鹼性磷酸酶已被廣泛用於去磷酸化反應中移除磷酸胜肽的磷酸根，而在質譜圖中比較去磷酸胜肽與原始磷酸胜肽的訊號表現會產生80 Da倍數的「質量偏移」現象，這種現象可用以尋找可能為磷酸胜肽的訊號位置。但鹼性磷酸酶試劑裡往往含有甘油的成分，還有幫助去磷酸化反應的反應用緩衝溶液所含有的多種鹽類，這兩類成分都會干擾液相層析結合質譜儀分析磷酸胜肽去磷酸化的訊號表現。因此本研究使用質譜分析經去磷酸化反應beta-酪蛋白的一段磷酸胜肽T6，去評估最恰當的鹼性磷酸酶使用量範圍及合適的反應用緩衝溶液，進而找出去磷酸化反應中去磷酸胜肽訊號的最佳化條件。藉由介質輔助雷射脫附離子化結合飛行時間質譜能快速得知1 pmol T6行去磷酸化反應的結果－在20 mM 碳酸氫銨緩衝溶液中，使用10的-5次方~1 U的鹼性磷酸酶是去磷酸化T6胜肽訊號的最佳化條件；由液相層析結合電噴灑離子化四極棒飛行時間質譜儀分析2 pmol T6行去磷酸化反應的結果－在20 mM 碳酸氫銨緩衝溶液中，使用10的-3次方~1 U的鹼性磷酸酶是去磷酸化T6胜肽訊號的最佳化條件。根據這些結果發現，相較在1X去磷酸化專用緩衝溶液中，行去磷酸化反應特定量的磷酸胜肽T6，在20 mM 碳酸氫銨緩衝溶液中所需的鹼性磷酸酶用量較少（為前者的1/600至1/10）。此外，若以超過1 U的AP使用量反應1 pmol的T6，那麼去磷酸化T6的胜肽訊號強度會有所減弱，這可能是因為AP試劑所含的甘油成分干擾所致。總結本研究描述在去磷酸化反應中，以不同的去磷酸化反應條件得到最佳化的去磷酸化T6胜肽質譜訊號，可幫助更精確且更有效率地探測到可能為磷酸胜肽的訊號。

Alkaline phosphatase（AP）has been wildly used for removing phosphate groups from phosphopeptides in dephosphorylation reaction, yielding a “mass shift” of 80n Da relative to the original mass of the phosphopeptides in MS spectra. The mass shift can be utilized for the localization of the potential
phosphopeptide signals. However, glycerol which is commonly contained in the AP reagent and additional reaction buffer which consists of various salts can interfere with LC-MS analysis. Therefore, we used a phosphopeptide T6 from beta-casein to evaluate an optimal amount range of AP and a suitable reaction buffer, and further to optimize dephosphorylated T6 signals in the
dephosphorylation reaction. The results of dephosphorylated T6 signals of the MALDI-TOF MS analysis showed the optimal conditions were used 10^-5 to 1
U of AP in 20 mM ammonium bicarbonate buffer per 1 pmol of T6; the results of the LC-ESI-Q-TOF MS analysis were used 10^-3 to 1 U of AP in the same reaction buffer per 2 pmol of T6. Based on these results, we observed the amount of AP using to dephosphorylate a particular amount of T6 in 20 mM ammonium buffer less than in 1X Dephosphorylation buffer. Besides, the intensity of dephosphorylated T6 signals in the MS spectra could be interfered with glycerol and decreased if 1 pmol of T6 is reacted with AP above 1 U. In conclusion, the study demonstrated the reaction condition for getting optimal dephosphorylated T6 signals in the dephosphorylation reaction, which is
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