CONSTRUCTION OF METHOD FOR PROTEOMICS RESEARCH

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Gene can display its function owing to translate correspond to protein, and the post-translational modification of protein is involved in a functional expression. Proteome research is a new field, which is starting to have a major impact in the post-genome era. For reconstruction of life system, proteomics focuses the structural and functional analysis of proteins, which exchange their expression dynamically and interact to other biological active substances.HeLa S3 cells were cultured with a medium containing 50 μM deoxycholic acid (DCA), and the effect of DCA stimulation to cells was investigated by the differential display. The clear protein expression change was observed in the cytosolic fraction, and the peptide fragments mixture was measured by MALDI-TOFMS on the heels of the in-gel digestion. According to the results using by peptide mass fingerprinting method, the interested protein was identified as peroxiredoxin I, which played an important role for the oxidative stress. On the other hand, an anti-DCA monoclonal antibody was produced using a DCA-bovine serum albumin immune complex. And it was immobilized to the agarose gel for production of immunoaffinity gel. The novel affinity labeling reagents, acyl adenylate and acyl 2-deoxyglucuronide, was developed for analysis of intramolecular interaction, and investigated their reactivity. The results indicated that the affinity labeling reagents developed could react selectively to interaction site on protein molecule by control of pH of reaction mixture. The peptide labeled DCA could be selectively extracted from enzyme digested peptide mixture using the immunoaffinity gel developed in this study.