新穎性金屬有機骨架生物催化器與有機高分子整體成形層析靜相材料於蛋白質體學的應用

Novel Metal-Organic Framework Biocatalyst and Organic Polymer Monolith Chromatographic Stationary Phase for Proteomics

劉婉舲(Wan-Ling Liu)

指導教授 ： 黃悉雅

中原大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6840/cycu201300303

摘要

本論文包含兩大方向的研究，第一部分為有機金屬骨架材料製備生物性催化器之研究，第二部分為有機高分子整體成形管柱的製備，最後將兩部份成果結合並應用至蛋白質體學研究。
在第一部份研究中，成功開發新穎的酵素固定化方法，將酵素有效地固定於有機金屬骨架材料並進行蛋白質水解反應。此方法僅需以渦旋震盪方式，利用酵素與有機金屬骨架基材間之主客作用力，即可在三十分鐘內將酵素固定化於基材中，無須額外進行基材表面的化學修飾。
本研究中，由於所選用的螢光染料異硫氰酸鹽螢光素(FITC)可透過親核反應與胰蛋白酶產生鍵結，且其分子尺寸與有機金屬骨架材料(CYCU-4 (Al))之孔洞相吻合，因此可將胰蛋白酶與異硫氰酸鹽螢光素結合後，再利用異硫氰酸鹽螢光素分子可吸附於有機金屬骨架材料孔洞之特性，製備出對於牛血清蛋白具有優異的生物催化活性的有機金屬骨架生物性催化器「胰蛋白酶結合異硫氰酸鹽螢光素之有機金屬骨架材料」(Trypsin-FITC@CYCU-4)。因有機金屬骨架材料(CYCU-4)經由胰蛋白酶結合異硫氰酸鹽螢光素之固定化步驟之後，同時具有微孔與中孔的孔洞特性，因此可造成胰蛋白酶結合異硫氰酸鹽螢光素分子與基材間除了具有主客作用力外還具有酵素與基材間特殊的空間拘限效應，可使胰蛋白酶分子能更有效且穩定的固定於基材上。
此外，我們更進一步的以另一種分子尺寸較小的螢光染料 “4-氯基-7-硝基苯並呋喃 (NBD)” 取代異硫氰酸鹽螢光素，製備另一種生物性催化器「胰蛋白酶結合4-氯基-7-硝基苯並呋喃之有機金屬骨架材料」(Trypsin-NBD@MOF)，進一步探討螢光素的分子尺寸與多種有機金屬骨架材料之孔洞大小的影響，並利用4-氯基-7-硝基苯並呋喃特殊的螢光性質直接測得基材上的酵素附載量(loading capacity)。而其蛋白質水解效率可與之前所開發之胰蛋白酶結合異硫氰酸鹽螢光素之有機金屬骨架材料相互媲美。
本研究所呈現的新穎性酵素固定化方法，除了具備快速及簡單的優點外，其水解效率在重複使用多次後仍是相當優異。
由於本研究需以奈升級液相層析串聯質譜儀系統 (NanoLC-MS)進行蛋白質水解效能之評估，因此如何製備高效能的層析管柱便有其必要性。在本研究的第二部分中，將製備聚甲基丙烯酸十八烷基酯-二乙烯苯之高分子整體成形管柱 (poly(SMA-DVB)，並於毛細管電層析及奈升級液相層析系統中評估其層析效能。首先在毛細管電層析系統中分離小分子分析物-盤尼西林化合物，探討以不同帶電荷單體所合成之高分子整體成形管柱對盤尼西林分離效果之影響，如陰離子帶電荷單體:對苯乙烯磺酸鈉鹽(VBSA)及陽離子帶電荷單體:對苯乙烯三甲基氯化銨(VBTA)。由測試結果得知，使用陽離子型管柱時，八種盤尼西林化合物具有良好的分離及再現性，陰離子型管柱對盤尼西林分離則有較差的效率及再現性。此外，為了提升偵測感度，於毛細管電層析串聯質譜儀系統中結合兩種線上濃縮方法對盤尼西林化合物進行分離，使偵測極限降低至0.05- 0.2 µg/L，最後，將此分法應用於牛奶樣品檢測微量盤尼西林化合物。由以上測試可得知poly(SMA-DVB)是穩定且高效率的靜相材料，因此將此管柱的單體比例稍作調整，將其應用於奈升級液相層析結合質譜儀系統中，對牛血清蛋白之水解物進行分離，並經由資料庫比對後，可得到72 % 符合率的胜肽序列，其層析效能與傳統的C18填充式層析管柱相當，並以經時達一個月之久的poly(SMA-DVB)管柱對牛血清蛋白之水解物再次進行分析，其胜肽序列符合率依舊可達76 %，因此由上述實驗結果得知，本實驗所開發之有機高分子整體成形管柱穩定性高且相當適合用來評估有機金屬骨架生物性催化器之效能。

關鍵字

有機高分子整體材料 ； 有機金屬骨架 ； 蛋白質體學 ； 生物催化器

並列摘要

This study comprises two parts. The first part presents the use of metal-organic frameworks (MOFs) for proteomics study and the second part studies the organic polymer monolith chromatographic stationary phase for proteomics.
Here, we show a new approach to biofunctionalization on metal-organic framework (MOF) via a 30 min vortex-assisted host-guest interaction without the necessity of chemical modification on the MOF surface. A fluorescein isothiocyanate (FITC) dye, whose molecular dimension is similar to the MOF’s channel pore size and can be reacted with trypsin by nucleophile attack then carried trypsin onto MOFs that creates a novel biocatalyst “trypsin-FITC@CYCU-4” with exemplary biocatalytic performance in bovine serum albumin (BSA) digestion. When a new MOF, CYCU-4 (Al), was functionalized with a conjugated trypsin-FITC catalyst, its structure obtained both micro- and mesopores permitting trypsin to reside on the surface with great stability. With this properties and the new synthesis strategy, a specific host-guest interaction combined with a space confinement effect was provided by the MOF.
Furthermore, FITC was replaced with 4-Chloro-7-nitrobenzofurazan (NBD) whose size is smaller than FITC which is more suitable to be anchored into most MOFs and the specific fluorescent feature could be used to calculate directly the loading capacity. In addition, the protein digestion efficiency of trypsin-NBD@MOF is as good as trypsin-FITC@CYCU-4 and this biocatalyst maintained its crystalline nature even after trypsin-NBD treatment.
Therefore, preparation of the proposed MOF biocatalyst is faster, easier and the digestion efficiency is exceptionally high even when reused many times.
However, before checking the performance of MOF-based biocatalyst, a protein identification method needs to be developed. Herein, a poly(stearyl methacrylate-divinylbenzene) (poly(SMA-DVB)) was synthesized and tested its performance in small molecules, penicillin antibiotics, on CEC system. First, the penicillin separation effect with different charged monomer was discussed. Results indicated that poly(SMA-DVB-VBTA) monolithic column provided reproducible performance, while the negatively charged poly(SMA-DVB-VBSA) column produced unstable separation. To enhance the sensitivity, on-line concentration steps of step-gradient elution combined with anion selective injection (ASEI) coupled with CEC-MS system was used for penicillin detection. Sensitivities were further improved to 0.05-0.2 µg/L. Then, this CEC-MS method was applied to trace penicillin analyses in milk samples. In addition, the proposed monolith with little monomer ratio adjustment was used to identify BSA digests in nano LC- MS2. The sequence coverage was 72 % which was as good as conventional C18 packing column, and the sequence coverage was still 76 % even when reused after one month, which demonstrated that poly(SMA-DVB) is very stable and suitable to evaluate the performance of MOF-based biocatalyst.

並列關鍵字

Proteomics ； Biocatalyst ； Organic Polymer Monolith ； Metal-Organic Framework

參考文獻

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