Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition

Article abstract of DOI:10.1021/ja1083909

The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na ₂S₂O₄, 2% HOCH₂CH₂SH, 10% HCO₂H, 95% CF₃CO₂H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO ₂H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.

Full text of DOI:10.1021/ja1083909

Published on Web 12/08/2010
Cleavable Biotin Probes for Labeling of Biomolecules via
Azide-Alkyne Cycloaddition
Janek Szychowski,† Alborz Mahdavi,† Jennifer J. L. Hodas,‡ John D. Bagert,†
John T. Ngo,† Peter Landgraf,§ Daniela C. Dieterich,‡,§ Erin M. Schuman,‡,| and
David A. Tirrell*,†
DiVision of Chemistry and Chemical Engineering and DiVision of Biology, California
Institute of Technology, 1200 East California BouleVard, Pasadena, California 91125,
Research Group Neuralomics, Leibniz-Institute for Neurobiology, Brenneckestrasse 6,
39118 Magdeburg, Germany, and Max Planck Institute for Brain Research,
Deutschordenstrasse 46, 60528 Frankfurt, Germany
Received September 16, 2010; E-mail: tirrell@caltech.edu
Abstract: The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins,
nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity
purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled
biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins
and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled
protein, these probes are subject to cleavage with either 50 mM Na2S2O4, 2% HOCH2CH2SH, 10% HCO2H,
95% CF3CO2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane
(DADPS) linker was found to be cleaved efficiently when treated with 10% HCO2H for 0.5 h. A model
green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective
conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features
make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.
Introduction
selective identification of newly synthesized proteins in cells.
In the BONCAT approach, newly synthesized proteins are
The azide-alkyne cycloaddition is selective, efficient, and
broad in scope: a paradigmatic example of a “click” reaction.1
The discovery that copper catalysis allows the cycloaddition to
be effected under mild conditions2,3 has stimulated numerous
studies of labeling of proteins,4,5 glycans,6,7 lipids,8,9 and RNA.10
For example, we recently reported the Bio-orthogonal Non-
Canonical Amino Acid Tagging4 (BONCAT) method for
distinguished from the preexisting pool of proteins by cotrans-
lational incorporation of a reactive noncanonical amino acid
(e.g., L-homopropargylglycine (Hpg, 1), Figure 1). Azide or
alkyne functional groups on the side chains of such noncanonical
amino acids11,12 can serve as bio-orthogonal handles for selective
conjugation to affinity probes through the copper-catalyzed
azide-alkyne cycloaddition reaction.13 Once conjugated, newly
synthesized proteins can be selectively enriched through con-
ventional affinity purification protocols that exploit the biotin-
streptavidin interaction. Proteins are released from the resin and
identified by mass spectrometry. Azide or alkyne functional
groups incorporated into proteins can also be used for cell-
selective labeling of newly synthesized proteins.14,15
† Division of Chemistry and Chemical Engineering, California Institute
of Technology.
‡ Division of Biology, California Institute of Technology.
§ Leibniz-Institute for Neurobiology.
| Max Planck Institute for Brain Research.
(1) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
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Chem., Int. Ed. 2002, 41, 2596–2599.
Avidin-based isolation of biotin-labeled proteins has found
widespread use; however, the strength of the biotin-avidin
interaction (Kd ∼ 10-15 mol/L) can make recovery of proteins
from affinity resins challenging. Conventional methods to elute
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Szychowski et al.
Figure 1. Schematic representation of the BONCAT method.
biotinylated proteins from immobilized avidin include the
following: (i) denaturation of streptavidin by boiling the resin
in a denaturing buffer16 that may include high concentrations
of chaotropic salts, (ii) trypsin digestion of proteins while they
are bound to the resin,17 or (iii) elution of proteins with excess
free biotin.18 These protocols can coelute contaminant proteins
by releasing nonspecifically bound proteins and/or naturally
biotinylated proteins concurrently with labeled proteins.19 In
addition, some of these methods can cause elution of high levels
of resin-based peptides along with the proteins of interest,
resulting in further sample contamination. For these reasons,
additional purification techniques can be required prior to
analysis by mass spectrometry. Furthermore, most biotin probes
require “spacers” to ensure accessibility of the biotin to the
avidin on the resin;20 thus these probes can be quite large
molecules. Ideally, any modification of the proteins of interest
should be small to facilitate protein identification by mass
spectrometry. These challenges have prompted the proteomics
community to explore the use of cleavable probes.21-25
between the cleavable moiety and biotin, and (v) the biotin
(Figure 2). In view of the importance of the copper-catalyzed
azide-alkyne cycloaddition in bioconjugation,4-10 we designed
a set of azide-functional, cleavable biotin probes, and we sought
to compare the performance of different cleavable moieties
embedded between the azide group and the biotin. An ideal
probe would leave behind a small modification on the protein
that would not complicate mass spectrometry; the spacer
between the azide and the cleavable moiety should be designed
accordingly. The probe should be cleavable under conditions
that are mild enough to avoid release of streptavidin or of
proteins nonspecifically bound to the resin, and that do not
modify the proteins in any way. Cleavage must also allow rapid,
efficient protein recovery in a manner that is compatible with
downstream analytical procedures.
Cleavable biotin probes often consist of five parts: (i) the
reactive handle, (ii) a spacer between the reactive handle and
the cleavable moiety (iii) the cleavable moiety, (iv) a spacer
Figure 2. Elements of a generic cleavable biotin probe.
Several cleavable biotin probes have been reported previously,
including the commercially available acid-cleavable probe used
in the Cleavable Isotope Coded Affinity Tag (cICAT) method.21
Other probes have used disulfides,22 diazobenzenes that are
cleavable under reducing conditions,15,23 or photocleavable
linkers.24 In order to design the most effective probe for use in
bioconjugation via the azide-alkyne cycloaddition, we explored
all of these cleavage approaches. In addition, we were tempted
by the acid- and fluoride-sensitive silicon-oxygen bond found
in a variety of protecting groups used in organic synthesis26 as
well as in the cleavable motifs used in solid phase synthesis.27
Five cleavable biotin probes of the general structure shown in
Figure 2, all bearing an azide group as the reactive site for
bioconjugation, were prepared. Probe performance was evalu-
ated by examining conjugation to and release from a test protein
(16) For example, see Leighton, B. H.; Seal, R. P.; Shimamoto, K.; Amara,
S. G. J. Biol. Chem. 2002, 277, 29847–29855.
(17) For example, see Dieterich, D. C.; Lee, J. J.; Link, A. J.; Graumann,
J.; Tirrell, D. A.; Schuman, E. M. Nat. Protoc. 2007, 2, 532–540.
(18) For example, see Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol.
2001, 19, 379–382.
(19) Fonovic, M.; Verhelst, S. H. L.; Sorum, M. T.; Bogyo, M. Mol. Cell.
Proteomics 2007, 6, 1761–1770.
(20) Hofmann, K.; Titus, G.; Montibeller, J. A.; Finn, F. M. Biochemistry
1982, 21, 978–984.
(21) Fauq, A. H.; Kache, R.; Khan, M. A.; Vega, I. E. Bioconjugate Chem.
2006, 17, 248–254.
(22) (a) Shimkus, M.; Levy, J.; Herman, T. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 2593–2597. (b) Merel, A.; Nessen, M. A.; Kramer, G.; Back,
J. W.; Baskin, J. M.; Smeenk, L. E. J.; de Koning, L. J.; van
Maarseveen, J. H.; de Jong, L.; Bertozzi, C. R.; Hiemstra, H.; de
Koster, C. G. J. Proteome Res. 2009, 8, 3709–3711.
(23) Verhelst, S. H. L.; Fonovic, M.; Bogyo, M. Angew. Chem., Int. Ed.
2007, 46, 1284–1286.
(24) (a) Olejnik, J.; Sonar, S.; Krzymanska-Olejnik, E.; Rothschild, K. J.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7590–7594. (b) Kim, H. H.;
Tallman, K. A.; Liebler, D. C.; Porter, N. A. Mol. Cell. Proteomics
2009, 8, 2080–2089.
(26) Wuts, P. G. M.; Greene, T. W. ProtectiVe Groups in Organic Synthesis,
4th ed.; John Wiley & Sons Inc.: Hoboken, NJ, 2007.
(27) Guillier, F.; Orain, D.; Bradley, M. Chem. ReV. 2000, 100, 2091–
2157.
(25) Park, K. D.; Liu, R.; Kohn, H. Chem. Biol. 2009, 16, 763–772.
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Cleavable Biotin Probes
A R T I C L E S
Scheme 1. Synthesis of a Photocleavable Biotin Probea
features make the DADPS probe especially attractive for use
in proteomics and other biomolecular labeling studies.
Results and Discussion
Synthesis of Cleavable Probes. In order to prepare a photo-
cleavable biotin probe, the methyl ketone 324 was obtained in
three steps from commercially available 5-methyl-2-nitrobenzoic
acid (2) (Scheme 1). Radical bromination at the benzylic position
followed by azide displacement gave compound 4 in 75%
overall yield. Reduction of the ketone by NaBH4 and reduction
of the azide under Staudinger conditions28 afforded compound
5. Amide formation using the commercially available NHS-
LC-biotin followed by derivatization of the benzylic alcohol
by treatment with disuccinimidyl carbonate and 3-azidopropyl-
amine afforded the desired photocleavable biotin probe (6).
We then turned our attention to the diazobenzene motif, which
is cleavable via reduction with sodium dithionite (Na2S2O4).
Compound 823 was prepared in two steps from 4-aminobenzoic
acid (7, Scheme 2). Amide formation with 3-azidopropylamine
introduced the azide handle, and following 9-fluorenylmethyl-
oxycarbonyl (Fmoc) deprotection with piperidine, the liberated
amine was coupled with NHS-LC-biotin to afford the desired
cleavable biotin probe (9).
a Reagents and conditions: (a) SOCl2; (b) Mg, diethyl malonate; (c) HCl,
83%, for three steps; (d) NBS, (BzO)2, 75%; (e) NaN3, quantitative; (f)
NaBH4, 91%; (g) PPh3, H2O, 70%; (h) NHS-LC-biotin, 80%; (i) DSC, then
3-azidopropylamine, 70%.
(green fluorescent protein, GFP) bearing a single copy of the
noncanonical amino acid 1. One probe, designed around the
acid-sensitive dialkoxydiphenylsilane (DADPS) motif, was
cleaved under especially mild conditions (10% HCO2H, 0.5 h).
This probe exhibits high labeling selectivity and leaves a small
(143 Da) mass tag on the labeled protein after cleavage. These
The acid and fluoride sensitivities of the silicon-oxygen bond
commonly found in cleavable protecting groups used in organic
synthesis can be tuned by varying the extent of steric hindrance
around the silicon atom.26 In designing a cleavable silane probe,
we placed two phenyl substituents on silicon, reasoning that
Scheme 2. Synthesis of a Na2S2O4 Cleavable Biotin Probea
a Reagents and conditions: (a) NaNO2, HCl; (b) tyramine, NaHCO3, then Fmoc-Cl, 55%, for two steps; (c) EDC, 3-azidopropylamine; (d) piperidine,
20%, for two steps; (e) NHS-LC-biotin, 80%.
Scheme 3. Synthesis of the DADPS Biotin Probea
a Reagents and conditions: (a) 1-amino-2-methyl-propan-2-ol, 85%; (b) Et3N, DCDPS, then 6-azidohexanol, 55%.
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Szychowski et al.
Scheme 4. Synthesis of Disulfide Biotin Probesa
Biotinylated proteins 19a-19e were obtained by treatment
of 18 with each of the biotin probes (6, 9, 12, 14a, and 16).
The Met form of the protein (17) was also treated under the
same reaction conditions with every probe as a selectivity
control. Western blot analysis with fluorescent anti-His antibody
was used as a measure of the total protein content of each
sample; fluorescent streptavidin was used to visualize biotiny-
lated protein. Comparison of the streptavidin signals obtained
from the control protein 17 with those from proteins 19a-19e
allowed quantitative comparison of the selectivity of the probes
(Figure 3 and Table 1). Probe 14a was significantly less selective
than the others (see Figure 3, lane 10b), likely due to disulfide
exchange with one of the three cysteine residues on the model
protein. The relative selectivity of probe 14a was therefore
arbitrarily designated as unity (see Table 1). Probes 6, 9, and
16 were of comparable selectivity, showing roughly 10-fold less
nonspecific labeling than probe 14a. Silane probe 12 was
remarkably more selective (see Table 1), exhibiting selectivity
2 orders of magnitude higher than that of 14a.
To determine whether cysteine alkylation prior to click
chemistry with probe 14b would reduce nonspecific protein
labeling by disulfide probes, we evaluated a reduction-alkylation
protocol widely used in mass spectrometry. Disulfide bonds were
reduced with ꢀ-mercaptoethanol, and the reduced samples were
alkylated with iodoacetamide. Using Western blot analysis, we
compared the biotinylation signal from COS7 cell lysates
incubated for 2 h in either Met or azidohomoalanine (Aha, 21)
and then alkylated, or not, prior to click chemistry with probe
14b. Addition of the alkylation step resulted in substantial
reduction of the biotin signal in the Met sample. The biotiny-
lation signal from the Aha sample, however, was not changed
by the alkylation step, suggesting that most, if not all, of the
biotinylation signal in Aha samples is derived from conjugation
to Aha residues (Figure 4). The orientation of the click reaction,
(i.e., whether the alkyne functionality was on the probe or on
the protein), did not affect the efficiency of the click chemistry
(see Supporting Information).
Labeled proteins 19a-19e were subjected to cleavage condi-
tions to yield 20a-20e (Scheme 6). Previously reported
conditions for cleavage of each motif were used.21-23 From a
comparison of streptavidin signals before (samples 19a-19e)
and after (samples 20a-20e) cleavage, the cleaving efficiency
for each probe was calculated as a percentage of the initial
streptavidin signal that remained after cleavage (Figure 3 and
Table 1). Probe 9 was the least efficiently cleaved (see Figure
3, lane 6b) with 23.5 ( 5.7% of the streptavidin signal remaining
after one treatment with 50 mM Na2S2O4 for 1 h. Successive
treatments with Na2S2O4 might have resulted in more complete
cleavage,30 but we were interested in comparing the probes after
a single exposure to the cleaving reagent. The streptavidin signal
remaining after cleavage was less than 3% for all of the other
probes (6, 12, 14a, 16). Remarkably, cleavage of the DADPS
probe 12 was efficient even upon treatment with 10% formic
acid. In contrast, the other acid-cleavable probe (16) required
treatment with 95% trifluoroacetic acid.
a Reagents and conditions: (a) 3-azidopropylamine, 80%; (b) propargy-
lamine, 81%.
smaller groups might render the probe prone to premature
cleavage. For the same reason, a tertiary alkoxy group was
placed on the biotin side of the silicon atom. The acid stability
of the dialkoxydiphenylsilane motif has been reported to increase
by 2 orders of magnitude upon replacement of a methoxy group
by a tert-butoxy substituent.29 The biotin NHS ester 10 was
treated with 1-amino-2-methylpropan-2-ol to afford the tertiary
alcohol 11 (Scheme 3). The latter was treated first with excess
dichlorodiphenylsilane (DCDPS) and then with excess 6-azi-
dohexanol to afford the acid cleavable biotin probe 12 after silica
gel chromatography.
The azido disulfide biotin probe 14a was prepared by treating
the commercial NHS ester biotin derivative 13 with 3-azidopro-
pylamine (Scheme 4). An alkynyl disulfide probe (14b) was
prepared by treatment of 13 with propargylamine. Acid-
cleavable probe 16 was prepared by treating the commercially
available iodoacetamide biotin derivative 15 of undisclosed
structure with 3-azidopropanethiol (Scheme 5).
Evaluation of Cleavable Probes. With the biotin probes in
hand, we proceeded to assess the labeling and cleaving
efficiencies of each probe with a model protein. A His-tagged
GFP 17 was engineered to contain a single methionine (Met)
site, at the initiator position (Scheme 6); replacement of Met
by 1 generated the alkyne-labeled protein 18. Use of a model
protein bearing a single alkyne site reduced complications that
might arise from multiple labeling and cleaving events. The
model protein was obtained by established protocols for in ViVo
synthesis of proteins containing noncanonical amino acids.17
Briefly, a Met-auxotrophic strain of Escherichia coli harboring
a plasmid coding for IPTG-inducible expression of the model
protein was grown in M9 medium supplemented with all 20
canonical amino acids and ampicillin. Upon reaching a cell
density corresponding to OD600 ) 1.0, cells were collected by
centrifugation and washed twice in chilled 0.9% NaCl solution.
Following resuspension in M9 medium supplemented with 1
mM 1 and all 20 canonical amino acids except Met, protein
synthesis was induced with IPTG. Following expression, His-
tagged GFP was isolated by nickel-affinity chromatography
under native conditions. Mass spectrometric analysis of protein
18 showed a single peak at 28 092 Da, indicating near-
quantitative replacement of Met by 1; no signal was detected
at 28 114 Da, the mass of the Met form of the protein (see
Supporting Information).
The acid sensitivity of probe 12 was evaluated by treatment
of labeled GFP with aqueous solutions of formic acid of
increasing concentration. Use of 10% HCO2H for 0.5 h at room
temperature resulted in >98% cleavage (Figure 5). Treatment
with 5% HCO2H cleaved the probe with 95% efficiency, see
(28) Gololobov, Y. G.; Kasukhin, L. F. Tetrahedron 1992, 48, 1353–1406.
(29) Gillard, J. W.; Fortin, R.; Morton, H. E.; Yoakim, C.; Quesnelle, C. A.;
Daignault, S.; Guindon, Y. J. Org. Chem. 1988, 53, 2602–2608.
(30) Denny, J. B.; Blobel, G. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 5286–
5290.
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A R T I C L E S
Scheme 5. Synthesis of the Acid Cleavable Biotin Probea
a Reagents and conditions: (a) 3-azidopropanethiol, Et3N, quantitative.
Scheme 6. Protein Labeling and Probe Cleavage
Table 1). No significant cleavage of probe 12 was observed
upon treatment with aqueous KF solutions of concentrations
up to 5 M (data not shown).
DADPS probe 12 was examined further with the objective
of developing optimized procedures for enrichment of Hpg-
tagged proteins. A GFP variant containing seven Hpg residues
was used in this experiment to enable direct observation of
labeled amino acid residues by mass spectrometry.31 The protein
was treated with probe 12 under the conditions recommended
by Finn and co-workers for copper-catalyzed azide-alkyne
bioconjugation.32 After successful click reaction, two additional
proteins were added to serve as positive and negative controls
for binding to the streptavidin resin. Bovine serum albumin
(BSA) was used as negative control to assess the removal of
nonspecifically bound proteins from the resin. Biotinylated
horseradish peroxidase (HRP) was used as a positive control
(31) Yoo, T. H.; Tirrell, D. A. Angew. Chem., Int. Ed. 2007, 46, 5340–
5343.
(32) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int.
Ed. 2009, 48, 9879–9883.
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Figure 3. Anti-His (a) and streptavidin (b) signals to determine the labeling and cleaving efficiencies of each probe. Lane 1, 17 + 6 treated with click
conditions; lane 2, 19a; lane 3, 20a; lane 4, 17 + 9 treated with click conditions; lane 5, 19b; lane 6, 20b; lane 7, 17 + 12 treated with click conditions; lane
8, 19c; lane 9, 20c; lane 10, 17 + 14a treated with click conditions; lane 11, 19d; lane 12, 20d; lane 13, 17 + 16 treated with click conditions; lane 14, 19e;
lane 15, 20e.
Table 1. Labeling and Cleaving Efficiencies Calculated for Each Probea
probe
6
9
12
14a
16
relative selectivityb
biotin remaining after
cleavage (%)
14.5 ( 2.2
1.1 ( 0.4
14.7 ( 3.4
23.5 ( 5.7
117.5 ( 18.3
1.6 ( 0.4 (5.4 ( 1.3)
1.0 ( 0.2c
2.4 ( 0.3
10.8 ( 2.5
2.5 ( 0.9
cleaving conditions
hν 365 nm, 30 min
Na2S2O4, 50 mM, 1 h
219
10% HCO2H, 0.5 h
(5% HCO2H, 0.5 h)
143
HO(CH2)2SH 2%, 1 h
188
CF3CO2H 95%, 2 h
344
MWd left behind (Da)
100
a All streptavidin and anti-His fluorescence signals were measured on a GE Healthcare Typhoon Trio Variable Mode Imager by integrating the peak
corresponding to the green fluorescent protein molecular weight. All streptavidin signals were normalized to the corresponding anti-His signals. All
numbers are based on triplicate analysis. b Relative selectivity ) (19a-19e streptavidin signal/19a-19e anti-His signal)/(17 streptavidin signal/17
anti-His signal), where the signals associated with 17 are those observed after treatment with the cleavable probe. c 14a relative selectivity was
arbitrarily designated as unity. d MW ) molecular weight.
Figure 5. Anti-His (a) and streptavidin (b) signals for the cleavage of probe
12 with 10% and 5% formic acid. Lane 1, 19c not treated with any acid
after 5 h; lane 2, 19c treated with 10% HCO2H for 0.5 h; lane 3, 19c treated
with 5% HCO2H for 0.5 h.
SDS to remove acid and four times with 1% SDS in PBS. These
elution fractions were combined. Finally, the resin was boiled
for 10 min in 2% SDS in PBS.
Figure 6A shows the results of the affinity purification of
Figure 4. Alkylation prior to click chemistry significantly reduces
7-Hpg-GFP from a solution containing BSA and biotinylated
HRP. As expected, 7-Hpg-GFP treated with probe 12 was bound
to the resin throughout all washes, and then efficiently recovered
after treatment with acid. Subsequent boiling of the resin with
2% SDS in PBS released no additional 7-Hpg-GFP, indicating
that the majority of the protein was eluted following acid
cleavage of the probe. In contrast, most of the biotinylated HRP
was recovered only by boiling the resin in 2% SDS. These
results show that Hpg-tagged proteins can be separated cleanly
from both nonspecifically bound (i.e., nonbiotinylated) and
endogenously biotinylated proteins.
Probe 12 was used to enrich Hpg-tagged GFP from a bacterial
cell lysate. 7-Hpg-GFP was added to a lysate of E. coli strain
DH10B cells; the lysate was then subjected to conjugation with
probe 12. The wash conditions described above were used to
remove nonspecifically bound proteins; the remaining bound
proteins were then eluted with 5% formic acid. Figure 6B shows
nonspecific protein labeling by probe 14b. COS7 cells incubated in either
Met or Aha were lysed and either not alkylated (lanes 1 and 2) or alkylated
(lanes 3 and 4) prior to reaction with 14b. Western blots were visualized
using an antibody against biotin.
for resin binding, and also served to test if treatment with 5%
formic acid would elute endogenous biotinylated proteins
commonly found in cellular proteomes. After click chemistry,
excess 12 was removed with a PD10 salt exchange column,
and proteins were resuspended in phosphate buffered saline
(PBS) at pH 7.4 with 0.1% SDS and treated with streptavidin
beads. After binding, the beads were washed sequentially with
1% SDS in PBS, 6 M urea in 250 mM ammonium bicarbonate,
and 1 M NaCl in PBS to remove nonspecifically bound proteins.
The last wash contained 0.1% SDS in water to remove
remaining salts from the resin before acid elution. Probe 12
was cleaved by incubating the resin in 5% formic acid in water
for 2 h. The resin was washed twice with water containing 0.1%
9
18356 J. AM. CHEM. SOC. VOL. 132, NO. 51, 2010

Cleavable Biotin Probes
A R T I C L E S
be of general interest with respect to the design of probes with
other reactive groups used in biomolecular labeling studies.
Experimental Section
Synthesis. General. Unless stated otherwise, reactions were
performed in flame-dried glassware under an argon or nitrogen
atmosphere using dry solvents. Commercially obtained reagents
were purchased from Sigma-Aldrich and used as received, unless
otherwise noted. NHS-LC-biotin was obtained from Toronto
Research Chemicals, homopropargylglycine (1) was purchased from
Chiralix Inc., compounds 10 and 13 were obtained from Pierce
Protein Research Products, and compound 15 was obtained from
Applied Biosystems. Thin-layer chromatography was performed
using Baker-Flex silica gel IB-F precoated plates and visualized
by UV fluorescence quenching, potassium permanganate, ceric
ammonium molybdate staining, or, when the biotin moiety was
present, with p-dimethylaminocinnamaldehyde33 staining solution.
EMD Silica gel 60 (particle size 0.040-0.063 mm) was used for
flash chromatography. 1H and 13C NMR spectra were recorded on
Varian instruments and chemical shifts are reported relative to
Me4Si. Data for 1H NMR spectra are reported as follows: chemical
shift (ppm), integration, multiplicity, and coupling constant (Hz).
Multiplicity and qualifier abbreviations are as follows: s ) singlet,
d ) doublet, t ) triplet, q ) quartet, m ) multiplet, comp )
complex, br ) broad, and app ) apparent. Low- and high-resolution
mass spectra were obtained from the Mass Spectrometry Facility
in the Division of Chemistry and Chemical Engineering at Caltech.
1-(5-Methyl-2-nitrophenyl)ethanone (4). Compound 324 (3.51
g, 19.6 mmol), N-bromosuccinimide (3.66 g, 20.6 mmol), and
benzoyl peroxide (475 mg, 1.96 mmol) were refluxed in CC14 (50
mL) overnight. Benzoyl peroxide (238 mg, 0.98 mmol) was added.
After 8 h, the precipitate was filtered off, the filtrate was evaporated
under reduced pressure, and the residue was dissolved in EtOAc.
The organic phase was washed with saturated NaHCO3 and with
brine, and dried over Na2SO4. After the solvent was evaporated
under reduced pressure, purification by silica gel chromatography
using a 10-20% gradient of EtOAc in hexane gave the benzylic
Figure 6. Polyacrylamide gel analysis of affinity purification of 7-Hpg-
GFP. (A) After click reaction with probe 12, affinity purification from
a solution containing BSA (band I), biotinylated HRP (band II), and
7-Hpg-GFP (band III), was performed on a streptavidin agarose resin.
Wash 1 was performed with 1% SDS in PBS, wash 2 was performed
with 6 M urea in 250 mM ammonium bicarbonate, wash 3 was performed
with 1 M NaCl in PBS, and wash 4 was performed with 0.1% SDS in
water to remove PBS. Elution with 5% formic acid was followed by
boiling of resin in 2% SDS in PBS to remove remaining proteins. (B)
Affinity purification of 7-Hpg-GFP from a DH10B lysate. Wash and
elution conditions were identical to those in (A). Gels were stained with
Coomassie Brilliant Blue.
the result. The GFP lane shows the position of GFP before probe
conjugation. The elution lane shows clean enrichment of GFP
from the complex lysate. After boiling the resin with 2% SDS,
we did not observe elution of more GFP, indicating efficient
cleavage of 12 and elution of proteins upon treatment with 5%
formic acid. The absence of significant bands corresponding to
lysate proteins in both elution fractions indicates the specificity
of the enrichment strategy.
Tandem mass spectrometry was used to verify directly the
conjugation and cleavage of probe 12. Peptides resulting from
tryptic digestion of eluted proteins were desalted by HPLC prior
to analysis by mass spectrometry. Peptides derived from the
control protein (7-Met-GFP) showed mass shifts of +16 Da,
consistent with oxidation of Met. Peptides derived from 7-Hpg-
GFP showed shifts of +121 Da, corresponding to replacement
of Met by Hpg and subsequent conjugation and cleavage of 12
at Hpg sites (see Supporting Information). The use of formic
acid to cleave probe 12 works well for mass spectrometry by
reducing sample complexity and the need for gel extraction.
The small mass modification resulting from attachment and
cleavage of the probe can serve as an aid in identification of
Hpg-tagged proteins.
1
bromide (3.61 g, 75%). H NMR (300 MHz, CDCl3), δ (ppm):
8.13-7.91 (1H, m), 7.62 (1H, ddd, J ) 20.21, 8.50, 2.00 Hz),
7.49-7.37 (1H, m), 4.55-4.41 (2H, m), 2.65-2.37 (3H, m). 13C
NMR (75 MHz, CDCl3), δ (ppm): 199.3, 144.8, 138.4, 131.0-124.8
(m, 4C), 77.2, 30.3. m/z calcd for C9H8BrNO3 [M + H]+: 257.9766.
MS found: 257.0. HRMS found: 257.9773. The benzylic bromide
(2.00 g, 7.75 mmol) was dissolved in acetone:H2O (5:1 by volume,
50 mL). Sodium azide (756 mg, 11.6 mmol) was added, and the
mixture was heated to 75 °C overnight in a flask equipped with a
reflux condenser. After acetone evaporation under reduced pressure,
the aqueous phase was extracted with EtOAc, washed with brine,
and dried with Na2SO4, and the solvent was evaporated under
reduced pressure. Purification by silica gel chromatography using
a 10-20% gradient of EtOAc in hexane gave compound 4 (1.65
Conclusion
1
g, quantitative). H NMR (300 MHz, CDCl3), δ (ppm): 8.10 (1H,
d, J ) 8.44 Hz), 7.54 (1H, dd, J ) 8.44, 1.92 Hz), 7.35 (1H, d, J
) 1.89 Hz), 4.52 (2H, s), 2.61-2.50 (3H, m). 13C NMR (75 MHz,
CDCl3), δ (ppm): 199.5, 142.9, 138.4, 129.6-124.6 (m, 4C), 53.3,
30.1. m/z calcd for C9H8N4O3 [M + H]+: 221.0675. MS found:
211.3. HRMS found: 221.0679.
Five cleavable biotin probes were synthesized and their
protein labeling selectivity and cleaving efficiencies were
compared. Probe 12, designed around the acid-sensitive DADPS
motif, was found to be the most selective. Efficient cleavage
under mild conditions (10% formic acid, 0.5 h) and the small
(143 Da) molecular fragment left on the labeled protein
following cleavage make this probe especially attractive for use
in proteomic studies. Probes 6, 9, 14, and 16 labeled less
selectively than 12 in the experiments reported here, but may
prove useful in analyses of other protein populations. The
selectivity of the disulfide probes 14 was improved by a simple
reduction-alkylation protocol that is compatible with proteomic
analysis by mass spectrometry. The reactive probes described
here are azides because we are interested in bioconjugation
through the azide-alkyne cycloaddition, but the results should
1-(5-(Aminomethyl)-2-nitrophenyl)ethanol (5). Compound 4
(1.50 g, 6.81 mmol) was dissolved in MeOH:dioxane (3:2 by
volume, 30 mL), and NaBH4 (378 mg, 10 mmol) was added slowly.
After 30 min, water (50 mL) and 2 M HCl (1 mL) were added and
the suspension was extracted twice with EtOAc, washed with brine,
dried over Na2SO4, and evaporated under reduced pressure and light
protection. The resulting brown oil was purified by silica gel
chromatography using a 10-20% gradient of EtOAc in hexane to
1
give the desired alcohol as a pale yellow oil (1.38 g, 91%). H
NMR (300 MHz, CDCl3), δ (ppm): 7.89 (1H, dd, J ) 12.06, 8.10
(33) McCormick, D. B.; Roth, J. A. Anal. Biochem. 1970, 34, 226–236.
9
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A R T I C L E S
Szychowski et al.
Hz), 7.79 (1H, dd, J ) 8.48, 5.24 Hz), 7.36 (1H, dd, J ) 8.37,
2.00 Hz), 5.42 (1H, dd, J ) 12.64, 6.33 Hz), 4.47 (2H, s),
1.63-1.43 (3H, m). 13C NMR (75 MHz, CDCl3), δ (ppm): 141.8,
127.4-124.9 (m, 5C), 65.5, 53.9, 24.3. m/z calcd for C9H10N4O3
[M + H]+: 223.0831. MS found: 222.6. HRMS found: 223.0831.
To a solution of the alcohol (1.00 g, 4.54 mmol) in THF (30 mL),
PPh3 (1.31 g, 5.00 mmol) and H2O (0.5 mL) were added and the
mixture was heated at 60 °C for 4 h. Evaporation of the solvent
under reduced pressure gave a residue that was dissolved in CH2Cl2
and purified by silica gel chromatography with 5-15% MeOH in
CH2Cl2 to give compound 5 (612 mg, 70%). 1H NMR (300 MHz,
CDCl3), δ (ppm): 7.92-7.72 (2H, m), 7.30 (1H, dd, J ) 8.35, 1.88
Hz), 5.39 (1H, q, J ) 6.32, 6.32, 6.32 Hz), 4.01-3.83 (2H, m),
1.67-1.37 (3H, m). 13C NMR (75 MHz, CDCl3), δ (ppm): 147.9,
142.2, 126.1-124.4 (m, 4C), 64.8, 45.2, 24.4. m/z calcd for
C9H12N2O3 [M + H]+: 197.0926. MS found: 196.8. HRMS found:
197.0931.
chromatography with a 5-10% gradient of MeOH in CH2Cl2 to
1
give compound 9 (92 mg, 80%). H NMR (300 MHz, MeOH), δ
(ppm): 8.82-8.55 (m, 1H), 8.02 (m, 4H), 7.93-7.79 (m, 1H),
7.79-7.65 (m, 1H), 7.56 (s, 1H), 7.42-7.16 (m, 1H), 7.16-6.89
(m, 1H), 6.39 (d, J ) 19.06 Hz, 2H), 4.39-4.18 (m, 1H), 4.18-3.99
(m, 1H), 3.71-3.15 (m, 4H), 2.95 (m, 4H), 2.62 (m, 6H), 2.01 (m,
4H), 1.79 (m, 2H), 1.34 (m, 8H). 13C NMR (75 MHz, MeOH), δ
(ppm): 173.5, 172.6, 166.3, 163.4, 154.2 153.6, 138.9, 136.9, 135.0,
131.6-129.1 (m, 5C), 123.1, 118.9, 61.7, 59.9, 56.1, 49.2, 37.5,
36.1, 35.9, 34.8, 29.1-28.7 (m, 4C), 26.8, 26.0-25.8 (m, 3C). m/z
calcd for C34H46N10O5S [M + H]+: 707.3452. MS found: 731.1
(M + Na). HRMS found: 707.3462.
N-(2-Hydroxy-2-methylpropyl)-1-(5-((3aS,4S,6aR)-2-oxo-
hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9,12-
tetraoxapentadecan-15-amide (11). To a solution of compound
10 (150 mg, 0.255 mmol) in CH2Cl2 (5 mL), 1-amino-2-methyl-
propan-2-ol (27 mg, 0.31 mmol) was added followed by Et3N (42
µL, 0.31 mmol). After 4 h, the solvent was evaporated under
reduced pressure and the residue was taken up in CH2Cl2 and
purified by silica gel chromatography with a 15-20% gradient of
1-(2-Nitro-5-((6-(5-((3aS,4S,6aR)-2-oxo-hexahydro-1H-
thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)meth-
yl)phenyl)ethyl 3-azidopropylcarbamate (6). To a solution of
NHS-LC-biotin (150 mg, 0.330 mmol) in DMF (5 mL), compound
5 (78 mg, 0.40 mmol) was added followed by Et3N (69 µL, 0.50
mmol). The reaction mixture was stirred overnight at room
temperature, the solvent was evaporated under reduced pressure,
and the residue was taken up in CH2Cl2 and purified by silica gel
chromatography with a 5-15% gradient of MeOH in CH2Cl2 to
give the desired amide (144 mg, 81%). m/z calcd for C25H38N5O6S
[M + H]+: 536.2543. MS found: 536.2. To a solution of the amide
(117 mg, 0.218 mmol) in DMF (5 mL), N,N′-disuccinimidyl
carbonate (84 mg, 0.33 mmol) and Et3N (91 µL, 0.66 mmol) were
added. The next morning, 3-azidopropylamine (110 mg, 1.09 mmol)
was added and the mixture was stirred at room temperature for
24 h. The solvent was evaporated under reduced pressure; the
residue was taken up in CH2Cl2 and purified by silica gel
chromatography using a 5-10% gradient of MeOH in CH2Cl2. An
impurity that gave a white spot with DACA staining was eluted
with 8% MeOH, and the desired compound 6 (101 mg, 70%) was
1
MeOH in CH2Cl2 to give compound 11 (121 mg, 85%). H NMR
(300 MHz, CDCl3), δ (ppm): 7.20 (s, 1H), 7.00 (s, 1H), 6.56 (s,
1H), 5.89 (s, 1H), 5.31 (s, 1H), 4.52 (s, 1H), 4.34 (d, J ) 4.58 Hz,
1H), 3.85-3.51 (m, 20H), 3.51-3.36 (m, 3H), 3.26 (d, J ) 5.96
Hz, 2H), 3.15 (d, J ) 4.36 Hz, 1H), 2.89 (d, J ) 4.78 Hz, 1H),
2.52 (t, J ) 5.84, 5.84 Hz, 2H), 2.24 (t, J ) 7.41, 7.41 Hz, 2H),
1.67 (dd, J ) 14.92, 7.45 Hz, 4H), 1.42-1.09 (m, 8H). m/z calcd
for C25H46N4O8S [M + H]+: 563.3115. MS found: 563.2. HRMS
found: 563.3089.
N-(4-((6-Azidohexyloxy)diphenylsilyloxy)butyl)-1-(5-
((3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide (12). To a
solution of dichlorodiphenylsilane (0.19 mL, 0.88 mmol) and Et3N
(0.34 mL, 2.4 mmol) in anhydrous CH2Cl2 (20 mL) was added
compound 11 (0.10 g, 0.18 mmol). After 10 h, 6-azidohexanol (0.30
g, 2.2 mmol) was added. The solvent was evaporated 16 h later,
and the residue was taken up in CH2Cl2 and passed through a silica
gel column with 10% MeOH in CH2Cl2. The solvents were
evaporated under reduced pressure, and the residue dissolved in
CH2Cl2 was washed with a saturated solution of NaHCO3. The
NaHCO3 solution was extracted with CH2Cl2 three times. The
CH2Cl2 fractions were combined and dried over Na2SO4. After
removal of CH2Cl2 under reduced pressure, a second purification
by silica gel chromatography with a 2-10% gradient of MeOH in
1
eluted with 10% MeOH. H NMR (300 MHz, MeOH), δ (ppm):
7.99-7.86 (1H, m), 7.61 (1H, s), 7.38 (1H, dd, J ) 8.44, 1.74
Hz), 6.16 (1H, dd, J ) 10.82, 4.39 Hz), 4.48 (2H, dd, J ) 9.05,
5.91 Hz), 4.29 (1H, dd, J ) 7.88, 4.44 Hz), 3.55-3.03 (6H, m),
3.03-2.81 (1H, m), 2.69 (1H, d, J ) 12.74 Hz), 2.48 (1H, dd, J )
10.93, 4.01 Hz), 2.42-2.33 (1H, m), 2.28 (2H, t, J ) 7.45, 7.45
Hz), 2.18 (2H, dd, J ) 12.35, 5.04 Hz), 1.88-1.26 (17H, m). 13C
NMR (75 MHz, MeOH), δ (ppm): 174.9, 173.0, 170.3, 164.9,
156.6, 146.7, 145.8, 139.1, 127.1, 125.9, 124.6, 68.3, 62.2, 60.4,
55.9, 42.3, 39.9, 39.0, 37.9, 36.6, 35.7, 30.8, 29.0, 28.9, 28.6, 28.5,
28.3, 28.0, 26.4, 25.8, 25.5, 21.3. m/z calcd for C29H43N9O7S [M
+ H]+: 662.3084. MS found: 684.3 (M + Na). HRMS found:
662.3072.
1
CH2Cl2 gave compound 12 (86 mg, 55%). H NMR (300 MHz,
CDCl3), δ (ppm): 7.62 (dd, J ) 7.74, 1.54 Hz, 4H), 7.54-7.20
(m, 6H), 6.83-6.66 (m, 1H), 6.56-6.39 (m, 1H), 5.71-5.47 (m,
1H), 4.54-4.35 (m, 1H), 4.35-4.17 (m, 1H), 3.87-3.45 (m, 20H),
3.41 (dd, J ) 5.57, 2.80 Hz, 3H), 3.31 (d, J ) 5.98 Hz, 2H),
3.22-3.06 (m, 2H), 2.96-2.78 (m, 1H), 2.72 (s, 1H), 2.40 (t, J )
6.07, 6.07 Hz, 2H), 2.19 (d, J ) 7.34 Hz, 2H), 1.82-1.49 (m, 6H),
1.37 (m, 6H), 1.31-1.10 (m, 8H). 13C NMR (126 MHz, CDCl3), δ
(ppm): 173.3, 171.2, 164.0, 134.7 (m, 6C), 130.2 (m, 2C), 127.8
(m, 4C), 75.7, 70.3 (m, 8C), 67.3, 63.0, 61.8, 60.2, 55.6, 51.4, 50.4,
40.5, 39.1, 37.1, 35.9, 32.2, 28.8, 28.2, 27.6, 26.4, 25.6 (2C), 25.3.
m/z calcd for C43H67N7O9SSi [M + H]+: 886.4569. MS found: 908.2
(M + Na). HRMS found: 886.4601.
N-(2-((3-(3-Azidopropylamino)-3-oxopropyl)disulfanyl)ethyl)-
1-(5-((3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-
4-yl)pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide (14a).
To a solution of compound 13 (50 mg, 0.067 mmol) in CH2Cl2 (5
mL), 3-azidopropylamine (8.7 mg, 0.086 mmol) was added followed
by Et3N (12 µL, 0.086 mmol). After 2 h, the solvent was evaporated
under reduced pressure and the residue was taken up in CH2Cl2
and purified by silica gel chromatography with a 5-12% gradient
of MeOH in CH2Cl2 to give compound 14a (39 mg, 80%). 1H NMR
(300 MHz, CDCl3), δ (ppm): 7.40 (s, 1H), 7.19 (s, 1H), 7.10-7.01
(m, 1H), 6.55 (s, 1H), 5.99-5.78 (m, 1H), 4.60-4.44 (m, 1H),
N-(3-Azidopropyl)-4-((2-hydroxy-5-(2-(6-(5-((3aS,4S,6aR)-2-
oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hex-
anamido)ethyl)phenyl)diazenyl)benzamide (9). To a solution of
compound 823 (1.50 g, 2.96 mmol) and 3-azidopropylamine (355
mg, 3.55 mmol) in CH2Cl2 (50 mL) was added EDC (850 mg, 4.43
mmol). The next morning, the organic phase was washed twice
with 1 M HCl (50 mL) and once with brine, and dried over Na2SO4.
The solvent was evaporated under reduced pressure to give a brown
oil that was dissolved in DMF (25 mL). Piperidine (2.5 mL) was
added, and the next morning the solvent was evaporated under
reduced pressure. The residue was taken up in CH2Cl2 and purified
by silica gel chromatography using a 10-50% gradient of MeOH
in CH2Cl2 to give the desired amine (220 mg, 20% for two steps).
m/z calcd for C18H22N7O2 [M + H]+: 368.1835. MS found: 368.0.
HRMS found: 368.1823. To a solution of NHS-LC-biotin (75 mg,
0.17 mmol) in DMF (5 mL), the above-described amine (73 mg,
0.20 mmol) was added followed by Et3N (35 µL, 0.25 mmol). The
next morning, the solvent was evaporated under reduced pressure
and the residue was taken up in CH2Cl2 and purified by silica gel
9
18358 J. AM. CHEM. SOC. VOL. 132, NO. 51, 2010

Cleavable Biotin Probes
A R T I C L E S
4.34 (d, J ) 4.42 Hz, 1H), 3.91-3.47 (m, 20H), 3.47-3.23 (m,
5H), 3.15 (d, J ) 4.46 Hz, 1H), 3.00 (t, J ) 7.00, 7.00 Hz, 2H),
2.95-2.72 (m, 3H), 2.63 (dd, J ) 15.62, 8.69 Hz, 2H), 2.51 (t, J
) 5.81, 5.81 Hz, 2H), 2.24 (t, J ) 7.27, 7.27 Hz, 2H), 2.03-1.56
(m, 6H), 1.45 (d, J ) 6.94 Hz, 2H). 13C NMR (75 MHz, CDCl3),
δ (ppm): 173.6, 172.0, 171.4, 164.1, 70.2 (m 8C), 67.2, 61.8, 60.2,
55.7, 49.2, 40.5, 39.2, 38.5, 38.1, 36.8 (2C), 36.4, 35.8, 35.0, 28.7,
28.1, 25.6. m/z calcd for C29H52N8O8S3 [M + H]+: 737.3149. MS
found: 759.2 (M + Na). HRMS found: 737.3148.
membrane in 10 mL of PBS, pH 7.4, containing 0.5% TWEEN
20. The membrane was kept in the dark under gentle shaking for
45 min. The residual antibodies were washed five times with 10
mL of PBS, pH 7.4, containing 0.5% TWEEN 20; fluorescence
was measured on a GE Healthcare Typhoon Trio Variable Mode
Imager.
Cloning. The previously reported expression vector pQE-80/
GFPrmAM30 was subjected to site-directed mutagenesis to generate
the M13L mutation of GFPrmAM, yielding pJS2. pJS2 encodes a
GFP variant that contains only one Met site, at the N-terminus.
Constructs were confirmed by sequencing.
N-(2-((3-(Propargylamino)-3-oxopropyl)disulfanyl)ethyl)-1-(5-
((3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide (14b). The
procedure described above for the preparation of compound 14a
was followed except that 3-azidopropylamine was replaced by
Protein Expression and Purification. pJS2 was transformed
into the Met auxotrophic E. coli strain M15MA/pREP4 and isolated
on LB-agar plates containing 35 µg/mL kanamycin and 200 µg/
mL ampicillin. A single colony was used to inoculate an overnight
5 mL culture in LB containing kanamycin and ampicillin. The
following morning, 1 mL of the culture was diluted in 49 mL of
M9 minimal medium (M9 salts, 0.2% glucose, 1 mM MgSO4, 0.1
mM CaCl2, 25 mg/mL thiamine) and supplemented with 40 mg/L
of each of the 20 natural amino acids in addition to kanamycin
and ampicillin. Cells were grown until OD600 reached 1.0 (4-5 h),
pelleted at 2000g for 10 min at 4 °C, and washed twice in cold,
sterile 0.9% NaCl. Cells were resuspended in M9 (-Met) medium
(M9 medium supplemented with 40 mg/L of each of the natural
amino acids except Met). Either Hpg or Met was added to a final
concentration of 1 mM. IPTG was added to a final concentration
of 1 mM to induce synthesis of GFP. Cells were induced for 4 h
at 37 °C and pelleted at 2000g for 10 min at 4 °C; the supernatant
was removed and cells were frozen at -80 °C overnight. The His-
tagged GFP was purified on a Qiagen NiNTA resin under native
conditions according to the manufacturer’s recommendations. The
purified protein was subjected to buffer exchange into PBS, pH
7.8, on GE Healthcare PD10 columns according to the manufac-
turer’s protocol. Glycerol was added to the protein sample at a final
concentration of 50%, and the protein was stored in 50% glycerol
at -20 °C until used.
Protein Digestion and Mass Spectrometry. GFP samples (20
µg) were dissolved in 8 M urea in 100 mM Tris ·HCl, pH 8.5, and
incubated in 3 mM tris(2-carboxyethyl)phosphine hydrochloride for
20 min at room temperature. The samples were then treated with
11.5 mM iodoacetamide for 15 min, followed by Endoprotease
Lys-C (Promega) digestion according to the manufacturer’s recom-
mendations. The sample was diluted to 2 M urea with 100 mM
Tris ·HCl, pH 8.5, and adjusted to 1 mM CaCl2. Tryptic digestion
(Promega) was carried out according to the manufacturer’s recom-
mendations, and was followed by addition of 5% formic acid to
stop trypsin activity. The resulting peptides were desalted by HPLC
(Waters Corp.) on a C8 peptide microtrap (Michrom Bioresources,
Inc.). The peptides were analyzed on a Thermo Finnigan Orbitrap
FT mass spectrometer in positive ion mode with collision-induced
dissociation (CID) for MS/MS analysis.
1
propargylamine, and afforded 14b in 81% yield. H NMR (500
MHz, CDCl3), δ (ppm): 6.97 (s, 1H), 6.35 (s, 1H), 5.52 (s, 1H),
4.57-4.44 (m, 1H), 4.33 (dd, J ) 7.29, 4.87 Hz, 1H), 4.05 (dd, J
) 5.22, 2.49 Hz, 2H), 3.75 (t, J ) 5.81, 5.81 Hz, 2H), 3.69-3.60
(m, 16H), 3.56 (m, 4H), 3.44 (dd, J ) 7.15, 3.78 Hz, 2H),
3.20-3.07 (m, 2H), 3.03-2.97 (m, 2H), 2.91 (dd, J ) 12.80, 4.88
Hz, 1H), 2.85 (t, J ) 6.51, 6.51 Hz, 2H), 2.75 (d, J ) 12.76 Hz,
1H), 2.65 (t, J ) 7.17, 7.17 Hz, 2H), 2.51 (t, J ) 5.81, 5.81 Hz,
2H), 2.37-2.16 (m, 4H), 1.82-1.60 (m, 4H), 1.43 (td, J ) 21.27,
7.37, 7.37 Hz, 2H), 1.25 (s, 1H). 13C NMR (126 MHz, CDCl3), δ
(ppm): 173.5, 172.0, 171.0, 163.9, 80.0, 71.3, 70.2 (m 8C), 67.3,
61.8, 60.2, 55.6, 45.9, 40.6, 39.2, 38.4, 36.9, 36.0, 34.4, 29.1, 28.1,
25.6, 8.6. m/z calcd for C29H49N5O8S3 [M + H]+: 692.2822. MS
found: 714.2 (M + Na). HRMS found: 692.2822.
Probe 16. To a solution of 15 (0.5 mg, 0.45 µmol) in acetonitrile
(1 mL), 3-azidopropanethiol (85 µg, 0.7 µmol) was added followed
by Et3N (0.10 µL, 0.7 µmol). The next morning, the solvent was
evaporated and H2O (1 mL) was added. Lyophilization of the
sample gave 16 (approximately 0.5 mg, quantitative). m/z calcd
for C48H85N11O9S2 [M + H]+: 1024.6051. MS found: 1024.1.
HRMS found: 1024.6043.
General Protocol for Click Chemistry. Protein 17 or 18 (100
µL of a 0.50 mg/mL 50% glycerol stock solution) was added to
800 µL of PBS, pH 7.8, followed by SDS (100 µL of a 10% w/v
stock solution). The biotin probe (10 µL of a 5 mM stock solution
in DMSO) and the tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-
amine ligand (10 µL of a 20 mM stock solution in DMSO) were
added followed by a freshly prepared and vigorously vortexed CuBr
solution (20 µL of a 3 mg of CuBr/mL in H2O stock solution, made
from CuBr 99.999% purchased from Sigma-Aldrich). The mixture
was stirred vigorously at 15 °C for 4 h, added dropwise to a
centrifugation tube containing acetone (5 mL) and kept at -20 °C
for 2 h. Centrifugation at 2000g for 10 min at 4 °C yielded a pellet
that contained protein and copper; the pellet was resuspended by
sonication in 100 µL of PBS, pH 7.8, containing 3% SDS.
Centrifugation at 2000g for 10 min at 4 °C sedimented the copper
in the form of a blue pellet. The supernatant containing the labeled
protein was stored at -20 °C until needed.
General Protocol for Probe Cleavage. The reagent required
for cleavage of each probe was added to the corresponding labeled
protein sample at room temperature. After the desired reaction time
(see Table 1), aliquots were mixed with SDS gel loading buffer,
loaded on an SDS gel, and subjected to Western blot analysis as
described below.
Western Blot Analysis. Tris-glycine SDS gels (12% acryla-
mide) were run (without preboiling the samples) using a Fisher
Scientific FB3000 instrument at 165 V/250 mA for 45 min. Proteins
were transferred to a GE Healthcare Life Science Hybond ECL
membrane using a Hoefer Scientific Instruments TE50X at 80 V
for 1 h at 4 °C. After the membrane was blocked with 5% milk
powder in PBS, pH 7.4, containing 0.5% TWEEN 20, the residual
milk solution was removed by three washes with 10 mL of PBS,
pH 7.4, containing 0.5% TWEEN 20. Anti penta-his alexa fluor
488 conjugate (Qiagen, 2 µL, 1:5000) and streptavidin alexa fluor
633 conjugate (Invitrogen, 4 µL, 1:2500) were added to the
Iodoacetamide Alkylation. COS7 cells were grown to 80%
confluence in DMEM++ media. Cells were washed and incubated
for 30 min in Hepes-buffered saline, pH 7.4 (HBS), followed by
2 h incubation in either 4 mM Met in HBS or 4 mM Aha in HBS.
Cells were pelleted and lysate was prepared as described previ-
ously.17 Cell lysate was reduced in 2% ꢀ-mercaptoethanol in PBS,
pH 7.8, for 1 h in the dark at 25 °C, and then proteins were
precipitated in acetone. Upon resuspension in 250 µL of PBS, pH
7.8, proteins were alkylated by treatment with 11 mM iodoaceta-
mide for 1 h in the dark at 25 °C, and then again precipitated in
acetone. After a second resuspension in 500 µL of PBS, pH 7.8,
the click chemistry reaction was assembled as described previ-
ously.17
Acknowledgment. This work was supported by NIH GM62523
(DAT), NIH MH065537 (EMS), the ARO Institute for Collaborative
Biotechnologies, and Fonds Que´be´cois de la Recherche sur la
Nature et les Technologies (as a postdoctoral fellowship to J.S.).
9
J. AM. CHEM. SOC. VOL. 132, NO. 51, 2010 18359

A R T I C L E S
Szychowski et al.
6, 9, 11, 12, 14a, and 14b; 13C NMR spectra for compounds 4,
5, 6, 9, 12, 14a, and 14b; MS/MS spectra of tryptic fragments.
This material is available free of charge via the Internet at http://
pubs.acs.org.
A.M. was supported in part by a National Science and Engineering
Council of Canada (NSERC) postgraduate scholarship. We thank
Sonja Hess, Jacob Bitterman, Eva Wurster, and Christiane Kowatsch
for helpful discussions, as well as the Proteome Exploration
Laboratory (PEL), MS, and NMR facilities of the California Institute
of Technology.
Supporting Information Available: Sequence and mass
1
spectrum of protein 18; H NMR spectra for compounds 4, 5,
JA1083909
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18360 J. AM. CHEM. SOC. VOL. 132, NO. 51, 2010