Cantone Joseph L, Xu-Lin Alan, Toyn Jeremy H, Drexler Dieter M
Bristol-Myers Squibb Company, Research and Development, Pharmaceutical Candidate Optimization, Discovery Analytical Sciences, 5 Research Parkway, Wallingford, CT 06492, United States.
J Neurosci Methods. 2009 Jun 15;180(2):255-60. doi: 10.1016/j.jneumeth.2009.03.018. Epub 2009 Mar 31.
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.
阿尔茨海默病（AD）当前的一个研究领域是淀粉样β肽（Aβ）的生物合成途径，该途径通过β-分泌酶（BACE）和γ-分泌酶对淀粉样β前体蛋白（APP）进行连续的蛋白水解切割。APP首先被BACE切割形成C端片段APP-βCTF，也称为C99，然后C99再被γ-分泌酶进一步切割形成Aβ。已观察到γ-分泌酶抑制剂会产生一种所谓的“Aβ升高”现象，即低抑制剂浓度会导致Aβ水平升高，而高抑制剂浓度会导致Aβ水平降低。我们实验室之前的一份报告表明，这种现象与APP-βCTF底物与γ-分泌酶的比例有关。使用重组C100蛋白作为校准标准进行定量蛋白质印迹分析，以评估APP-βCTF、γ-分泌酶和导致“Aβ升高”的各种抑制剂之间的关系。开发了一种采用“替代肽”方法的在线液相色谱-质谱联用（LC-MS）方法，以准确定量蛋白质印迹分析中使用的重组C100。替代肽方法利用蛋白质的胰蛋白酶消化，以化学计量方式产生一个独特的肽片段，在这种情况下为（C100）Aβ17-28（LVFFAEDVGSNK），它可以很容易地通过LC-MS检测到。使用合成的Aβ17-28在0.001-1 microM范围内生成校准曲线，并使用15N同位素标记的Aβ1-40作为酶消化的内标及其蛋白水解肽[15N]-Aβ17-28进行分析，从而完成对C100的绝对定量评估。
