title: 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.
authors.forename: Alborz
authors.initials: A
authors.lastname: Mahdavi
authors.forename: Graham D
authors.initials: GD
authors.lastname: Hamblin
authors.forename: Granton A
authors.initials: GA
authors.lastname: Jindal
authors.forename: John D
authors.initials: JD
authors.lastname: Bagert
authors.forename: Cathy
authors.initials: C
authors.lastname: Dong
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.affiliation: Max Planck Institute for Brain Research , Frankfurt am Main 60438, Germany.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Schuman
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D006224:Cricetinae; D004926:Escherichia coli / Q000378:metabolism; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D008322:Mammals; D008718:Methionine-tRNA Ligase / Q000378:metabolism; D051379:Mice; D014176:Protein Biosynthesis; D020543:Proteome / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism
chemical_list: D000596:Amino Acids; D020543:Proteome; D012358:RNA, Transfer, Met; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase