Optimization of Dephosphorylated Peptide Signals inDephosphorylation Reaction

• Main Authors: Jing-Yun Wu, 吳璟昀
• Other Authors: Pao-Chi Liao
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/25165875111316597656

碩士 === 國立成功大學 === 環境醫學研究所 === 97 === 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 contributive to the mining of potential phosphopeptide signals more accurate and efficient.