Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis
Abstract
Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNA^(Met) in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.
Additional Information
© 2016 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: August 24, 2015. Publication Date (Web): March 18, 2016. We are grateful for financial support by National Institutes of Health grant NIH R01 GM062523 and the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation, the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from U.S. Army Research Office. M.S. and S.H. were supported by the Gordon and Betty Moore Foundation through grant GMBF775 and NIH grant 1S10RR029594-01A1. A.M. was supported by a scholarship from the National Science and Engineering Research Council of Canada and by a postgraduate scholarship from the Donna and Benjamin M. Rosen Center for Bioengineering at Caltech. G.D.H. was supported by a postdoctoral fellowship from the National Science and Engineering Research Council of Canada. The authors declare no competing financial interest.Attached Files
Published - ja5b08980.pdf
Supplemental Material - ja5b08980_si_001.pdf
Supplemental Material - ja5b08980_si_002.xlsx
Supplemental Material - ja5b08980_si_003.xlsx
Files
Additional details
- PMCID
- PMC4825725
- Eprint ID
- 65873
- DOI
- 10.1021/jacs.5b08980
- Resolver ID
- CaltechAUTHORS:20160404-075529150
- R01 GM062523
- NIH
- Gordon and Betty Moore Foundation
- W911NF-09-0001
- Army Research Office (ARO)
- GMBF775
- Gordon and Betty Moore Foundation
- 1S10RR029594-01A1
- NIH
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- Donna and Benjamin M. Rosen Bioengineering Center
- Created
-
2016-04-04Created from EPrint's datestamp field
- Updated
-
2022-05-05Created from EPrint's last_modified field
- Caltech groups
- Rosen Bioengineering Center