Computational protocol: Persistent inhibition of pore-based cell migration by sub-toxic doses of miuraenamide, an actin filament stabilizer

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[…] Cells were seeded in 10 cm dishes and treated with 20 nM miuraenamide for 4, 8, 16, 24, 32, 48, 56, 72 and 96 h and harvested simultaneously. We can not completely exclude that some cyclic behavior of the cells (e.g. depending on cell cycle) might have altered protein expression in addition to treatment. Subsequently, samples were lysed in 8 M urea with 40 mm Tris/HCl (pH 7.6), 1 × EDTA-free protease inhibitor cocktail (Complete Mini, Roche), and 1 × phosphatase inhibitor mixture (Sigma-Aldrich). The lysate was centrifuged at 10,000 × g for 10 min and the supernatant was kept at −80 °C before further processing. It was then reduced with 10 mM DTT and alkylated with 50 mM chloroacetamide before digested overnight with trypsin (Promega, Madison, WI). The digest was then desalted with C18 SepPak columns (Waters, MA). TMT labeling of desalted digest was carried out with TMT10 reagents (ThermoFisher Scientific) according to the manufacturer’s instruction. Peptide mixture was then fractionated with a Trinity P1 column on a Dionex Ultimate 3000 HPLC system (both ThermoFisher Scientific) into 32 fractions. These fractions were then vacuum dried and reconstituted in 0.1% formic acid (FA) for LC-MS/MS measurement.The samples were measured on a Dionex Ultimate 3000 nanoLC (ThermoFisher Scientific) coupled to an Orbitrap Q Exactive HF mass spectrometer (ThermoFisher Scientific). NanoLC separation was carried out using an in-house packed capillary column (75 μm × 45 cm) filled with 3 μm Reprosil Gold C18 particles (Dr. Maisch GmbH, Ammerbuch, Germany) at 300 nL·min-1. Sample was loaded onto a trap column (75 μm × 2 cm) in 0.1% formic acid at 5 μL·min−1 for 10 min. The trap column was packed with 5 μm Reprosil ODS-3 particles. The analytical column was heated to 50 °C using a 30-cm capillary column heater (ASI, Pompton Plains, NJ). Solvent A was 0.1% FA, 5% DMSO in water; solvent B was 0.1% FA, 5% DMSO in acetonitrile,. For the analysis of regular peptides, the gradient was 0–1 min, 2–4% B; 1–52 min, 4–32% B; 52–54 min, 35–80% B; 54–56 min, 80% B; 56–58 min, 80–2% B; 58–60 min, 2% B. A top 25 data dependent acquisition method was used for MS. The survey scan was acquired at 60,000 resolution with a mass range of 360–1300 m/z and AGC target value of 3e6. The maximum injection time (max IT) was 100 ms. MS/MS spectra were acquired at 30,000 resolution with fixed first mass at 120 m/z. AGC target was 2e5 and max IT was 57 ms. Isolation window was 1.7 m/z and normalized collision (NCE) energy was 33. An underfill ratio of 1.0% was used and + 1, + 7 and higher, and unknown charge states were excluded. Further setting included “peptide match preferred” and the “exclude isotopes” option was turned on. Dynamic exclusion was set to 20 s.The raw files were searched using MaxQuant (v1.5.3.30) against the UniProtKB Human Reference Proteome database (v22.07.13, 88,381 entries). Default MaxQuant parameters were used and the match-between-runs feature was enabled. The results were further processed in Perseus (v1.5.5.3). The detected proteins were grouped into ten clusters based on Pearson correlation. The clusters with down- and up-regulated proteins after stimulation for 56 h were depicted in plots with the statistic software R (R version 3.3.2). GOBPs (Gene Ontology and Biological Pathways) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were plotted with a threshold of FDR < 0.5. Pathway with FDR < 0.2 (plotted in orange) and the highest enrichment values (enrichment > 2) were depicted in red with annotations. […]

Pipeline specifications

Software tools Trinity, MaxQuant, Perseus
Databases UniProtKB KEGG
Application MS-based untargeted proteomics
Diseases Neoplasms, Drug-Related Side Effects and Adverse Reactions