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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

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.
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pone.0142253.g002: 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.

Mentions: Composition of intact MSC component is still uncertain due to high sensitivity caused by uncontrolled proteolysis and various transient interactions of ARS proteins. Additionally, a little clue about MSC composition concerning the assembly of its components has been obtained by reconstitution of sub-complex from recombinant proteins and by affinity purification [10, 23, 24]. In this study, we have established a new workflow to detect and quantify MSC components and free ARSs, which are not included in MSC. We used both HEK 293T and lysyl-tRNA synthetase (KARS)-overexpressing (KARSoe) cells. KARSoe cells had also been used in our previous work (Fig 1B), where we identified TARSL2 and AIMP2-DX2 in KARS precipitate [9]. Cell lysates prepared from the two cell lines were fractionated by SEC into 20 consecutive fractions of equal volume. Subsequently, each fraction was analyzed by MRM-MS after digestion with trypsin. In a parallel experiment, we purified MSC by streptavidin affinity chromatography from KARSoe cells (KARSoe-AP) before SEC (Fig 1). In summary, HEK 293T (3 mg), KARSoe (3 mg) cell lysate and KARSoe-AP eluate (200 μg) were subjected to SEC (Fig 2A). SEC is a well-established technique used to separate proteins and protein complexes in solution based on their size and shape. Therefore, complex-forming protein will elute in the earlier fraction than the protein when it does not form a complex.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142253.g002: 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.
Mentions: Composition of intact MSC component is still uncertain due to high sensitivity caused by uncontrolled proteolysis and various transient interactions of ARS proteins. Additionally, a little clue about MSC composition concerning the assembly of its components has been obtained by reconstitution of sub-complex from recombinant proteins and by affinity purification [10, 23, 24]. In this study, we have established a new workflow to detect and quantify MSC components and free ARSs, which are not included in MSC. We used both HEK 293T and lysyl-tRNA synthetase (KARS)-overexpressing (KARSoe) cells. KARSoe cells had also been used in our previous work (Fig 1B), where we identified TARSL2 and AIMP2-DX2 in KARS precipitate [9]. Cell lysates prepared from the two cell lines were fractionated by SEC into 20 consecutive fractions of equal volume. Subsequently, each fraction was analyzed by MRM-MS after digestion with trypsin. In a parallel experiment, we purified MSC by streptavidin affinity chromatography from KARSoe cells (KARSoe-AP) before SEC (Fig 1). In summary, HEK 293T (3 mg), KARSoe (3 mg) cell lysate and KARSoe-AP eluate (200 μg) were subjected to SEC (Fig 2A). SEC is a well-established technique used to separate proteins and protein complexes in solution based on their size and shape. Therefore, complex-forming protein will elute in the earlier fraction than the protein when it does not form a complex.

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