Fig 1.
Overall workflow for detection of ARS proteins.
(A) Overall workflow. Two cell lysates (HEK 293T and KARSoe) and affinity purification eluate (KARSoe-AP) were fractionated using SEC. Each fraction was digested with trypsin and spiked with the SIS peptide. After C18 clean-up, the digest was analyzed by LC-MRM-MS. All the data were analyzed by Skyline. (B) Overexpression of KARS tagged with S/FLAG/SBP in HEK 293T cell was detected by western blot using anti-FLAG antibody. Actin was used as a loading control. N, HEK 293T cell; Koe, KARS-overexpressing cell.
Fig 2.
Two-dimensional MRM profile of ARS proteins in the fractions of size exclusion chromatography.
(A) Two cell lysates and affinity purification eluate were injected on a Superdex 200 column and the elution profile was recorded by following the 280 nm absorbance. Red: HEK 293T cell lysate; Blue: KARSoe cell lysate; Green; KARSoe-AP eluate. (B, C and D) The average amount of 25 ARS proteins in 20 fractions of HEK 293T (B), KARSoe (C) and KARSoe-AP (D) from duplicated LC-MRM runs is represented as heat maps. The average value is normalized against the largest value among the 20 fractions (S2, S3 and S4 Tables). In the heat maps, each row represents an ARS protein; each column represents an SEC fraction. (D, right panel) The histogram represents recovery rate (%) of each ARS protein after affinity purification. The recovery rate is a relative value to that of KARS which was used as a bait.
Table 1.
MRM conditions for quantified proteotypic peptides representing 25 ARS proteins.
Fig 3.
Expression level of endogenous ARS proteins in HEK 293T fractions by western blot analysis.
All fractions (5 μL, respectively) were analyzed by western blot with selected ARS antibodies (left panel). Total cell lysate (10 μg) of HEK 293T (H) and KARSoe (Koe), and 5μg of affinity-purified proteins of KARSoe cells (AP) were used as a positive control (right panel). Actin was used as a loading control for total cell lysate. MSC, multi-tRNA synthetase complex; TCL, total cell lysate. Black arrow indicates EPRS.
Fig 4.
MS/MS spectra, extracted ion chromatograms, and calibration curves of TARSL2 and AIMP2-DX2 surrogate peptides.
(A) MS/MS spectra of SIS peptides for TARSL2 (left) and AIMP2-DX2 (right) after optimization of MRM parameters (Heavy isotope-labeled amino acid is colored red in the peptide sequence). Three fragment y-ions of TARSL 2 (y7+, y5+, y6+) and five fragment y-ions of AIMP2-DX2 (y16++, y13++, y16+++, y15+++, y15++) used for peak assignment and quantitation are annotated within the spectra. (B and C) MRM chromatograms and calibration curves for quantifying TARSL2 (B) and AIMP2-DX2 (C). XIC of the TARSL2 (three y-ions) and AIMP2-DX2 (five y-ions) for SIS peptides (left panel) and for endogenous peptides in seventh SEC fraction of HEK 293T (middle panel) are presented. A dilution series of the SIS peptides for TARSL2 (y7+) and AIMP2-DX2 (y16++) (right panel) were analyzed in triplicated MRM runs and the resultant MRM peak areas are plotted as a function of peptide amount (right panel). The straight line within the plots represents linear response range with R2 ≥0.99 and CV ≤ 20% in which the lowest value corresponds to LOQ. Blue circles represent endogenous peptides in seventh fraction.