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.

Highlights

  • Aminoacyl tRNA synthetases (ARSs) are key enzymes that catalyze the attachment of specific amino acid to their cognate tRNAs, which is the first step of protein synthesis [1]

  • Composition of intact multi-tRNA synthetase complex (MSC) component is still uncertain due to high sensitivity caused by uncontrolled proteolysis and various transient interactions of ARS proteins

  • We have established a new workflow to detect and quantify MSC components and free ARSs, which are not included in MSC

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Summary

Introduction

Aminoacyl tRNA synthetases (ARSs) are key enzymes that catalyze the attachment of specific amino acid to their cognate tRNAs, which is the first step of protein synthesis [1]. Because the aminoacylation catalyzed by ARSs prevails in every living organism, ARSs are essential component for protein synthesis. ARSs encompass editing processes which hydrolyze misactivated amino acids or mischarged tRNAs [2]. In addition to their canonical functions in translation and editing, recent studies suggest that non-canonical. Analysis of Multi-tRNA Synthetase Complex by MRM-MS. Research Project funded by National Research Council of Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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