| 摘要: | 本研究主要是利用胰蛋白酶水解蛋白質，以液相層析儀(HPLC)、毛細管電泳儀(CE)和MALDI-TOF質譜儀對蛋白質水解產物進行純化和分析。以HPLC的分離純化與MALDI的胜肽序列鑑定，我們完成了α-酪蛋白和β-酪蛋白經胰蛋白酶水解所得胜肽樣品在毛細管電泳圖譜中的析出順序。根據此CE圖譜，我們分析了在不同離子強度的緩衝溶液中，以及添加不同濃度的不同種類金屬離子於電泳緩衝溶液中，α-酪蛋白胜肽片段的電泳遷移行為。其中添加適當濃度Ca2+、Ba2+離子會對含磷酸化胜肽有較大的電泳遷移變化。另外，改變蛋白質水解反應條件，包括反應緩衝溶液的離子強度、金屬離子的添加、有機溶劑的添加、以及反應時間的長短…...等，會影響所得胜肽產物的正確切和錯誤切比例和分布情形，也會影響以MALDI進行蛋白質鑑定的結果。
另外，我們發現利用銨鹽與酸混合添加於樣品，可以顯著的改善以MALDI質譜儀偵測磷酸化胜肽片段時，所得訊號偏低的現象，其中以100 mM 亞甲基二磷酸(Methylenediphosphonic acid, MDPNA)與25 mM 磷酸二氫銨混合後添加於樣品之偵測效果最佳。我們也發現此類添加劑不但對磷酸化胜肽片段的偵測有幫助，也可有效提高親水性胜肽片段質譜訊號。2,5-二羫基苯甲酸(2,5-Dihydroxy benzoic acid, DHB)基質對於胜肽有良好的偵測效果，但其所形成的針狀結晶之均勻度不佳，會影響定量分析的再現性。我們開發以冷凍抽氣(Ice vacuum)的方式進行MALDI樣品製備，可以大幅提高樣品的均勻度以及質譜訊號的再現性。不過，冷凍抽氣後樣品的訊號值通常都比室溫空氣乾燥訊號值小，我們使用添加劑以及小幅提高雷射強度，可以有效提高MALDI訊號，並維持良好的再現性。此外，我們也分別以C18和幾丁聚醣(Chitosan)對蛋白質水解之胜肽片段進行固相微萃取實驗，兩者皆可達到純化、濃縮胜肽樣品的效果。
In this thesis we mainly utilize high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF MS) to purify and analyze the hydrolysis products of proteins digested by trypsin. With the sample purification by HPLC and sequence identification of peptides by MALDI, we have established the electrophoretic elution orders of the tryptic digests in the respective electropherograms of α-casein and β-casein. Based on the resulting electropherograms we are able to analyze the effects of ionic strength and metal ion addition in CE buffer on the electrophoretic migration behaviors of the peptide fragments generated from the tryptic digests of α-casein and β-casein. We found that the addition of adequate concentrations of Ca2 + and Ba2 + ions would result in larger electrophoretic mobility shifts for the phosphopeptides. Besides, varying protein digestion conditions such as ionic strength of reaction buffer, addition of metal ions and organic solvents, and reaction duration would alter the product ratio of the correct cut to the wrong cut peptide fragments, and thus influence the results of protein identification by MALDI.
Moreover, we found that with the addition of certain ammonium salt and acid in peptide sample, the usually inferior MALDI signals of phosphopeptides can be largely enhanced. Among several additives investigated, the mixture of 100 mM methylenediphosphonic acid (MDPNA) and 25 mM ammonium dihydrogen phosphate (ADP) has the best detection results for phosphopeptides. We also observed signal enhancement for the hydrophilic peptides by this MDPNA-ADP additive. 2,5-dihydroxy benzoic acid (DHB) is a commonly used matrix and provides relatively good MALDI detection for peptide fragments. However, its needle-like crystals formed after air-drying sample would generate an inhomogeneous sample spot, making quantitative analysis unfeasible. In order to prepare a homogeneous sample spot for MALD measurement, we developed an ice-vacuum method for MALDI sample preparation and greatly improve the reproducibility of MALDI signals for peptide and protein samples. However, the MALDI signal of the ice-vacuum prepared sample is generally smaller than that of the air-dry prepared sample. Utilizing MDPNA-ADP additive and slightly increasing laser power, we can effectively improve the MALDI signals of ice-vacuum prepared sample and in the same time maintain good reproducibility. In addition, C18 and Chitosan were demonstrated as good absorbents in solid phase micro-extraction experiments for the purification and concentration of peptide samples. |
