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Non-canonical Amino Acid Labeling in Proteomics and Biotechnology

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Review

Non-canonical Amino Acid Labeling in Proteomics and Biotechnology

Aya M Saleh et al. J Biol Eng.

Abstract

Metabolic labeling of proteins with non-canonical amino acids (ncAAs) provides unique bioorthogonal chemical groups during de novo synthesis by taking advantage of both endogenous and heterologous protein synthesis machineries. Labeled proteins can then be selectively conjugated to fluorophores, affinity reagents, peptides, polymers, nanoparticles or surfaces for a wide variety of downstream applications in proteomics and biotechnology. In this review, we focus on techniques in which proteins are residue- and site-specifically labeled with ncAAs containing bioorthogonal handles. These ncAA-labeled proteins are: readily enriched from cells and tissues for identification via mass spectrometry-based proteomic analysis; selectively purified for downstream biotechnology applications; or labeled with fluorophores for in situ analysis. To facilitate the wider use of these techniques, we provide decision trees to help guide the design of future experiments. It is expected that the use of ncAA labeling will continue to expand into new application areas where spatial and temporal analysis of proteome dynamics and engineering new chemistries and new function into proteins are desired.

Keywords: Bioorthogonal chemistry; Biotechnology; Metabolic labeling; Proteomics; Residue-specific labeling; Site-specific labeling.

Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Azide-alkyne cycloaddition reactions. a Copper(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC). b [3 + 2] cycloaddition of azides and strain-promoted alkynes (cyclooctynes) (SPAAC)
Fig. 2
Fig. 2
Incorporation of ncAAs by native cellular machinery. Non-canonical amino acids (ncAAs) are incorporated into the growing polypeptide chain as the protein is synthesized at the ribosome. a ncAA is covalently attached to a tRNA by aminoacyl tRNA synthetase (aaRS). b The tRNA, charged with the ncAA (ncAA-tRNA, ncAA in blue), recognizes mRNA codons in the ribosome and the ncAA is added to the growing polypeptide chain
Fig. 3
Fig. 3
Examples of non-canonical amino acids. Chemical structures of amino acids highlighted in this review: methionine (Met), homoallylglycine (Hag), homopropargylglycine (Hpg), azidohomoalanine (Aha) and azidonorleucine (Anl). Azidophenylalanine (Azf), and acetylphenylalanine (Acf) are analogs of phenylalanine. Propargyloxyphenylalanine (Pxf) is a tyrosine analog (See Table 1 for more discussion of these ncAAs)
Fig. 4
Fig. 4
Overview of residue-specific protein labeling. a A ncAA (red sphere) is added to the system (cell culture or animal model). Native translational machinery incorporates the ncAA into the newly synthesized proteins. b An example of the codon sequence and corresponding peptides that result from either natural synthesis or synthesis in the presence of the ncAA. c A peptide labeled at two residue-specific sites with a ncAA carrying an alkyne functional group is conjugated to a azide-containing fluorophore via CuAAC
Fig. 5
Fig. 5
Overview of site-specific ncAA incorporation using orthogonal tRNA/aminoacyl synthetase pair. a A plasmid that expresses the desired orthogonal tRNA and tRNA synthetase is transfected into cells along with the plasmid containing the protein of interest that has been engineered to carry the suppressed codon sequence at a specific site. ncAA is added to the system and the protein of interest is labeled site-specfically with the ncAA. b An example of the codon sequence and corresponding peptides that result from either natural synthesis or synthesis in the presence of the orthogonal tRNA/tRNA synthetase and ncAA. c A peptide labeled site-specifically with a ncAA carrying an alkyne functional group is conjugated to a azide-containing fluorophore via CuAAC
Fig. 6
Fig. 6
Decision tree for ncAA labeling in proteomics applications. If global proteome labeling is desired, consider residue-specific labeling. Residue-specific ncAA labeling is designed to replace a specific natural amino acid of interest in the entire proteome. Several natural amino acids analogs have been utilized (See Fig. 3 and Table 1). No genetic modification is needed for global proteome labeling with ncAA. Nevertheless, the labeling efficiency in bacterial cells is greatly enhanced if auxotrophic mutants are used. Similarly, labeling of cultured mammalian cells and non-mammalian animal models (e.g. nematodes) can be achieved by adding the ncAA directly to the culture/feeding media. However, if higher degree of labeling is required, consider using culture media that lacks the natural amino acid to be replaced. For in vivo labeling of small animal models (e.g. rodents), the ncAA can be injected or added to animal diet and/or drinking water. If embryonic labeling is desired, consider ncAA injection since it has been demonstrated that ncAAs are effectively incorporated into embryos when injected into pregnant animals without disturbing normal development [87]. If labeling of specific cell types in a mixed culture system is desired, consider using transgenic lines that express a mutant aaRS designed to charge the ncAA of interest. Since the ncAA is not a substrate of endogenous aaRSs, only cells expressing the mutant aaRS in the mixed culture system are labeled. Similarly, if cell-selective labeling of animals is required, consider use transgenic animals that express the mutant aaRS under cell-specific promoters. If specific protein labeling rather than global proteome labeling is needed, ncAA can be incorporated site-specifically in the polypeptide chain in response to an amber stop codon. This requires introducing the amber codon into the gene of interest and using an orthogonal aaRS/amber suppressor tRNA pair evolved for charging the desired ncAA
Fig. 7
Fig. 7
Decision tree for ncAA biotechnology applications. For bioconjugation, it is easiest to target natural amino acids such as lysine, however, this approach provides minimal control over the conjugation site. In addition, the conjugation chemistry is not biorthogonal such that other proteins in the sample will also be conjugated. If biorthogonality is not necessary, the natural N- or C- terminus of the protein can also be targeted. Cysteine can also be targeted, but this can interfere with disulfide bonds if present in the protein. In addition, cysteine conjugation may require some mutagenesis for site-specific conjugation as native surface-exposed cysteines need to be removed and replaced with cysteine at the desired conjugation location. If biorthogonal conjugation is desired and/or greater control over the conjugation site is desired, then first consider residue-specific ncAA incorporation. This has some of the same limitations as targeting natural amino acids as this method replaces a natural amino acid with an analog. However, for proteins with a small number of methionines, this could work well for the desired application. In some studies partial ncAA incorporation at the N-terminus has been observed. If precise predetermined control of the exact locations for conjugation is desired, consider site-specific ncAA incorporation using orthogonal aaRS/tRNA pairs. If aaRS/tRNA have not been engineered to incorporate the desired ncAA for the desired conjugation reaction, chemically aminoacylated tRNA can be used at the small scale. Otherwise, an aaRS/tRNA pair will need to be engineered. Fortunately, a number of aaRS/tRNA pairs have already been engineered for site-specifically incorporating click-chemistry reactive ncAAs

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