| Summary: | 碩士 === 國立臺北科技大學 === 化學工程所 === 93 === Two-dimensional electrophoresis (2DE) combined with mass spectrometry has significantly improved the possibilities of large-scale identification of proteins.
However, 2DE is limited by their inability to speed up the in-gel digestion process. We developed a new approach to speed up protein identification process utilizing microwave technology. Proteins excised from gels are subjected to in-gel digestion with endoprotease trypsin under microwave irradiation, which rapidly produces
peptide fragments. The peptide fragments were further analyzed by matrix-assisted laser desorption ionization (MALDI) technique for protein identification. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several proteins, including lysozyme, albumin, conalbumin, and ribonuclease A. The method reduced the required time for in-gel digestion of proteins from 16 hours to as short as 5 minutes. This new application of microwave technology to protein identification will be an important advancement in biotechnology and proteome research. Therefore, we applied this microwave method to investigate the differentiation of HL-60 to macrophages.
Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. HL-60, a human myeloid cancer cell line, can be induced to differentiate into benign noncancerous macrophages by 12-O-tetradecanoylphorphorbol-13- acetate (TPA). The differentiation of HL-60 can be mediated not only by the cell surface receptors but also by numerous soluble and membrane anchored mediators. Concerning specific subcellular compartments and macromolecular structures of the cell can not only provide information about the possible cell surface receptors and mediators during this differentiation, but also tell us
subcellular location of certain proteins and help understand their structures and biological functions. In this study, we used a new approach to speed up subcellular protein identification process utilizing microwave technology combined with mass spectrometry. Therefore, we apply a new method to remove sodium dedecyl sulfate (SDS) before in-gel digestion. We have identified thirteen proteins in the differentiation process. In our results, we found four proteins have isoforms; these proteins are peptidyl-prolyl cis-trans isomerase A, prohibitin, enolase 1, myeloperoxidase precursor. It is likely that characterization of the changes that occur at the subcellular components during differentiation in response to TPA will lead to a better understanding of these processes and further expand the repertoire of diagnostic and therapeutic target.
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