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

Park SJ, Ahn HS, Kim JS, Lee C - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4636271&req=5

pone.0142253.g003: 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.

Mentions: The heat maps shown in Fig 2 present the amount of 25 ARS proteins (20 synthetases, 3 auxiliary proteins, and 2 variants) in HEK 293T, KARSoe and KARSoe-AP fractions (Fig 2B, 2C and 2D). In case of HEK 293T and KARSoe, 8 ARS proteins and 3 auxiliary proteins, which are known as MSC components, were detected in the earlier fractions (from 6 to 10). The elution volume of these fractions was close to the void volume of the SEC column employed in the study. Free ARSs (such as TARS, NARS, etc.) were detected in the later fractions (from 13 to 23), the exact position depending on their molecular weight. The overall MRM profile of ARSs was quite similar between HEK 293T and KARSoe except for those of KARS and leucyl-tRNA synthetase (LARS) (Fig 2B and 2C). We calculated correlation coefficient between HEK 293T and KARSoe. For this, we took the MRM signals of 10 MSC components except KARS in the fractions 6 to 10 (S2 and S3 Tables). The values we found were >0.91 for all the five fractions indicating that KARS overexpression did not affect the overall stoichiometry of MSC severely. When KARS was overexpressed, a large portion of KARS was eluted around fraction 16, presumably due to exclusion of extra KARS from MSC formation. Some portions of LARS were also eluted around fraction 16 in case of KARSoe. The mechanism for this phenomenon is yet unknown. Even though KARS and LARS participate in MSC formation in executing their canonical function, various non-canonical functions have also been investigated for which the ARSs need not form MSC. It is hypothesized that the large amount of extra KARS may be related with non-canonical function of LARS by hitherto unknown mechanism. To confirm the MRM-MS data and to detect the expression level of selected MSC components (MARS, KARS, LARS, RARS, EPRS, AIMP1, AIMP2 and TARSL2) and free ARSs (AARS, FARSA, VARS, TARS and YARS), western blot was executed. The western blot results were highly consistent with the MRM-MS profiles in HEK 293T and KARSoe (Fig 3, left panel and S3 Fig). This result suggests that MRM-MS analysis based on SEC is effective for detection and quantification of intact protein complex in native condition.


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

Park SJ, Ahn HS, Kim JS, Lee C - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4636271&req=5

pone.0142253.g003: 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.
Mentions: The heat maps shown in Fig 2 present the amount of 25 ARS proteins (20 synthetases, 3 auxiliary proteins, and 2 variants) in HEK 293T, KARSoe and KARSoe-AP fractions (Fig 2B, 2C and 2D). In case of HEK 293T and KARSoe, 8 ARS proteins and 3 auxiliary proteins, which are known as MSC components, were detected in the earlier fractions (from 6 to 10). The elution volume of these fractions was close to the void volume of the SEC column employed in the study. Free ARSs (such as TARS, NARS, etc.) were detected in the later fractions (from 13 to 23), the exact position depending on their molecular weight. The overall MRM profile of ARSs was quite similar between HEK 293T and KARSoe except for those of KARS and leucyl-tRNA synthetase (LARS) (Fig 2B and 2C). We calculated correlation coefficient between HEK 293T and KARSoe. For this, we took the MRM signals of 10 MSC components except KARS in the fractions 6 to 10 (S2 and S3 Tables). The values we found were >0.91 for all the five fractions indicating that KARS overexpression did not affect the overall stoichiometry of MSC severely. When KARS was overexpressed, a large portion of KARS was eluted around fraction 16, presumably due to exclusion of extra KARS from MSC formation. Some portions of LARS were also eluted around fraction 16 in case of KARSoe. The mechanism for this phenomenon is yet unknown. Even though KARS and LARS participate in MSC formation in executing their canonical function, various non-canonical functions have also been investigated for which the ARSs need not form MSC. It is hypothesized that the large amount of extra KARS may be related with non-canonical function of LARS by hitherto unknown mechanism. To confirm the MRM-MS data and to detect the expression level of selected MSC components (MARS, KARS, LARS, RARS, EPRS, AIMP1, AIMP2 and TARSL2) and free ARSs (AARS, FARSA, VARS, TARS and YARS), western blot was executed. The western blot results were highly consistent with the MRM-MS profiles in HEK 293T and KARSoe (Fig 3, left panel and S3 Fig). This result suggests that MRM-MS analysis based on SEC is effective for detection and quantification of intact protein complex in native condition.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus