silver stain (right):
enriched PC3 cell lysate treated with ManNAcyne then clicked with
probe II. (B) Fluorescence image (left) and silver stain (right): enriched
PC3 cell lysate clicked with probe II, PC3 cell (Ex: 419 nm; Em:
490 Æ 25 nm).
We thank Dr Chung-Yi Wu for critical comments on this
manuscript, and Chien-Hung Chen for technical support on
MS analysis.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 5575–5577 | 5577