1. Light-Activated Proteomic Labeling via Photocaged Bioorthogonal Non-Canonical Amino Acids
- Author
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Steven M Adelmund, Cole A. DeForest, Emily R. Ruskowitz, Julie V Wolfe, and Payam E. Farahani
- Subjects
Proteomics ,0301 basic medicine ,Azides ,Light ,Quantitative proteomics ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Tissue culture ,Protein biosynthesis ,Humans ,Amino Acids ,chemistry.chemical_classification ,Staining and Labeling ,Proteins ,Translation (biology) ,General Medicine ,0104 chemical sciences ,Amino acid ,030104 developmental biology ,chemistry ,Protein Biosynthesis ,Molecular Medicine ,Azide ,Bioorthogonal chemistry - Abstract
This work introduces light-activated bioorthogonal non-canonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging L-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compound remains stable for many hours in culture, but can be photochemically liberated rapidly and on demand with spatial control. Upon directed light exposure, the uncaged amino acid is available for local translation, enabling downstream proteomic interrogation via bioorthogonal conjugation. Exploiting the reactive azide moiety present on Aha’s amino acid side chain, we demonstrate that newly synthesized proteins can be purified for quantitative proteomics or visualized in synthetic tissues with a new level of spatiotemporal control. Shedding light on when and where proteins are translated within living samples, we anticipate that laBONCAT will aid in understanding the progression of complex protein-related disorders.
- Published
- 2018