Tyrosine Kinase Inhibitor-induced cardiotoxicity in hiPSC derived cardiomyocytes - Proteomics - Dataset (ID:20349)
| HMS Dataset ID: | 20349 |
| Dataset Title: | Tyrosine Kinase Inhibitor-induced cardiotoxicity in hiPSC derived cardiomyocytes - Proteomics |
| Screening Lab Investigator: | Huan (Sharon) Wang, Robert Everley, Alison Erickson |
| Screening Principal Investigator: | Peter K. Sorger |
| Assay Description: |
To define protein markers of tyrosine kinase inhibitor-induced cardiotoxicity, we treated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with tyrosine kinase inhibitors (TKIs) displaying a range of mild to severe effects on heart function. Two independent cell experiments were performed. In the first experiment, hiPSC-CMs were treated with one of four TKIs (Erlotinib, Lapatinib, Sorafenib and Sunitinib) at 3 μM or a DMSO vehicle-only control in biological replicates (10 samples in total) and harvested at 72 hours for tandem mass tag mass spectrometry. In the second experiment, a different lot of hiPSC-CMs were treated the same way, but samples were collected at both 24 and 72 hours (20 samples in total). Corresponding gene expression data can be found in datasets 20324 and 20325. |
| Assay Protocol: |
1. Cor.4U hiPSC-CMs were cultured on 60mm plates in BMCC media (Ncardia, previously Axiogenesis) containing 1% fetal bovine serum during drug treatment. Media was changed every other day during culture. 2. At 24 or 72 hours after treatment, cells were rinsed with PBS and gently scraped off plates into lysis buffer (2% SDS, 150 mM NaCl, 50 mM Tris, pH 8.5) containing protease and phosphatase inhibitors (Roche). Cell lysates were then vortexed and passed through QIAshredder columns (Qiagen). Protein concentration was determined for each sample by BCA assay (Thermo Fisher Scientific). 3. After cell lysates were reduced with 5 mM DTT and alkylated with 15 mM iodoacetamide (Sigma-Aldrich), 0.15mg of protein from each sample (as determined by BCA) underwent methanol/chloroform precipitation. 4. Precipitates were re-solubilized in urea in EPPS (Sigma-Aldrich, pH 8.5) and further digested using Lys-C (Wako Chemicals) and Trypsin (Promega). 5. Samples (60 μg each) were then dissolved into 10% (v/v) acetonitrile (ACN) and labeled 2:1 (TMT:Peptide ratio) with TMT-10 reagent (Thermo Fisher Scientific). TMT-labeling reactions were quenched by adding hydroxylamine (0.5% final volume, Sigma-Aldrich). 6. The samples were combined, acidified with formic acid (FA, Thermo Fisher Scientific) to 2% final volume, and desalted using a C18 Sep-Pak (Waters). 7. The combined sample was fractionated based on basic pH reversed phase chromatography using an Agilent 1200 HPLC equipped with a UV-DAD detector and fraction collection system (Agilent). A total of 12 fractions were collected and desalted using the C18 StageTip procedure. 8. Each fraction was loaded onto a column (35 cm long x 100 µm inner diameter) packed with 1.8 μm beads (Sepax) and separated using a 3-hour gradient from 8% Buffer B and 92% Buffer A to 27% Buffer B and 73% Buffer A (Buffer B: 99% ACN and 1% FA. Buffer A: 96% water, 3% ACN and 1% FA) using an Easy 1000 nano-LC (Thermo Fisher Scientific) before injection into an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) using a multi-notch MS3 method. 9. Raw data were converted to mzXML and searched via Sequest (version 28) against a concatenated Uniprot database downloaded on 02/04/2014. Variable modifications of oxidized Methionine and over-labeling of TMT on Serine, Threonine and Tyrosine were considered. Linear discriminant analysis was used to distinguish forward and reverse hits, and reverse hits were filtered to a false discovery rate of 1% at the protein level. Shared peptides were collapsed into the minimally sufficient number of proteins using rules of parsimony. Quantitation filters of > 200 sum reporter ion S:N and > 0.7 isolation specificity were incorporated. After converting raw data into values of signal intensity based on these pre-processing steps, data were further normalized such that protein expression values for each protein summed to 100 for the 10 samples in each 10-plex TMT experiment. 10. The two 10-plex TMT experiments for 72-hour treatments were combined by calculating the mean of one replicate from each of the two experiments. To confirm the validity of combining the results from the two independent experiments, we performed a hierarchical clustering analysis based on the average unweighted distance between each replicate, as implemented in the linkage method in MATLAB software. As expected, the four replicates for each condition, spanning both experiments, consistently cluster together. |
| HMS Dataset Type: | Proteomics |
| Date Publicly Available: | 2018-07-18 |
| Most Recent Update: | 2018-07-18 |