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