title: Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography.
abstract: Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea.
authors.forename: Seong-Jun
authors.initials: SJ
authors.lastname: Park
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea.|Department of Biological Chemistry, University of Science and Technology, Daejeon 305-333, Republic of Korea.
authors.forename: Hee-Sung
authors.initials: HS
authors.lastname: Ahn
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea.
authors.forename: Jun Seok
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea.|Department of Biological Chemistry, University of Science and Technology, Daejeon 305-333, Republic of Korea.
authors.forename: Cheolju
authors.initials: C
authors.lastname: Lee
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D007527:Isoenzymes; D000604:Amino Acyl-tRNA Synthetases