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

<div><p>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.</p></div

Expression level of endogenous ARS proteins in HEK 293T fractions by western blot analysis.

<p>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 KARS^oe (K^oe), and 5μg of affinity-purified proteins of KARS^oe 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.</p

MS/MS spectra, extracted ion chromatograms, and calibration curves of TARSL2 and AIMP2-DX2 surrogate peptides.

<p><b>(A)</b> 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>(B and C)</b> 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.</p

MRM conditions for quantified proteotypic peptides representing 25 ARS proteins.

<p>^a Light peptides represent endogenous peptides.</p><p>^b Heavy peptides represent SIS peptides.</p><p>^c Declustering potential.</p><p>^d Collisional energy.</p><p>^e Collisional cell exit potential.</p><p>^f. Retention time.</p><p>*: Carbamidomethylated cysteine</p><p>MRM conditions for quantified proteotypic peptides representing 25 ARS proteins.</p

Two-dimensional MRM profile of ARS proteins in the fractions of size exclusion chromatography.

<p><b>(A)</b> 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: KARS^oe cell lysate; Green; KARS^oe-AP eluate. <b>(B, C and D)</b> The average amount of 25 ARS proteins in 20 fractions of HEK 293T (B), KARS^oe (C) and KARS^oe-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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142253#pone.0142253.s007" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142253#pone.0142253.s008" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142253#pone.0142253.s009" target="_blank">S4</a> Tables). In the heat maps, each row represents an ARS protein; each column represents an SEC fraction. <b>(D, right panel)</b> 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.</p

Integrative analysis for the discovery of lung cancer serological markers and validation by MRM-MS

<div><p>Non-small-cell lung cancer (NSCLC) constitutes approximately 80% of all diagnosed lung cancers, and diagnostic markers detectable in the plasma/serum of NSCLC patients are greatly needed. In this study, we established a pipeline for the discovery of markers using 9 transcriptome datasets from publicly available databases and profiling of six lung cancer cell secretomes. Thirty-one out of 312 proteins that overlapped between two-fold differentially expressed genes and identified cell secretome proteins were detected in the pooled plasma of lung cancer patients. To quantify the candidates in the serum of NSCLC patients, multiple-reaction-monitoring mass spectrometry (MRM-MS) was performed for five candidate biomarkers. Finally, two potential biomarkers (BCHE and GPx3; AUC = 0.713 and 0.673, respectively) and one two-marker panel generated by logistic regression (BCHE/GPx3; AUC = 0.773) were identified. A validation test was performed by ELISA to evaluate the reproducibility of GPx3 and BCHE expression in an independent set of samples (BCHE and GPx3; AUC = 0.630 and 0.759, respectively, BCHE/GPx3 panel; AUC = 0.788). Collectively, these results demonstrate the feasibility of using our pipeline for marker discovery and our MRM-MS platform for verifying potential biomarkers of human diseases.</p></div

Analyses of proteomes from pooled plasma by mass spectrometry.

<p><b>(A)</b> SDS-PAGE (protein 10 μg) of plasma pooled from 10 healthy control patients and 10 lung cancer patients, divided into 25 fractions. <b>(B)</b> Schematic diagram of the high-pH RPLC fractionation (protein 10 μg) setup. The eluates were combined by column (1–12 columns, 12 fractions). The surrogate peptides were monitored by measuring the UV absorbance of the eluates at 215 nm. <b>(C)</b> Venn diagram of the number of proteins identified by GeLC-MS/MS and high-pH RPLC fractionation. <b>(D)</b> Venn diagram of the number of analyzed molecules among DEGs, secretomes, and plasma proteome.</p

Analyses of conditioned media harvested from NSCLC cell lines.

<p><b>(A)</b> Proteins (10 μg) in the conditioned media (CM) and cell extracts (CE) were analyzed by Western blot analysis using an anti-α-tubulin antibody. <b>(B)</b> The number of identified proteins in the cell secretome (FDR a 1%). Secretion pathways were predicted by SignalP, SecretomeP, and TMHMM. Bovine contaminants were distinguished using the human-FBS database. <b>(C)</b> Venn diagram of DEGs in tissues and identified proteins from cell secretomes. <b>(D)</b> Predicted secretion pathways of identified secretome proteins in all cell lines.</p

Systematic evaluation of serum MRM assays.

<p><b>(A)</b> Total ion chromatogram (TIC) of endogenous (red) peptides and their respective SIS peptides (blue). <b>(B)</b> Circular heatmap of the relative expression of four proteins in the two groups. The 46 clinical samples are shown in the circular heat map, clockwise from the top: 23 controls and 23 NSCLCs (16 adenocarcinomas and 7 squamous cell carcinomas). Indexing was followed by the sequence of LC-MRM runs. <b>(C)</b> Serum levels of BCHE in the control and NSCLC groups <b>(D)</b> Serum levels of GPx3 in the control and NSCLC groups. <b>(E)</b> ROC curves of BCHE, GPx3, and the combination of the two proteins. NSCLC: non-small cell lung cancer</p

Analyses of 9 transcriptome datasets from the GEO.

<p><b>(A)</b> A heat map of 2,696 differentially expressed probes between tumor (n = 669) and non-tumor tissues (n = 218) collected from 9 GEO data sets (p-values < 1 x 10⁻⁶ and over two-fold changes; red: up-regulation; green: down-regulation) <b>(B)</b> Classification of DEGs based on their molecular function as suggested by DAVID. <b>(C)</b> Subcellular locations of DEGs (grey: up-regulated genes in NSCLC; black: down-regulated genes in NSCLC).</p