Live‐Cell Imaging of Cellular Proteins by a Strain‐Promoted Azide–Alkyne Cycloaddition

Beatty, Kimberly E.; Fisk, John D.; Smart, Brian P.; Lü, Ying; Szychowski, Janek; Hangauer, Matthew J.; Baskin, Jeremy M.; Bertozzi, Carolyn R.; Tirrell, David A.

doi:10.1002/cbic.201000419

Cited by 150 publications

(139 citation statements)

References 53 publications

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“…For live cell applications, cyclooctyne-functionalized reagents can be used to tag the azide side chain of Anl residues in a copper-free manner (39,40). Our chemical tagging strategy is compatible with routine MS sample preparation methods such as gel electrophoresis liquid chromatography-mass spectrometry (GeLC-MS), filter-aided sample preparation (FASP) (41,42), and multidimensional protein identification technology (MudPIT) (43), and is easily combined with SILAC, isobaric tag for relative and absolute quantification (iTRAQ), and multiple-reaction monitoring (MRM) quantitative MS methods (44,45).…”

Section: Discussionmentioning

confidence: 99%

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Mahdavi

Szychowski

Ngo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.proteomics | click chemistry | BONCAT | Yop

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Section: Discussionmentioning

confidence: 99%

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Mahdavi

Szychowski

Ngo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…10,11 While the [3 + 2] Huisgen cycloaddition and its catalyzed version are not completely compatible with AuNPs chemistry because the high temperature or the Cu(I) required to push the reaction to completion cause severe nanoparticle aggregation, 12 the SPAAC reaction presents limited chemoselectivity due to the possibility of nucleophilic attacks (especially from thiols and amines largely present in biomolecules) to the highly reactive strained triple bond. [13][14][15] A recent work of ours highlighted this issue showing how post assembly deprotection of peptides once "clicked" on the AuNP surface was necessary to efficiently synthesize a nanoparticle bioconjugate through the SPAAC reaction. 16 The Bertozzi-Staudinger ligation is a reaction that was specifically developed for investigating the metabolic engineering of cell surfaces 17 and takes place between the azide and a substituted triphenylphosphine.…”

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confidence: 98%

Small gold nanoparticles for interfacial Staudinger–Bertozzi ligation

et al. 2015

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Small gold nanoparticles (AuNPs) that possess interfacial methyl-2-(diphenylphosphino)benzoate moieties have been successfully synthesized (Staudinger-AuNPs) and characterized by multi-nuclear MR spectroscopy, transmission electron microscopy (TEM), UV-Vis spectroscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy (XPS). In particular, XPS was remarkably sensitive for characterization of the novel nanomaterial, and in furnishing proof of its interfacial reactivity. These Staudinger-AuNPs were found to be stable to the oxidation of the phosphine center. The reaction with benzyl azide in a Staudinger-Bertozzi ligation, as a model system, was investigated using (31)P NMR spectroscopy. This demonstrated that the interfacial reaction was clean and quantitative. To showcase the potential utility of these Staudinger-AuNPs in bioorganic chemistry, a AuNP bioconjugate was prepared by reacting the Staudinger-AuNPs with a novel azide-labeled CRGDK peptide. The CRGDK peptide could be covalently attached to the AuNP efficiently, chemoselectively, and with a high loading.

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“…Since it does not require a Cu catalyst, SPAAC is quite biocompatible allowing both in vivo and in vitro bioconjugation without deleterious effects (Beatty et al, 2010;Hui et al, 2014;. To overcome a relatively slow reaction rate of a simple cyclooctyne (0.0024 M À1 s À1 ), a benzene ring or a cyclopropane was fused to yield dibenzoazacyclooctyne (DIBAC) or bicyclononyne (BCN), each of which exhibits a rate constant of 0.31 and 0.14 M À1 s À1 , respectively (Dommerholt et al, 2010;Jewett et al, 2010), and is most commonly employed in SPAAC.…”

Section: Azide-alkyne Cycloadditionmentioning

confidence: 99%

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

Lim

Kwon

2015

Critical Reviews in Biotechnology

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The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.

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Live‐Cell Imaging of Cellular Proteins by a Strain‐Promoted Azide–Alkyne Cycloaddition

Cited by 150 publications

References 53 publications

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Small gold nanoparticles for interfacial Staudinger–Bertozzi ligation

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

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