Chemistry & Biology
Azobenzene-Based Affinity Tags for Proteomics
digestion (w/w = 1/100) being carried out at room temperature for 18 hr. The
resulting protease-digested peptides were then incubated with prewashed
streptavidin beads at room temperature for 1.5 hr on end-over-end rotator.
The beads were sequentially washed trice with 1.5 M urea/10 mM HEPES
(pH 8.0) and 10 mM HEPES (pH 8.0). Bound peptides were cleaved from the
beads by treating with freshly made elution buffer (25 mM sodium dithionite,
1 3 GIBCO’s PBS) for 1 hr. Repeat this cleavage step by treating the beads
with freshly made elution buffer for 10 min twice. Collect and combine the
eluants. The cleaved peptides were cleaned up by using C8 cartridge (Waters)
and eluted with 70% CH3CN/20% H2O/0.1% TFA. Eluted peptides were dried
in SpeedVac and then resolubilized in H2O (with 0.1% TFA) for nano-HPLC/
MS/MS analysis.
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On-Bead Protease Digestion of AOA-Labeled Proteins
for MS Analysis
Air-dried pellets were resuspended in 6 M urea/2 M thiourea/10 mM HEPES
(pH 8.0) (Choudhary et al., 2009). Proteins were reduced with 1 mM DTT
(100 mM stock) for 40 min and then alkylated with 5.5 mM iodoacetamide
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on end-over-end rotator. The beads were sequentially washed trice with 6 M
urea/2 M thiourea/10 mM HEPES (pH 8.0), 0.2% SDS (in PBS), and PBS.
The beads were then resuspended in 4.5 M urea/1.5 M thiourea/10 mM HEPES
(pH 8.0) and incubated with Lys-C (w/w = 1/100) at room temperature for 4 hr.
Urea concentration was further diluted by adding 3 volumes of 10 mM HEPES
(pH 8.0) to enable trypsin digestion (w/w = 1/100) being carried out at room
temperature for 18 hr. The beads were then spun down at 2000 3 g for 1
min. Supernatant (tryptic solution) was collected, and the beads were sub-
jected to Na2S2O4 cleavage. Both the protease-digestion fraction and eluant
collected from Na2S2O4 cleavage were cleaned up using C8 cartridge, eluted
by 70% CH3CN/20% H2O/0.1% TFA, and dried in SpeedVac for mass spec-
trometric analysis.
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teomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc.
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SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
six figures, and five tables and can be found with this article online at doi:10.
ꢀ
Fonovic, M., Verhelst, S.H.L., Sorum, M.T., and Bogyo, M. (2007). Proteomics
evaluation of chemically cleavable activity-based probes. Mol. Cell.
Proteomics 6, 1761–1770.
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Q., Karakossian, M.H., Otis, T.S., Kristan, W.B., Trauner, D., and Kramer, R.H.
(2008). Photochemical control of endogenous ion channels and cellular excit-
ability. Nat. Methods 5, 331–338.
ACKNOWLEDGMENTS
We thank the Rockefeller University Proteomics Resource Center for MS anal-
ysis. G.C. is a graduate fellow in the Rockefeller/Sloan-Kettering/Weill-Cornell
Tri-Institutional Program in Chemical Biology. H.C.H. acknowledges support
from NIH/NIDA (1R21DA025751-01), Northeastern Biodefense Center
NIH/NIAID (2 U54 AI057158-06), Irma T. Hirschl/Monique Weill-Caulier Trust,
and Lerner Trust.
Gartner, C.A., Elias, J.E., Bakalarski, C.E., and Gygi, S.P. (2007). Catch-and-
release reagents for broadscale quantitative proteomics analyses.
J. Proteome Res. 6, 1482–1491.
Grammel, M., Zhang, M.M., and Hang, H.C. (2010). Orthogonal alkynyl amino
acid reporter for selective labeling of bacterial proteomes during infection.
Angew. Chem. Int. Ed. Engl. 49, 5970–5974.
Received: July 27, 2010
Revised: September 5, 2010
Accepted: September 14, 2010
Published: November 23, 2010
Huang, Z.P., Park, J.I., Watson, D.S., Hwang, P., and Szoka, F.C., Jr. (2006).
Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates
for analytical and drug delivery applications. Bioconjug. Chem. 17, 1592–1600.
Jewett, J.C., and Bertozzi, C.R. (2010). Cu-free click cycloaddition reactions in
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