| Metadata |
| datasetIdentifier | PASS00731 |
| datasetType | SRM |
| submitter | Heesung Ahn <zaulim3@gmail.com> |
| submitter_organization | Korea Institute of Science and Technology |
| lab_head_full_name | Cheolju Lee |
| lab_head_email | clee270@kist.re.kr |
| lab_head_organization | Korea Institute of Science and Technology |
| lab_head_country | Republic of Korea |
| datasetTag | ARS_SEC_AP |
| datasetTitle | Evaluation of multi-tRNA synthetase complex by multiple reaction monitoring mass spectrometry coupled with size exclusion chromatography |
| publicReleaseDate | 2015-08-04 00:00:00 |
| finalizedDate | |
| summary | MRM quantification of ARS family proteins in 20 SEC fractions of HEK 293T, KARS-overexpressed HEK 293T (KARSoe), and elute of KARSoe-AP (KARSoe-AP).
|
| contributors | Seong-Jun Park,
Hee-Sung Ahn,
Jun Seok Kim,
Cheolju Lee |
| publication | Seong-Jun Park, Hee-Sung Ahn, Jun Seok Kim, and Cheolju Lee, Evaluation of multi-tRNA synthetase complex by multiple reaction monitoring mass spectrometry coupled with size exclusion chromatography, PLOS ONE, accepted |
| growth | The HEK 293T cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) in a humidified incubator in an atmosphere of 95% air and 5% CO2 at 37 °C. Cells were grown to ~80% confluence at the initiation of the experiment. KARS was cloned into a vector, pIRES2-EGFP-SBP, engineered to express fusion proteins with N-terminal S, FLAG and streptavidin binding peptide (SBP) tags. The construct was transiently transfected into HEK 293T cell line using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics, Indianapolis, IN, USA) for 36 h at 37 °C. The HEK 293T and transfected cells (KARSoe) were harvested at a confluence of 80~90% and lysed using NETN buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.1 % Nonidet P-40, and Protease Inhibitor Cocktail (Roche Diagnostics). Cell lysate was pelleted by centrifugation (12,000 rpm, 10 min, 4 °C) and the supernatant was collected. |
| treatment | |
| extraction | Briefly, 50 µL of streptavidin agarose beads (Thermo Scientific) were activated and equilibrated with 500 µL of NETN buffer twice. KARSoe cell lysate (2 mg) was added to the agarose beads and subjected to rotation (10 rpm, 2 h, 4 °C). After incubation, the mixtures were washed three times with NETN buffer. The bound proteins were eluted employing 30 µL of NETN buffer containing biotin (0.82 mM) using a 0.22 µm centrifugal filter device (Millipore, Billerica, MA, USA). The elution step was repeated twice. |
| separation | The supernatant from HEK 293T (3 mg), KARSoe (3 mg) and streptavidin affinity purification from KARSoe cells (denoted as KARSoe-AP afterwards) (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 (Millipore). |
| digestion | 40 µL of protein mixture 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. |
| acquisition | To stop the tryptic reaction, 0.3% trifluoroacetic acid (TFA) (pH < 3.0) was added. 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 mL of 2% acetonitrile and 0.1% formic acid, injected with a full sample loop injection of 1 µL.Raw files of MRM-MS data (*.wiff) from Analyst software (Version 1.5.2, ABSciex) were processed in Skyline (version 2.6.0). The program was designed to extract quantitative information from MRM measurements based on extracted ion chromatogram (XIC). The most intense transition of each peptide was selected for the quantification and the other transitions together with the most intense transition were used for peak assignment and validation. The identification and quantification of endogenous peptides were based on the corresponding SIS peptides. |
| informatics | All statistical data were analyzed by using R package (version 3.0.3) and Excel 2010 (version 14.0, Microsoft Office). |
| instruments | QTRAP 5500 |
| species | Human |
| massModifications | static: C+57.021464, variable: variable: K+8.014199, R+10.008269 |
