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.

MRM quantification of ARS family proteins in 20 SEC (Size Exclusion Chromatography) fractions of HEK 293T, KARS-overexpressed HEK 293T (KARSoe), and elute of KARSoe-affinity purified sample (KARSoe-AP). Protein mixture in each fraction was digested by trypsin. Stable isotope standard (SIS) peptides were spiked into the digests. In the fractions of HEK 239T and KARSoe, 4 pmol of SIS peptides were added, while 2 pmol of SIS peptides were added to all fractions of KARSoe-AP eluate. The digests were then desalted with a C-18 spin column cartridge (Nest group, Southborough, MA, USA), and the eluates were dried in a vacuum centrifuge (miVac Duo Concentrator, Genevac, Suffolk, UK) and stored at -20 °C until use. Samples were reconstituted with 20 uL of 2% acetonitrile and 0.1% formic acid, injected with a full sample loop injection of 1 µL. Each samples were executed 8-min scheduled MRM in duplicate.

For using the SEC technique, the supernatant from HEK 293T (3 mg), KARSoe (3 mg) and streptavidin affinity purification from KARSoe cells (200 µg) prepared in 500 µL solution were loaded onto a Superdex 200 10/300 GL column in an AKTA FPLC system (GE healthcare, Uppsala, Sweden), and eluted with PBS buffer at an optimal flow rate of 0.5 mL/min. The eluate was monitored by UV absorption at 280 nm, collected in 500 µl fractions, and exchanged with 50 mM Tris-Cl (pH 7.5) plus 6 M urea employing 3K Amicon ultra centrifugal filter. Equal volume of buffer-exchanged fractions (40 µL) was reduced with 5 mM dithiothreitol (DTT) at 25 °C for 1 h and alkylated with 15 mM iodoacetamide (IAA) at 25 °C for 1 h in the dark. The samples were diluted 10-fold with 50 mM Tris-Cl (pH 7.5) to decrease the concentration of urea in the sample to less than 1 M. For tryptic digestion, sequencing-grade modified trypsin (Promega, Madison, WI, USA) was added to the sample with an enzyme to protein ratio of 1/50 (w/w) and incubated at 37 °C for 16 h. To stop the reaction, 0.3% trifluoroacetic acid (TFA) (pH < 3.0) was added.