Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography
- Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography
- 박성준; 안희성; 김준석; 이철주
- Issue Date
- PLoS ONE
- VOL 10, NO 11, e0142253-1-e0142253-16
- 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 copurified 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.
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