Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

PLoS One. 2015 Nov 6;10(11):e0142253. doi: 10.1371/journal.pone.0142253. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acyl-tRNA Synthetases / isolation & purification
  • Amino Acyl-tRNA Synthetases / metabolism*
  • Chromatography, Gel / methods*
  • Chromatography, Reverse-Phase
  • Gene Expression Regulation, Enzymologic
  • HEK293 Cells
  • Humans
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Mass Spectrometry / methods*

Substances

  • Isoenzymes
  • Amino Acyl-tRNA Synthetases

Grants and funding

The study was supported by a grant from the National Research Foundation (Next-Generation Applied Omics program: 2013M3A9B6076414) funded by the Korean Ministry of Science, ICT and Future Planning, a grant from the Korea Health Industry Development Institute (Research Hospital program: HI14C3484) funded by the Korean Ministry of Health & Welfare, and a grant for the Joint Research Project funded by National Research Council of Science and Technology. The study was also supported by an intramural program of the Korea Institute of Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.