應用微波技術探討人類骨髓癌細胞分化為巨噬細胞之次細胞蛋白質體學分析

Application of Microwave Technology to Investigate a Subcellular Proteomics Analysis of Human Myeloid Leukemia Cells Differentiation to Macrophages

張幸娟(Shing-Chuan Chang)

指導教授 ： 陳灝平 ； 阮雪芬

國立臺北科技大學/工程學院/化學工程所/碩士(2005年)

摘要

二維膠體電泳（two-dimensional electrophoresis, 2-DE）結合質譜儀（mass spectrometry）很明顯地已改善大規模蛋白質的身分鑑定（protein identification）。然而，由於膠體內水解（in-gel digestion）步驟之故，使得二維膠體電泳無法快速完成。我們發展了一個新方法，即是利用微波（microwave）技術來加速蛋白質身份鑑定。藉由微波照射加速內切胰蛋白酶（endoprotease trypsin）膠體內水解反應，使蛋白質能迅速地分解成胜肽片段。這些胜肽片段（peptide fragments）進一步以基質輔助雷射脫附離子化（matrix-assisted laser desorption/ionization, MALDI）方法來鑑定蛋白質身份。我們利用四種蛋白質，溶菌蛋白（lysozyme）、白蛋白（albumin）、伴蛋白（conalbumin）和核醣核酸酶A（ribonuclease A）的胜肽片段之質譜分析而證明這個方法是非常有效率的。此方法明顯地使膠體內水解反應所需的時間從16小時縮短成5分鐘。微波技術應用於蛋白質身份鑑定的新方法將會使得生物科技及蛋白質體研究有重大的進展。所以我們應用此方法於結合微波技術和質譜儀探討人類骨髓細胞分化為巨噬細胞之次細胞蛋白質體學分析。
在血液學中，用來治療血癌的方法之一就是重要癌細胞誘導分化療法（differentiation therapy）。HL-60是人類骨髓白血癌細胞株經由12-O-tetradecanoylphorphorbol-13-acetate（TPA）能誘導分化為良性非癌化巨噬細胞。HL-60分化機制的調制，不僅經由細胞表面受體，而且也透過許多可溶性及膜蛋白質傳遞。這是考慮細胞的特殊次細胞組成及巨分子結構，不僅可以提供分化期間可能的細胞表面受體及傳遞的相關資訊，而且也可告訴我們某些蛋白質在次細胞的位置，並進而幫助了解它們的結構與生物功能。本研究我們應用微波技術結合質譜儀來加速次細胞（subcellular）蛋白質身份鑑定的步驟，並以新方法應用於將膠體內的十二烷基硫酸鈉移除，以進一步進行膠體內水解。在本研究中，我們共鑑定出13個蛋白質。這些蛋白質可能是在TPA誘發HL-60分化成巨噬細胞時，使次細胞構成的特性改變。同時，我們發現肽基輔胺酸順反異構酶（peptidyl-prolyl cis-trans isomerase A）、抑制素（prohibitin）、烯醇酶（enolase 1）、過氧化骨髓酶（myeloperoxidase precursor）有異構體的產生。這些結果將使我們更了解這些分化的過程，並進而促進診斷與治療標的物的發展。

關鍵字

微波爐 ； 蛋白質體學 ； 膠體內水解 ； TPA ； 次細胞蛋白質體學 ； 質譜

並列摘要

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.

並列關鍵字

Microwave ； Proteomics ； In-gel digestion ； TPA ； Subcellular Proteomics ； Mass Spectrometry

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