Park, Seong-Jun Ahn, Hee-Sung Kim, Jun Seok Lee, Cheolju 2024-01-20T05:33:38Z 2024-01-20T05:33:38Z 2021-09-03 2015-11-06 1932-6203 https://pubs.kist.re.kr/handle/201004/124762 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. English PUBLIC LIBRARY SCIENCE PROTEIN-PROTEIN INTERACTIONS ELONGATION FACTOR-I MACROMOLECULAR ASSEMBLAGE CANCER PURIFICATION CELLS QUANTIFICATION TRANSLATION DISSECTION PROTEOMICS Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography Article 10.1371/journal.pone.0142253 1 PLoS One, v.10, no.11 PLoS One 10 11 scie scopus 000364398700101 2-s2.0-84952683826 Multidisciplinary Sciences Science & Technology - Other Topics Article PROTEIN-PROTEIN INTERACTIONS ELONGATION FACTOR-I MACROMOLECULAR ASSEMBLAGE CANCER PURIFICATION CELLS QUANTIFICATION TRANSLATION DISSECTION PROTEOMICS