Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Cell-type-specific proteomics is crucial for understanding complex biological processes in multicellular organisms.
• Existing bioorthogonal tools have limitations in mammalian systems.

Purpose of the Study:

• To develop novel engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling.
• To enable cell-type-specific proteomics in live mammals.

Main Methods:

• Engineered a tyrosyl (ScTyrY43G) and a phenylalanyl (MmPheT413G) tRNA synthetase.
• Incorporated azide-bearing noncanonical amino acids into nascent proteomes.
• Utilized azide-alkyne cycloadditions for chemoselective tagging with fluorophores or affinity resins.

Main Results:

• Both ScTyrY43G and MmPheT413G successfully labeled proteins in human, hamster, and mouse cell lines.
• Mutant synthetases selectively activated azido-bearing amino acids over 10-fold compared to canonical ones.
• In vivo labeling of melanoma tumor proteome and plasma secretome in mice was achieved.

Conclusions:

• The engineered synthetases provide new tools for mammalian residue-specific bioorthogonal chemistry.
• These tools facilitate more robust and comprehensive cell-type-specific proteomics in live mammals.
• Enables advanced imaging and mass spectrometric characterization of proteomes.