Multifactorial Comparative Proteomic Study of Cytochrome P450 2E1 Function in Chronic Alcohol Administration

With the use of iTRAQ technique, a multifactorial comparative proteomic study can be performed. In this study, to obtain an overview of ethanol, CYP2E1 and gender effects on liver injury and gain more insight into the underlying molecular mechanism, mouse liver proteomes were quantitatively analyzed using iTRAQ under eight conditions including mice of different genders, wild type versus CYP2E1 knockout, and normal versus alcohol diet. A series of statistical and bioinformatic analyses were explored to simplify and clarify multifactorial comparative proteomic data. MALDI plates were analyzed with a TOF/TOF 5800 mass spectrometer (AB Sciex). The instrument was calibrated using the 4700 mass calibration standard. MS spectra between m/z 800 and 4,000 were acquired for every spot using 1,000 laser shots in reflector mode. The 20 most intense ion signals per spot having a S/N > 10 were selected as precursors for MS/MS acquisition using 2,000 laser shots. Peptide and protein identifications were performed with the ProteinPilot Software 4.5 (AB Sciex) using the Paragon algorithm. Combined data and spectra from all 24 OFFGEL fractions were searched against the UniProt mouse database (release-2010_11). The following search parameters were selected: iTRAQ 8-plex peptide label, cysteine alkylation, trypsin specificity, ID focus on biological modifications, and processing including quantitation and thorough ID. A protein with a confidence threshold of 95% (unused confidence threshold ProtScore>1.3) was reported and the corresponding False Discovery Rate (FDR) was less than 1%. In protein grouping, competitor threshold was set as 2.00 in ProtScore.