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Response Profiling Using Shotgun Proteomics Enables Establishing Global Metallodrug Mechanisms of Action - cytoplasmic proteins of KP1537-treated SW480 epithelial cells
Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, was applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not in HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80% convergence of protein identifications, but with significant expression differences, which in turn seem affect the extent of protein regulation. For example, while a clear-cut redox response was observed in SW480 cells upon arsenic treatment, such an effect was lacking in HCT116 cells and the latter express drastically higher levels of the involved redox proteins. Response profiling was then used to investigate four anticancer metallodrugs (KP46, KP772, KP1339 and KP1537) by means of functional groups of proteins and mapped their effects according to DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. We were able to characterize the global effects of these metallodrugs on the proteome and generate hypotheses on hitherto unrecognized mechanisms. Significant differences in the two colon cell lines strongly suggest that knowledge of mechanistic hallmarks of anticancer metallodrug action are imperative for the design of clinical studies and that outcome may be enhanced by means of patient stratification strategies according to these hallmarks.
Sample Processing Protocol
SW480, kindly provided by Michael Jakupec (Department of Inorganic Chemistry, University of Vienna, Austria), were cultured in EMEM (Sigma-Aldrich, USA) supplemented with 10% FCS, 100U/ml penicillin/streptomycin, 0.1mM non-essential amino acids and 1mM sodium pyruvate at 37°C and 5% CO2. Cells were treated with 0.5µM KP1537. Experiments were conducted for 24 hours in T25 flasks in triplicates with on average 107 cells per flasks. With the combination of an isotonic lysis buffer containing protease inhibitors and shear stress cytoplasmic proteins were released and after centrifugation (2300xg, 5min, 4°C) precipitated with ice-cold ethanol at -20°C over night. Precipitated proteins were dissolved in sample buffer (7.5M urea, 1.5M thiourea, 4% CHAPS, 0.05% SDS, 100mM dithiothreitol (DTT)) and a Bradford assay (Bio-Rad-Laboratories, Germany) was employed to determine protein concentrations. In solution digestions were performed with 25µg of protein. The samples were pre-concentrated on 10kDa filters (Pall Austria Filter GmbH), reduced with DTT (56°C, 30 min) and carbamidomethylated with iodacetamide (45 min in the dark). To receive peptides, samples were digested with a trypsin/lys-c mixture (Promega, Germany) for 18h at 37°C. Eluates were dried and stored at -20°C until analysis. For analysis a nanoLC-system (Dionex Ultimate 3000, Thermo Scientific, Austria) was coupled to a high resolution QExactive orbitrap mass spectrometer (Thermo Scientific, Austria). Dried samples were resolved in 5µL 30% formic acid (FA) containing four synthetic peptides (10fmol each) and diluted with 40µL mobile phase A (98% H2O, 2% ACN, 0.1% FA). Each sample was recorded twice, 5µL were injected per analysis. The LC-method lasted 135 minutes with a 90min long gradient from 8-40% solvent B (80%ACN, 20%H¬2O, 0.1% FA) running over a 75µm x 50cm C-18 separation column (Dionex, Acclaim PepMap RSCL). The MS-method lasted 115min with the resolution set to 70000 and 17500, maximum injection time to 50ms and 75ms for MS1 and MS2, respectively. M/z was scanned from 400-1400 and a top 8 method was chosen for MS/MS analysis.
Data Processing Protocol
Proteome Discoverer 1.4 (Thermo Fisher Scientific, Austria) running Mascot 2.4 (Matrix Science, UK) was used for protein identification. Protein identification was achieved searching against the SwissProt Database (version August 2014 with 20 194 entries) allowing a mass tolerance of 10ppm for MS spectra and 50mmu for MS/MS spectra as well as a maximum of 2 missed cleavages. Furthermore, search criteria included carbamidomethylation on cysteins as fixed modification and methionine oxidation as well as N-terminal protein acetylation as variable modifications.
Kreutz D, Bileck A, Plessl K, Wolrab D, Groessl M, Keppler BK, Meier SM, Gerner C. Response Profiling Using Shotgun Proteomics Enables Global Metallodrug Mechanisms of Action To Be Established. Chemistry. 2016 Nov 7 PubMed: 28071820
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||67366||no assay title provided (mzIdentML)||5727||36438||25083||34213||24022||
|2||67367||no assay title provided (mzIdentML)||5581||35398||24163||33546||23693||
|3||67364||no assay title provided (mzIdentML)||5401||34129||23679||32512||22842||
|4||67365||no assay title provided (mzIdentML)||5470||34165||23687||32528||22656||
|5||67362||no assay title provided (mzIdentML)||4248||26575||17500||24928||17414||
|6||67363||no assay title provided (mzIdentML)||4816||30859||21406||28599||20587||