|摘要: ||本研究主要在開發以毛細管電泳(CE)及質譜(MALDI-TOF/MS)技術做為偵測磷酸化蛋白質的新方法。我們以HPLC和MALDI確認β-酪蛋白經胰蛋白酶消化後的胜肽片段之CE析出順序。在電泳緩衝溶液(10 mM H3BO4-Na2B4O7，pH 9.35)中添加兩價金屬離子Ca2+、Sr2+、Ba2+，利用磷酸化胜肽片段與金屬離子結合而造成較大電泳遷移率變動的現象，可有效區別α-酪蛋白及β-酪蛋白的磷酸化與非磷酸化胜肽片段，當添加3 mM Ba2+時，效果最佳。此外，蛋白質經酵素消化反應(在50 mM NH4HCO3樣品緩衝溶液中，於37 ℃反應18小時)時，常會產生多切或少切的錯誤切片段。我們發現，當改變反應條件時(10 mM NH4HCO3樣品緩衝溶液，添加0.1 mM Ba2+和0.1 mM Ca2+，在37 ℃反應18小時)，可得較多正確切產物，所得MALDI質譜圖數據經蛋白質資料庫比對，進行peptide mapping時，亦可得到較佳吻合度。|
此外，我們發現，磷酸化胜肽片段通常偏低的MALDI訊號，可藉由在製備MALDI樣品時將添加物(酸及其銨鹽)與樣品和基質混合，而得到明顯的改善。使用添加物100 mM HCl + 100 mM NH4Cl的混合溶液可大幅提高α-酪蛋白及β-酪蛋白上磷酸化胜肽片段的MALDI訊號。另外，以0.1 % PEO (Mwt：106)及100 mM蜜二糖(Melibiose)做為MALDI樣品添加物，亦能提高不同分子量的Polylysine在質譜上的訊號強度。最後，我們發展出新的MALDI樣品製備方法，將最被廣泛應用，但也卻最易形成不均勻結晶的2,5-Dihydroxybenzoic acid(2,5-DHB)基質所製備樣品之結晶均勻化；在基質及樣品混合點盤後，急速冷凍(-20 ℃)，並經真空乾燥。此樣品製備方法可大幅提高MALDI訊號的再現性，並增加利用MALDI進行定量分析的應用性。
In this research we mainly aimed to develop capillary electrophoresis (CE) and MALDI-TOF/MS based techniques for the detection of phosphoproteins. We established the elution orders of the tryptic digest of β-casein for CE electropherogram by HPLC and MALDI. With the addition of divalent metal ion Ba2+, Ca2+, and Sr2+ into the CE buffer (10 mM H3BO4-Na2B4O7，pH 9.35), and owing to the preferential binding of metal ions onto phosphopeptides, larger mobility shifts could be observed for phosphopeptides, and thus it was feasible to distinguish between phosphorylated and non-phosphorylated peptide fragments for the tryptic digests of α-casein and β-casein. The best result could be obtained when 3 mM Ba2+ was added. In addition, in the reaction conditions of 50 mM NH4HCO3 sample buffer, 37 ℃ reaction temperature, and 18-hr reaction duration, some false peptide fragments of miss-cut or over-cut could be found from the products of enzymatic digestion of proteins. According to our experimental results, more correctly digested fragments could be produced when the reaction conditions were changed to 10 mM NH4HCO3 sample buffer with the addition of 0.1 mM Ba2+ and 0.1 mM Ca2+, 37 ℃ reaction temperature, and 18-hr reaction duration. Better agreement with the on-line protein data bank could be achieved when performing peptide mapping process for the MALDI spectrum obtained from the above-mentioned new reaction conditions.
Furthermore, we found that the usually low MALDI signals of the phosphorylated peptide fragments could be improved by mixing the additives, including acids and ammonium salts, with sample and matrix while preparing MALDI sample. The MALDI signals of the phosphorylated peptide fragments in trypic digest of α-casein and β-casein could be largely raised by the 100 mM HCl + 100 mM NH4Cl additive. Moreover, both MALDI sample additives, 0.1 percentage PEO (Mwt : 106) and 100 mM melibiose, proved to be able to increase the MALDI signals of polylysine samples with different molecular weights. Finally, we developed a new sample preparation method to make homogenous sample when using 2,5-Dihydroxybenzoic acid (2,5-DHB) as MALDI matrix, which usually resulted in hetergenous MALDI sample. After placing the mixture of matrix and sample on the sample plate, the sample was frozen quickly at -20 ℃ and then dried with the vacuum. This new sample preparation method could largely enhance the reproducibility of measured MALDI signals and increase the applicability of MALDI on quantitative analysis.