奈米碳管與雙胺基苯甲酸/溶膠凝膠材料輔助雷射脫附游離質譜法的研究

Abstract

基質輔助雷射脫附游離質譜法 (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry, MALDI-MS) 目前已被廣泛地使用於各類型樣品的分析。在進行傳統MALDI分析時，樣品需與有機基質混合形成良好的共結晶化，使得具吸收雷射能量的有機基質在吸收雷射能量後，可有效地傳遞能量給待測樣品分子並提供氫質子為樣品游離的來源，而讓樣品分子可被脫附游離成氣相離子而經由質譜分析儀所偵測。但是傳統有機基質需與樣品形成良好結晶化的要求，常造成在分析上的困擾，因此我們使用無機材料為基質發展適當的方法，避免傳統有機基質在分析時會產生的問題，並將發展出的方法用於胜肽、蛋白質及DNA等樣品的分析。

本論文使用具活性的陽極氧化鋁為基板合成奈米碳管，利用此材料具有吸收雷射能量的特性，研究其當做輔助樣品在雷射脫附游離中為基質的效果。由於奈米碳管材料中含有大量的鈉、鉀離子，形成大量分析物的鈉、鉀離子加成物並造成高偵測極限、低解析度等問題，因此本實驗在樣品製備中加入了檸檬酸做為共基質 (co-matrix)，提供了質子化來源，而能有效地幫助分析物游離化且降低鈉、鉀加成物的形成，使得可偵測質量上限可達12 k Da左右。

此外，將奈米碳管表面以檸檬酸修飾成帶負電性之探針，可用於吸附水中微量的正電性樣品。如利用胜肽或蛋白質具有不同等電點的特性，可在不同pH值的環境下使其帶有不同的正負電性，可藉著pH值之改變而利用檸檬酸修飾的奈米碳管篩選濃縮特定的目標生化分子，並可直接以MALDI-MS分析吸在奈米碳管上之分析物，此方法可有效用於蛋白質酵素消化產物的濃縮與分析。

本論文也利用雙胺基苯甲酸與溶膠凝膠混成產物當做MALDI基質來分析DNA分子，利用雙胺基苯甲酸本身具有吸收雷射的特性，且其含胺之官能基可和鈉、鉀離子競爭DNA上磷酸根的負離子結合部位，因此可和DNA有較強的親合力，而在脫附游離的過程中並可轉移質子給磷酸根，可有效降低鹽類的干擾，此一基質系統可取代傳統MALDI基質混合胺鹽的效果，適合用於DNA樣品的分析。

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used in the analysis of various types of analytes. In conventional MALDI-MS analysis, good co-crystallization of analyte/matrix is required, which mainly relied on the good solubility between the analytes and the MALDI matrix. MALDI matrices play the roles as the light absorber and the proton donor to assist the desorption/ionization of analytes during MADLI-MS analysis. However, the requirement of good co-crystallization of matrix/analyte always causes some difficulties during analysis. In this thesis, inorganic materials were alternatively used as the MALDI matrices for the analysis of peptide, proteins, and DNA to avoid the problems arising in the conventional MALDI-MS analysis. Carbon nanotubes (CNTs) generated from using anodic aluminum oxide (AAO) as a reactive template were examined as the MALDI matrix because CNTs have good absorption capacity in the laser wavelength that used for the MALDI-MS analysis. Owing to high amounts of sodium and potassium ions presenting in the CNTs, sodium and potassium adducts of analytes dominated the CNT MALDI mass spectra, which resulted in high detection limits and poor mass resolution. The addition of a high concentration of citrate buffer to the MALDI sample preparation can effectively render the protonation of analytes and also effectively suppress the sodium and potassium adducts of analytes. The largest detectable mass is therefore extended to ca. 12,000 Da. Additionally, citric acid treated CNTs were employed as affinity probes to selectively concentrate traces of positively charged analytes from sample solutions through electrostatic interactions. Because amphiprotic species, such as peptides and proteins, have unique isoelectric points (pI), by varying the values of pH of the sample solution, specific target proteins or peptides can be concentrated selectively on the surface of the citric acid-treated CNTs. Using this approach, low concentrations of protein enzymatic digest products can be concentrated and characterized by using CNT MALDI-MS analysis. Sol–gel/diaminobenzoic acid (DABA) hybrid materials were also generated to be used as the MALDI matrices for the analysis of oligonucleotides. It is because that DABAs have good molar absorptivity at wavelength of 337 nm. Furthermore, two amino functional groups attached to the aromatic ring in DABAs can also suppress the cation adductions of oligonucleotides and leave the protons to the oligonucleotides during MALDI-MS analysis. This matrix system can be used as an alternative for the analysis of oligonucleotides.