Here, we define the spectrum of functional CHR elements

. The equilibrated strips were transferred onto 12% homogenous polyacrylamide gels casted in low fluorescence glass plates using an Ettan-DALT six system. Electrophoresis was run at 2 watts/gel and 20 C for about 17 h. The differentially labeled co-resolved proteins within each gel were imaged at 100 dots/inch resolution using a DIGE Imager scanner. Cy2-, Cy3-, and Cy5-labeled images of each gel were acquired at excitation/emission values of 488/520, 523/580, and 633/670 nm, respectively. Gels were scanned directly between the glass plates, and the 16-bit image file format images were exported for data analysis. After imaging for Cy-Dyes, the gels were removed from the plates and stained with colloidal Coomassie, following standard procedures. 3.5. Image Acquisition and DIGE Data Analysis Semi-automated image analysis was performed with Progenesis SameSpots V3.2 software. Image quality control was first performed to identify saturated spots. Multiplexed analysis was selected for DIGE experiments and a representative gel image was chosen as reference. Spots were detected and their normalized volumes were ranked on the basis of ANOVA p-values and fold changes. In the proteomic analyses, normalization tools and statistical package from SameSpots and ProteinPilot software were employed. 3.6. MS Analysis of the Gel PubMed ID: Spots The gel spots of interest were manually excised and transferred to microcentrifuge tubes. Samples selected for analysis were in-gel reduced, alkylated and digested with trypsin according to the method of Sechi and Chait. The samples were analyzed using the MALDI -TOF /TOF mass spectrometer 4800 Proteomics Analyzer and 4000 Series ExplorerTM Software. Data Explorer version 4.2 was used for spectra analyses and for generating peak-picking lists. All mass spectra were internally calibrated using autoproteolytic trypsin fragments and externally calibrated using a standard peptide mixture. TOF/TOF fragmentation spectra were acquired by selecting the 10 most abundant ions of each MALDI-TOF peptide mass map. 3.7. Database Search The mono-isotopic peptide mass fingerprinting data obtained by MS and the amino acid sequence tag obtained from each peptide fragmentation in MS/MS analyses were used to search for protein candidates using Mascot version 2.2 from Matrix Science. Peak intensity was used to select up to 50 peaks per spot for peptide mass fingerprinting, and 50 peaks per precursor for MS/MS Int. J. Mol. Sci. 2014, 15 12585 identification. Tryptic autolytic fragments, keratin and matrix-derived peaks were removed from the dataset used for the database search. The searches for peptide mass fingerprints and tandem MS spectra were performed in the UniProt knowledgebase, by searching in the UniProtKB/Swiss-Prot database, containing 519348 entries. Fixed and variable modifications were considered, allowing one trypsin missed cleavage site and a mass tolerance of 50 ppm. For MS/MS identifications, a precursor tolerance of 50 ppm and MS/MS fragment tolerance of 0.3 Da were used. Identifications were accepted as JW 55 positive when at least five peptides matched and at least 20% of the peptide coverage of the theoretical sequences matched within a mass accuracy of 50 or 25 ppm with internal calibration. Probability scores were significant at p < 0.01 for all matches. 3.8. Cisplatin Resistance Sensitivity to the cytotoxic effect of cisplatin was assessed using a colony formation assay. Cultures in 1 mL SD containing a total of 6 106 cells wer

Comments are closed.