for 10 minutes at 4 °C. The supernatant was collected and the
protein concentration determined by Bradford assay. 5 μg of
lysate was incubated with 1.8–0.2 μg of either Cy 5 probe 3 or
HA-Ub-VME probe 6 in the presence or absence of NEM (200
nM) in a total volume of 20 μL. Reactions were incubated at
3
7 °C for 3 hours prior to addition of 20 μL of reducing sample
buffer and heating to 95 °C for 5 min. Samples were resolved by
–12% reducing SDS-PAGE gel and detected by both fluor-
4
escence detection using a GE Healthcare Typhoon™ 9400
system with a red laser (633 nm) for excitation and a 670 nm
wavelength filter, and western blotting methods using a directly
coupled HA-HRP antibody (1 : 10 000 dilution, Sigma Aldrich
H6533). PR619 (Progenra/Lifesensors) (0–50 μg) was incubated
with cell lysate for 30 min at 0 °C prior to probe labelling and
detection as described above.
Scheme 1 Synthesis of 11.
3
7
60 μL of a solution containing 2 M NHS and 1 M NaOH,
5 μL of 1 M Tris base (pH 7.4) was added followed by 360 μL
of 1 : 1 MeCN–0.5 M NaOH containing 0.25 M alkyne 11. The
reaction mixture was incubated at 37 °C for three hours, prior to
buffer exchange to 50 mM NaOAc (pH 4.5) and concentration.
Alkyne probe 2 was purified to >95% purity using a SCX bio-
monolith column (5.2 × 4.95 mm, Agilent) with a linear gradient
from 0% to 100% B, 50 mM NaOAc (pH 4.5, buffer A), 50 mM
Acknowledgements
B.M.K is supported by the NIHR Biomedical Research Centre,
Oxford, UK. M.A. is supported by the Swedish Research
Council, the Loo and Hans Ostermanns Foundation for Geriatric
Research and the Foundation for Geriatric Diseases at the Karo-
linska Institutet. We thank Dr Benjamin Nicholson (Progenra
Inc.) for providing us with recombinant UCH-L3 enzyme.
NaOAc (pH 4.5) and 1 M NaCl (buffer B) at a flow rate of
.5ml min− using HPLC (Agilent 1100) (Fig. S1†). Product
1
0
containing fractions were concentrated and probe 2 was analyzed
by MALDI-TOF (Bruker) in linear mode using an α-cyano-4-
hydroxycinnamic acid matrix (Fig. 2). A further aliquot was sub-
jected to digestion by trypsin and the resulting peptides analyzed
by LC-MS/MS (Fig. S2†).
Notes and references
1
O. Kerscher, R. Felberbaum and M. Hochstrasser, Annu. Rev. Cell Dev.
Biol., 2006, 22, 159.
2
A. Borodovsky, H. Ovaa, N. Kolli, T. Gan-Erdene, K. D. Wilkinson, H.
L. Ploegh and B. M. Kessler, Chem. Biol., 2002, 9, 1149.
D. Finley, Annu. Rev. Biochem., 2009, 78, 477.
F. E. Reyes-Turcu, K. H. Ventii and K. D. Wilkinson, Annu. Rev.
Biochem., 2009, 78, 363.
Synthesis of fluorescent Ub probes 3–4
3
4
7
50 μL of Na PO /NaH PO (50 mM, pH 8) containing a final
2 4 2 4
−1
concentration of 1.1 mg mL of alkyne probe 2 was treated
with the corresponding fluorescein (synthesis described in ESI†)
or Cy 5 azide (Lumiprobe) (1.24 μmol in 25 μL MeCN) fol-
lowed by tris[(1-ethoxycarbonylmethyl-1H-1,2,3-triazol-4-yl)-
5 L. Daviet and F. Colland, Biochimie, 2008, 90, 270.
6
7
8
A. Shanmugham, A. Fish, M. P. Luna-Vargas, A. C. Faesen, F. El Oualid,
T. K. Sixma and H. Ovaa, J. Am. Chem. Soc., 2010, 132, 8834.
S. Virdee, Y. Ye, D. P. Nguyen, D. Komander and J. W. Chin, Nat. Chem.
Biol., 2010, 10, 750.
X. Tian, N. S. Isamiddinova, R. J. Peroutka, S. J. Goldenberg, M.
R. Mattern, B. Nicholson and C. Leach, Assay Drug Dev. Technol., 2011,
22
methyl] amine (0.5 mg in 25 μL MeCN) then Cu(I)Br (0.6 mg,
.2 μmol in 25 μL MeCN). The reaction mixtures were shaken at
4
room temperature for 30 min prior to desalting (PD-10, GE
Healthcare) and concentration. Analysis was carried out by
linear mode MALDI-TOF analysis (Bruker).
9, 165.
9
A. Borodovsky, B. M. Kessler, R. Casagrande, H. S. Overkleeft, K.
D. Wilkinson and H. L. Ploegh, EMBO J., 2001, 20, 5187.
10 K. R. Love, R. K. Pandya, E. Spooner and H. L. Ploegh, ACS Chem.
Biol., 2009, 4, 275.
DUB labelling assays. Recombinant UCH-L3 (Progenra/Life-
sensors, 200 ng) was resuspended in 2 μL of Tris buffer (0.5 M,
pH 7.4). Varying concentrations of either HA-Ub-Br probe 5 or
fluorescent probe 4 were added and the total volume adjusted to
1
1 B. F. Cravatt, A. T. Wright and J. W. Kozarich, Annu. Rev. Biochem.,
008, 77, 383.
12 H. Ovaa, Nat. Rev. Cancer, 2007, 7, 613.
2
1
1
3 M. Zorko and U. Langel, Adv. Drug Delivery Rev., 2005, 57, 529.
4 M. C. Morris, J. Depollier, J. Mery, F. Heitz and G. Divita, Nat. Biotech-
nol., 2001, 19, 1173.
1
5 μL with 0.5 M Tris buffer, pH 7.4 containing 1 mM DTT.
Solutions were incubated at 37 °C for 1 hour, followed by
addition of 15 μL of reducing sample buffer and heating to
15 S. O. Lo and S. Wang, Macromol. Rapid Commun., 2010, 31, 1134.
1
6 V. Kersemans, K. Kersemans and B. Cornelissen, Curr. Pharm. Des.,
008, 14, 2415.
2
9
5 °C for 5 min. Samples were resolved by 18% reducing
17 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew.
Chem., Int. Ed., 2002, 41, 2596.
SDS-PAGE gel and detected using an Autochemi™ UVP bio-
imaging system. Gels were scanned with UV transillumination
using a green (515–570) filter and images captured using a
Hamamatsu charged coupled device (CCD) camera. Subsequent
silver staining was conducted using standard protocols.
HEK293T cell pellets were lysed at 0 °C using glass beads in
homogenisation buffer (50 mM Tris, pH 7.5 containing 5 mM
18 M. Altun, H. B. Kramer, L. I. Willems, J. L. McDermott, C. A. Leach, S.
J. Goldenberg, K. G. S. Kumar, R. Konietzny, R. Fisher, E. Kogan, M.
M. Mackeen, J. McGouran, S. V. Khoronenkova, J. L. Parsons, G.
L. Dianov, B. Nicholson and B. M. Kessler, Chem. Biol., 2011, 18, 1401.
19 D. Volke and R. Hoffmann, Electrophoresis, 2008, 29, 4516–4526.
20 C. Grison, S. Genève, E. Halbin and P. Coutrot, Tetrahedron, 2001, 57,
4903.
21 R. P. Hanzlik and S. A. Thompson, J. Med. Chem., 1984, 27, 711.
MgCl , 250 mM sucrose, 1 mM DTT and 2 mM ATP) as
2
22 T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin, Org. Lett., 2004,
6, 2853.
9
described. The resulting suspension was centrifuged at 14 000 rpm
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 3379–3383 | 3383