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.

37 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neural plasticity requires protein synthesis, but the identity of newly synthesized proteins generated in response to plasticity-inducing stimuli remains unclear. We used in vivo bio-orthogonal noncanonical amino acid tagging (BONCAT) with the methionine analog azidohomoalanine (AHA) combined with the multidimensional protein identification technique (MudPIT) to identify proteins that are synthesized in the tadpole brain over 24 hr. We induced conditioning-dependent plasticity of visual avoidance behavior, which required N-methyl-D-aspartate (NMDA) and Ca(2+)-permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, αCaMKII, and rapid protein synthesis. Combining BONCAT with western blots revealed that proteins including αCaMKII, MEK1, CPEB, and GAD65 are synthesized during conditioning. Acute synthesis of CPEB during conditioning is required for behavioral plasticity as well as conditioning-induced synaptic and structural plasticity in the tectal circuit. We outline a signaling pathway that regulates protein-synthesis-dependent behavioral plasticity in intact animals, identify newly synthesized proteins induced by visual experience, and demonstrate a requirement for acute synthesis of CPEB in plasticity.
    Cell Reports 02/2014; 6(4). DOI:10.1016/j.celrep.2014.01.024 · 8.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In recent years, critical technical advances have profoundly enhanced the ability to perform global analysis of biological systems, including novel bioorthogonal labeling reactions and mass spectrometry-based proteomic techniques. For the former, the ability to achieve selective labeling of biomolecules within the extremely complex environments of organisms, cells, and cell extracts has opened numerous avenues for the efficient characterization of biological systems. Here, bioorthogonal reactions, including the azide−alkyne cycloadditions, either the copper-catalyzed or copper-free reactions, as well as the Staudinger Ligation, have emerged as vital chemical tools (Sletten and Bertozzi, 2009). In examples of powerful applications, the analogs of sugars (Saxon and Bertozzi, 2000), amino acids (Link et al., 2003 and Liu and Schultz, 2010), and lipids (Kho et al., 2004 and Hang et al., 2007) bearing diminutive reactive tags that represent only minor structural perturbations (azide or alkyne) have been shown to effectively infiltrate biosynthetic pathways. This has been exploited to achieve the bioorthogonal labeling of resulting cell surface glycoproteins, newly synthesized proteins, and posttranslationally lipidated proteins, respectively. In addition, the strategy of activity-based protein profiling has been advanced for the collective labeling of proteins using small molecule probes that label target proteins (Speers et al., 2003 and Cravatt et al., 2008).
    Chemistry & biology 11/2010; 17(11):1166-8. DOI:10.1016/j.chembiol.2010.11.001 · 6.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Chemical reporters are powerful tools for the detection and discovery of protein modifications following cellular labeling. The metabolism of alkyne- or azide-functionalized chemical reporters in cells can influence the efficiency and specificity of protein targeting. To evaluate the effect of degradation of chemical reporters of protein fatty acylation, we synthesized 15-hexadecynyloxyacetic acid (HDYOA), a reporter that was designed to be resistant to β-oxidation, and compared its ability to label palmitoylated proteins with an established reporter, 17-octadecynoic acid (ODYA). HDYOA was able to label known candidate S-palmitoylated proteins similarly to ODYA. Accordingly, bioorthogonal proteomic analysis demonstrated that 70% of proteins labeled with ODYA were also labeled with HDYOA. However, the proteins observed differentially in our proteomic studies suggested that a portion of ODYA protein labeling is a result of β-oxidation. In contrast, downstream enzymes involved in β-oxidation of fatty acids were not targeted by HDYOA. Since HDYOA can label S-palmitoylated proteins and is not utilized by downstream β-oxidation pathways, this fatty acid chemical reporter may be particularly useful for bioorthogonal proteomic studies in cell types metabolically skewed toward fatty acid breakdown.
    Bioorganic & medicinal chemistry 04/2011; 20(2):650-4. DOI:10.1016/j.bmc.2011.03.062 · 2.79 Impact Factor
Show more


37 Reads
Available from