Bioorthogonal Non-canonical Amino Acid Tagging (BONCAT)

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Bioorthogonal Non-canonical Amino Acid Tagging (BONCAT)

Introduction

The bioorthogonal non-canonical amino acid tagging (BONCAT), based on intracellular introducing orthogonal chemoselective groups of small molecules, can realize specifically labeling, qualitative and quantitative analysis of newly translated proteins. With BONCAT, it is also possible to focus on proteome homeostasis, enabling a higher-dimensional in-depth research of protein properties.

Technical Principles

BONCAT relies on cellular uptake of artifical amino acids bearing chemically modifiable groups, such as L-azidohomoalanine, L-homopropargylglycine, which can be integrated into the primary structure of the protein during translation process, however, free of obvious cytotoxicity and alteration of the rate of protein synthesis or degradation. In the first step of BONCAT, the host cells utilize the azide homoalanine with azide or homopropargyl glycine with alkyne as a substitute for methionine to synthesize newly protein. These small functional groups (eg. Azide, alkyne) provide unique chemical properties to their target molecules, which can be specifically labeled with proper probes for further detection or isolation. Copper-catalyzed azide-alkyne linkage (or "click chemistry") was used to allow the reactive azide group of L-azidohomoalanine or the alkyne group of homopropargylglycine to covalently interact with an alkyne-bearing affinity tag or an azide-bearing alkyne group, respectively. These functionalized tags enable subsequent detection, affinity purification, and mass spectrometry identification of tagging proteins.

Technical Advantages

BONCAT is not only restrict to identifying newly translated proteins, but also assessing variability in different proteomes between specific subpopulations of the translatome.

Non-canonical amino acid tagged cell populations with different fluorecent reporter can facilitate qualitative analysis of protein localization and fluorescent visualization of the spatiotemporal protein dynamics. Besides, BONCAT allows identification of low abundance, low copy number newly synthesized proteins with higher magnitude as compared to SILAC.

Technical Applications

BONCAT is widely used for the labeling of newly synthesized proteins in various primary cell cultures, biopsies, and zebrafish models in vivo. BONCAT has emerged as a cutting-edge tool to gain insight into the dynamics of protein turnover and storage in behavioral time windows.

In addition, BONCAT has been shown to correlate with other established methods for quantifying microbial activity, characterizing bacterial communities and organisms in native growth environments, and has been utilized for investigating the uncultured microorganisms in soil and marine samples.

BONCAT technology can also be combined with ribosomal RNA-targeted fluorescence in situ hybridization to directly link taxonomy to in situ activity. Using BONCAT and fluorescence-activated cell sorting technology, it is possible to isolate living cells from complex samples and further characterize them by DNA sequencing. Although BONCAT is somewhat limited due to differences in cellular amino acid uptake and metabolic disturbances, this technique provides a flexible tool for relatively simple, inexpensive, and high-throughput analysis of in situ activity at the single-cell level. Moreover, labeled proteins can be selectively enriched by affinity chromatography and subject to proteomic analysis. The combined application of these methods enables high-throughput tracing of proteins synthesized by uncultured microorganisms under different physicochemical conditions.

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