D in an Ettan Spot Handling Workstation (GE Healthcare). The selected
Post date July 18, 2017 Post last updated dateUpdated July 18, 2017 Post read time4 min read Post author
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D in an Ettan Spot Handling Workstation (GE Healthcare). The selected protein spots were washed with 15 mM ammonium bicarbonate and 50 methanol and then digested in 0.02 mg/mL sequencing grade trypsin solution (Promega, Madison, WI, USA) at 37uC for 2 h. The tryptic peptides were extracted with 50 (v/v) acetonitrile (ACN) and 0.5 (v/v) trifluoroacetic acid (TFA), dissolved in 5 mg/mL R-cyano-4-hydroxycinnamic acid (Amersham Bioscience) in 50 (v/v) ACN and 0.1 (v/v) TFA and then spotted on the MS sample plate.Matrix-assisted Laser PD-168393 Desorption/ionization Time-of-flight Mass BIBS39 Spectrometry (MALDI-TOF/TOF MS) AnalysisProtein identification was performed with the 12926553 ABI 4800 Proteomics MALDI-TOF/TOF Analyzer (Applied Biosystems, Foster City, CA, USA) in positive ion reflector mode. Monoisotopic peak masses were acquired in a range of 900?,000 Da with a signal-to-noise ratio (S/N) .200. Trypsin autolytic peptides of masses 842.5 and 2211.1 were used as internal standards. Five of the most intense ion signals were automatically selected as precursors for MS/MS acquisition, excluding the trypsin autolysis peaks and matrix ion signals. The peptide mass fingerprint (PMF) combined MS/MS spectra were searched against the NCBInr database using GPS ExplorerTM software (Version 3.6, Applied Biosystems) and MASCOT version 2.1 (Matrix Science). The search parameters were set as follows: Homo sapiens, trypsin cleavage (one missed cleavage allowed), carbamidomethylation as fixed modification, methionine oxidation as variable modification, peptide mass tolerance set at 75 ppm and fragment tolerance set at 0.2 Da. A significantly high MASCOT score that resulted in a confident interval (CI) greater than 95 for PMF or MS/MS data for a spot was considered as a credibly identified protein. The other criteria included a minimum of four peptides hits in PMF data-based identification and at least two peptides with distinct sequences identified in MS/MS analysis.2D-DIGE Analysis and MS/MS IdentificationAccording to our experimental design described 23727046 in Materials and methods, four 2D-DIGE gels in total were set up for proteomic analysis of chemoresistant versus chemoresistant patient ascites. For each gel a merged image was generated from three images of the chemosensitive, chemoresistant and internal standard samples. A representative DIGE gel showing the overlay of the Cy2, Cy3 and Cy5 labeled images is shown in Figure 2. A total of 1523 to 1711 spots were detected in different Differential In-gel Analysis (DIA) workspaces in all the gels using DeCyder software. In the Biological Variation Analysis (BVA) module, the Cy3 image from gel number four was chosen as the master gel as it had the maximum number of spots. Thirty-four spots were found to be differentially expressed based on the criteria of having an average ratio of more than +1.5 or less than 21.5 and a student t-test P value ,0.05. Among them, 14 spots were found to be down-regulated in the chemoresistant ascites, and 27 were up-regulated compared to the chemosensitive patients. Some of the differential spots detected by DeCyder software could not be visualized in the preparative gel stained with colloidal coomassie likely due to low abundance. After visual review, 20 protein spots showing high abundance and significantly altered expression in chemoresistant versus chemosensitive patients were selected for MALDI-TOF/TOF MS analysis. A total of 11 differentially expressed proteins, including 3 up-regulated.D in an Ettan Spot Handling Workstation (GE Healthcare). The selected protein spots were washed with 15 mM ammonium bicarbonate and 50 methanol and then digested in 0.02 mg/mL sequencing grade trypsin solution (Promega, Madison, WI, USA) at 37uC for 2 h. The tryptic peptides were extracted with 50 (v/v) acetonitrile (ACN) and 0.5 (v/v) trifluoroacetic acid (TFA), dissolved in 5 mg/mL R-cyano-4-hydroxycinnamic acid (Amersham Bioscience) in 50 (v/v) ACN and 0.1 (v/v) TFA and then spotted on the MS sample plate.Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry (MALDI-TOF/TOF MS) AnalysisProtein identification was performed with the 12926553 ABI 4800 Proteomics MALDI-TOF/TOF Analyzer (Applied Biosystems, Foster City, CA, USA) in positive ion reflector mode. Monoisotopic peak masses were acquired in a range of 900?,000 Da with a signal-to-noise ratio (S/N) .200. Trypsin autolytic peptides of masses 842.5 and 2211.1 were used as internal standards. Five of the most intense ion signals were automatically selected as precursors for MS/MS acquisition, excluding the trypsin autolysis peaks and matrix ion signals. The peptide mass fingerprint (PMF) combined MS/MS spectra were searched against the NCBInr database using GPS ExplorerTM software (Version 3.6, Applied Biosystems) and MASCOT version 2.1 (Matrix Science). The search parameters were set as follows: Homo sapiens, trypsin cleavage (one missed cleavage allowed), carbamidomethylation as fixed modification, methionine oxidation as variable modification, peptide mass tolerance set at 75 ppm and fragment tolerance set at 0.2 Da. A significantly high MASCOT score that resulted in a confident interval (CI) greater than 95 for PMF or MS/MS data for a spot was considered as a credibly identified protein. The other criteria included a minimum of four peptides hits in PMF data-based identification and at least two peptides with distinct sequences identified in MS/MS analysis.2D-DIGE Analysis and MS/MS IdentificationAccording to our experimental design described 23727046 in Materials and methods, four 2D-DIGE gels in total were set up for proteomic analysis of chemoresistant versus chemoresistant patient ascites. For each gel a merged image was generated from three images of the chemosensitive, chemoresistant and internal standard samples. A representative DIGE gel showing the overlay of the Cy2, Cy3 and Cy5 labeled images is shown in Figure 2. A total of 1523 to 1711 spots were detected in different Differential In-gel Analysis (DIA) workspaces in all the gels using DeCyder software. In the Biological Variation Analysis (BVA) module, the Cy3 image from gel number four was chosen as the master gel as it had the maximum number of spots. Thirty-four spots were found to be differentially expressed based on the criteria of having an average ratio of more than +1.5 or less than 21.5 and a student t-test P value ,0.05. Among them, 14 spots were found to be down-regulated in the chemoresistant ascites, and 27 were up-regulated compared to the chemosensitive patients. Some of the differential spots detected by DeCyder software could not be visualized in the preparative gel stained with colloidal coomassie likely due to low abundance. After visual review, 20 protein spots showing high abundance and significantly altered expression in chemoresistant versus chemosensitive patients were selected for MALDI-TOF/TOF MS analysis. A total of 11 differentially expressed proteins, including 3 up-regulated.
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D in an Ettan Spot Handling Workstation (GE Healthcare). The selected protein spots