Comparative Analysis of Cleavable Azobenzene-Based Affinity Tags for Bioorthogonal Chemical Proteomics

Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA.
Chemistry & biology (Impact Factor: 6.65). 11/2010; 17(11):1212-22. DOI: 10.1016/j.chembiol.2010.09.012
Source: PubMed


The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na₂S₂O₄-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.

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    • "AHA's highly reactive azide group does not react with functional groups in cells but efficiently reacts with biotin alkyne using copper-catalyzed azide-alkyne cycloaddition (CuAAC) in a click chemistry reaction. Furthermore, the small size of the reactive group does not interfere with protein function and is not toxic to cells or animals (Beatty and Tirrell, 2008; Best, 2009; Dieterich et al., 2006, 2010; Hinz et al., 2012; Melemedjian et al., 2010; Ngo and Tirrell, 2011; Yang et al., 2010). Because almost all proteins have at least one methionine (97.9% of Xenopus transcripts in RefSeq begin with methionine), this method can provide an accurate report of newly synthesized proteins. "
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