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.
Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites.
Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli
Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation
A designed phenylalanyl-tRNA synthetase variant allows efficient in vivo incorporation of aryl ketone functionality into proteins
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes
Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition
A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems
Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)
Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids
Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging
Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo
Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation
Orthogonal alkynyl amino acid reporter for selective labeling of bacterial proteomes during infection
Core-clickable PEG-branch-azide bivalent-bottle-brush polymers by ROMP: grafting-through and clicking-to
FGF21 analogs of sustained action enabled by orthogonal biosynthesis demonstrate enhanced antidiabetic pharmacology in rodents
Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish
Ontogeny informs regeneration: explant models to investigate the role of the extracellular matrix in cartilage tissue assembly and development.
Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse
Extracellular matrix at the muscle - tendon interface: functional roles, techniques to explore and implications for regenerative medicine
The Dynamic Relationship of Breast Cancer Cells and Fibroblasts in Fibronectin Accumulation at Primary and Metastatic Tumor Sites
A Picture Worth a Thousand Molecules-Integrative Technologies for Mapping Subcellular Molecular Organization and Plasticity in Developing Circuits.
A general asymmetric synthesis of artificial aliphatic and perfluoroalkylated α-amino acids by Luche's cross-electrophile coupling reaction.
Bioinformatics in Biomedicine
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