| Metadata |
| datasetIdentifier | PASS01484 |
| datasetType | MSMS |
| submitter | Lingli Liu <leonie@nwsuaf.edu.cn> |
| submitter_organization | HongKong University of Science and Technology |
| lab_head_full_name | PeiYuan Qian |
| lab_head_email | boqianpy@ust.hk |
| lab_head_organization | HKUST |
| lab_head_country | Canada |
| datasetTag | itraqlabledcell |
| datasetTitle | Proteomic comparison of the cytotoxicology of two diastereomers of citreamicin |
| publicReleaseDate | 2019-11-23 00:00:00 |
| finalizedDate | 2019-11-22 10:51:21 |
| summary | An iTRAQ based quantitative proteomic analysis reveal the mechanism of cytotoxicity of citreamicin ε A and B in PtK2 cells. A total of 1079 proteins were identified and quantified, among which 103 and 94 proteins displayed significantly changes in expression levels after the treatment of citreamicin ε A and B, respectively. |
| contributors | Ling-Li Liu, Jin Sun, Ying Xu, Li-Sheng He, Kondethimmahalli H. Chandramouli, Pei-Yuan Qian |
| publication | unpublished |
| growth | |
| treatment | PtK2 cells were seeded in plate 12 h before the treatment with 0.1 μM citreamicin ε A or B for 6 h, 12 h, and 24 h. DMSO (vehicle control) was used as a negative control. |
| extraction | Cells were harvested and lysed in lysis buffer (8 M urea and 40 mM HEPES, pH = 7.4) on ice, followed by centrifugation at 15000 × g for 15 min at 4 °C. Supernatants containing cytosolic and other cellular proteins were precipitated by the addition of 4 volumes of cold acetone at -20 °C for 2 h, and then the proteins were quantified using the RC-DC kit |
| separation | Two iTRAQ reagents multiplex kit were used to label samples treated with 0.1 μM citreamicin ε A and B, respectively. PtK2 cells treated with DMSO (iTRAQ 114), PtK2 cells treated with citreamicin ε A or B for 6 h (iTRAQ 115), PtK2 cells treated with citreamicin ε A or B for 12 h (iTRAQ 116), and PtK2 cells treated with citreamicin ε A or B for 24 h (iTRAQ 117). The treatment time-points and the concentration of the treatment compounds were determined based on the previous TUNEL assay. The labeled peptides were pooled together before analysis. All combined peptide mixture samples were dried using a SpeedVac, followed by reconstituting with 100 μL of buffer A [10 mM KH2PO4, pH 3.0, ACN / H2O 25 / 75 (v / v)] respectively, and then loaded onto a Polysulfoethyl A strong cation exchange (SCX) column (200 mm × 4.6 mm, 200-Å pore size, 5 μm particle size) (PolyLC, Columbia, MD, USA) on a Waters Delta 600 HPLC (Waters, Milford, MA, USA). The samples were fractionated using a gradient of 100% buffer A for 10 min, 0-30% buffer B [10 mM KH2PO4, pH 3.0, 500 mM KCl, ACN / H2O 25 / 75 (v / v)] for 25 min, 30-100% buffer B for 5 min and 100% buffer B for 10 min at a flow rate of 1 mL/min for 1 h. |
| digestion | A total of 200 μg of peptide from each prepared cell lysate sample was reduced with 5 mM triscarboxyethyl phosphine hydrochloride (TCEP) at 60 °C for 1 h and alkylated with 10 mM methylethanethiosulfonate (MMTS) at room temperature for 20 min (Han et al., 2013). Each sample was then digested overnight at 37 °C with sequencing-grade trypsin. |
| acquisition | All of the dried peptide fractions were reconstituted in 30 μL 0.1% formic acid respectively and then analyzed with a Waters nanoACQUITY UPLC System and a Waters Q-Tof Premier Mass Spectrometer equipped with a nano-ESI source (nano AcquityTM, Waters, Milford, MA, USA). Peptides were separated using buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile) with a nanoflow gradient from 5% buffer B to 80% buffer B over 110 min at a flow rate of 300 nL/min and then back to the initial gradient of 5% buffer B. For electrospray analysis, the ESI positive ion mode with a survey scans ranging from m/z 300 to 2000 was applied. |
| informatics | Peptide identification and quantification were performed using ProteinLynx Global SERVER 2.2.5 (Waters Corp., Milford, MA, USA). Raw MS/MS data were converted into .pkl files with and without MS/MS deisotoping. The 4 groups of reporter ions were extracted from the nondeisotoped files and the respective deisotoped files of the same mass range were replaced by using a python script. The combined files with all fractions in each replicate were merged and submitted to Mascot version 2.3.0 (Matrix Sciences, Ltd., London, UK) to search against the database which concatenated real and reversed Human sequences. Trypsin was selected as enzyme for digestion, cysteine carbamidomethyl was selected as fixed modification, and methionine oxidation was selected variable modification. |
| instruments | Waters Q-Tof Premier Mass Spectrometer nano AcquityTM, Waters, Milford, MA, USA |
| species | Ptk2 cells |
| massModifications | none |
