• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Neuroscience Methods >Application of quantitative LC-MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Abeta) biosynthetic intermediate protein APP-betaCTF.
【24h】

Application of quantitative LC-MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Abeta) biosynthetic intermediate protein APP-betaCTF.

机译：定量LC-MS替代肽方法在淀粉样β肽（Abeta）生物合成中间蛋白APP-betaCTF分析中的应用。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

An area of current research in Alzheimer's disease (AD) is the biosynthetic pathway of amyloid beta peptides (Abeta) via consecutive proteolytic cleavages of the amyloid beta precursor protein (APP) by BACE and gamma-secretase enzymes. APP is first cleaved by BACE to form a C-terminal fragment APP-betaCTF, or also called C99, which then undergoes further cleavage by gamma-secretase to form Abeta. Inhibitors of gamma-secretase have been observed to yield a so-called 'Abeta rise' phenomenon whereby low inhibitor concentrations result in an increase in Abeta levels while high inhibitor concentrations result in lower Abeta levels. A previous report from our labs indicated that this phenomenon was related to ratios of APP-betaCTF substrate relative to gamma-secretase enzyme. A quantitative Western blot analysis was used with a recombinant C100 protein as calibration standards to assess the relationship of APP-betaCTF, gamma-secretase enzyme and various inhibitors resulting in the 'Abeta rise'. An on-line liquid chromatography mass spectrometry (LC-MS) method employing the 'surrogate peptide' methodology was developed to accurately quantify the recombinant C100 used in the Western blot analyses. The surrogate peptide approach utilizes tryptic digestion of the protein to stoichiometrically yield a unique peptide fragment, in this case (C100)Abeta17-28 (LVFFAEDVGSNK) that can be readily detected by LC-MS. The absolute quantitative assessment of C100 was accomplished using synthetic Abeta17-28 to generate calibration curves over a 0.001-1 microM range and 15N isotopically labeled Abeta1-40 as the internal standard for enzymatic digestion and its proteolytic peptide [15N]-Abeta17-28 for the analysis.
机译：阿尔茨海默氏病（AD）的当前研究领域是通过BACE和γ-分泌酶对淀粉样β前体蛋白（APP）的连续蛋白水解切割，淀粉样β肽（Abeta）的生物合成途径。 APP首先被BACE切割形成C末端片段APP-betaCTF，也称为C99，然后被γ-分泌酶进一步切割形成Abeta。已经观察到γ-分泌酶的抑制剂产生所谓的“ Abeta升高”现象，由此低抑制剂浓度导致Abeta水平升高，而高抑制剂浓度导致较低Abeta水平。我们实验室的先前报告表明，这种现象与APP-betaCTF底物相对于γ-分泌酶的比率有关。定量蛋白质印迹分析与重组C100蛋白一起用作校准标准品，以评估APP-betaCTF，γ-分泌酶和各种导致“ Abeta升高”的抑制剂之间的关系。开发了一种使用“替代肽”方法的在线液相色谱质谱（LC-MS）方法，以准确定量Western blot分析中使用的重组C100。替代肽方法利用蛋白质的胰蛋白酶消化以化学计量产生独特的肽片段，在这种情况下（C100）Abeta17-28（LVFFAEDVGSNK）可以通过LC-MS轻松检测到。使用合成的Abeta17-28生成在0.001-1 microM范围内的校准曲线和15N同位素标记的Abeta1-40作为酶促消化的内标，并通过蛋白水解肽[15N] -Abeta17-28完成对C100的绝对定量评估。分析。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号