Study On The Application Of "Shotgun" Proteomic Strategies In Solving Several Bioengineering Problems

The"shotgun"proteomic technologies and strategies were introduced to solving the bioengineering problems. Firstly, it was used to investigate the antibiotic biosynthesis pathways. Secondly, it was used to seek candidate tumor biomarkers and developing of new drug targets. Thirdly, 18O stable isotope labeling technology was introduced to quantificationally investigate the proteome changes in process of apoptosis of k562 induced by docetaxel.For strain 103 and cnn1, a total of 726 and 809 proteins were identified respectively using 2D LC-MS/MS. By exploring the key enzymes of several probable antibiotic biosynthesis pathways in the identified proteins of strain 103, only polyketide synthase and other proteins associated with the polyketide pathways were found, indicating that Maituolaimysin might be synthesized through the polyketide pathway. Combining with the results of the inhibition experiments of beta-ketoacyl ACP synthase, the above conclusion was proved.SDS-PAGE and sub-cellular pre-fractionation were added to achieve a more comprehensive protein profile of k562 cell line. A total of 1707 non-redundant proteins were identified. Proteins like to be associated with cell cycle, apoptosis, tumor and leukemia were investigated. Many proteins that have the potential to be biomarkers or drug targets of leukemia therapy, including MLL3, SET, DEK, Stathmin and nucleophosmin were identified unambiguously to exist in k562 cell line. For the nuclear proteins of k562 cell line, a total of 334 proteins were identified. Proteins probably associated with tumor such as NF45 protein,PIBF1,DDX48,IKAP were found.A total of 86 proteins were analyzed in the experiment of 18O labeled quantitative proteomics. Forty-six proteins were equal-expressed. Fifteen proteins were down-expressed including SET, EEF1A1, etc. Twenty proteins were up-expressed including alpha enolase, Peroxiredoxin-2, etc. These repressed or activated proteins were the potential tumor biomarkers and drug targets, which would offer the candidate proteins for tumor diagnosis and treatments.