Computational protocol: A Proteomic-Based Approach to Study the Mechanism of Cytotoxicity Induced by Interleukin-1α and Cycloheximide

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Protocol publication

[…] The HPLC–MS/MS experiments were performed on the Finnigan LTQ (Thermo Finnigan, San Jose, CA, USA) instrument. The HPLC was equipped with a thermostated column oven and Surveyor autosampler controlled at 10 °C, a quaternary gradient Surveyor MS pump and a diode array detection (DAD) system. It was combined on-line with the LTQ linear ion trap MS system with electrospray (ESI) ion source controlled by Xcalibur software 1.4 (Thermo Finnigan, San Jose, CA, USA).The HPLC separation was performed on C-18 analytical column: XTerra MS C18 3.5 μm (2.1 × 100 mm) produced by Waters (Milford, MA, USA). Peptides elution was achieved using 120-min linear gradient from 0% B to 60% of solvent B at 200 μL min−1 flow rate. The mobile phases were: solvent A—0.1% FA in water, and solvent B—0.1% FA in acetonitrile, mixed on-line. All HPLC reagents were MS-grade and obtained from Sigma-Aldrich (Steinheim, Germany). The injection volume was 10 μL.The mass spectra were generated in positive ion mode, in the 500–2000 m/z range, under constant instrumental conditions: source voltage 4.6 kV, capillary voltage 41 V, sheath gas flow rate 40 (arbitrary units), auxiliary gas flow 10 (arbitrary units), sweep gas flow 1 (arbitrary units), capillary temperature 220 °C, and tube lens voltage −105 V. MS/MS spectra were obtained by collision-induced dissociation in the linear ion trap, with an isolation width of 3 Da (m/z); the activation amplitude was 35% of ejection RF amplitude (this corresponds to 1.58 V). m/z values, measured for the most intense peaks in acquired MS/MS spectra, were automatically searched against the protein database using the Sequest algorithm, incorporated into BioWorks 3.1 (Thermo Finnigan, San Jose, CA, USA). The human ‘.fasta’ format was downloaded from UniProtKB []. Trypsin was defined as the cleavage enzyme and up to three missed cleavages were allowed. As a result of this search we obtained peptides scoring parameters—the difference between normalized cross-correlation functions for the first and second ranked results (ΔC n) and cross-correlation score between the observed peptide fragment mass spectrum and the theoretically predicted one (X corr). To correctly identify peptides, we applied the levels of these filtering parameters evaluated by Washburn and co-workers [] Accepted X corr values for +1 charged fully tryptic peptides were higher than 1.9, over 2.2 or 3.3 for fully and partially tryptic +2 and +3 charged peptides. The ΔC n values were above 0.08 for all analyzed spectra. Basing on the results obtained for each HeLa sample type, the proteins, detected only in one of four of them, were distinguished (Table , Online Resources ESM_1). [...] The bioinformatic analysis of the HeLa differentiating proteins was performed with the use of PANTHER Classification System (Protein ANalysis THrough Evolutionary Relationships). The gene names of these proteins were uploaded to the PANTHER Gene List Analysis tool, freely available at []. The proteins were classified and organized according to their gene ontology (GO) molecular function (Fig. ) and PANTHER protein class (Fig. ) categories. The results were depicted in pie charts. The assigned to each category proteins were also presented on sides of the pie charts.Fig. 1 Fig. 2 [...] The verification of protein disulphide isomerase (PDI) levels in the investigated HeLa cell cultures was performed using Enzyme-linked Immunosorbent Assay (ELISA) Kit for Human Protein Disulphide Isomerase from Uscn Life Science Inc. (Wuhan, Hubei, PRC), according to the producer’s instructions. The colorimetric measurements were performed on Multiskan FC Microplate Photometer (Thermo Scientific, Rockford, IL, USA) at the wavelength of 450 nm. The assay detection range was from 0.312 to 20 ng mL−1. The PDI concentrations were determined then by comparing the samples O.D. to the standard curve and normalized to total protein concentration for each sample. The data were calculated as nanograms of PDI per milligram of total protein.The obtained PDI concentrations for each type of differently stimulated HeLa cells, namely HeLaIL-1, HeLaCHX and HeLaIL-1/CHX, were then compared to the PDI concentration in the control sample (HeLaK). Statistical significance of the differences in PDI concentrations between these samples was verified using t Student test on Statistica 10.0 software (Statsoft, Tulsa, OK, USA). The differences were considered as statistically significant, when obtained for them p values were below 0.05. […]

Pipeline specifications

Software tools Comet, PANTHER, Statistica
Databases UniProtKB
Applications Miscellaneous, MS-based untargeted proteomics, Protein sequence analysis
Diseases Neoplasms, Drug-Related Side Effects and Adverse Reactions
Chemicals Cycloheximide