在实验室中确定乙氨基对蛋白的共价结合的位点占用率
Timon Geib1, Cristina Lento2, Vanessa Marensi3
1Chemistry Department Université du Québec à Montréal Montréal Canada.
Analytical science advances
|May 8, 2024
概括
乙氨基的毒性源于NAPQI与蛋白质结合. 一种新的LC-MS/MS方法量化了这种与GST等特定蛋白质的结合,揭示了修饰部位并协助了毒性研究.
科学领域:
- 生物化学 生物化学
- 药理学 药理学是指药理学的学科.
- 分析化学 分析化学
背景情况:
- 过量服用乙氨基 (APAP) 会引起肝脏毒性,原因是反应性代谢物N-乙p-二胺 (NAPQI).
- 纳普基与细胞蛋白结合,破坏它们的功能,导致细胞死亡.
- 量化蛋白质添加物对于理解APAP毒性机制至关重要.
研究的目的:
- 开发和验证一种向液体染色学-并联质谱法 (LC-MS/MS) 方法,用于量化APAP与特定蛋白质的结合.
- 识别和测量谷氨S转移酶 (GSTs) 和其他蛋白质上的APAP修饰位.
- 建立一种新的方法来确定多种蛋白质硫醇的位点占用率.
主要方法:
- 开发一种使用多重反应监测 (MRM) 的向LC-MS/MS方法.
- 用CYP3A4进行体外化以产生APAP及其活性代谢物NAPQI,在纯化的GST异酶和肝脏显微体的存在下.
- 通过同位素稀释合成定制的同位素标记的化剂,以量化通过同位素稀释对囊残留的APAP添加物.
- 在LC-MRM分析之前,通过HPLC进行蛋白质消化 (trypsin或pepsin) 和分化.
- 使用人类血清白蛋白作为模型系统验证相对量化.
主要成果:
- 在GSTM2,GSTP1,人类MGST1和老鼠MGST1.1.上对七个APAP修饰位点的量化.
- 成功量化了来自肝脏显微体的三种蛋白质的APAP位点占用率.
- 使用同位素标记的化试剂来确定地点占用率的新方法的演示.
- 相对地点占用分析提供了不同地点和蛋白质的比较修改水平.
结论:
- 开发的LC-MS/MS方法准确量化了APAP与特定蛋白质硫醇的结合.
- 这种方法可以识别和测量APAP蛋白添加物,进步了解乙氨基诱导的毒性.
- 新型的同位素标记化方法为涉及活性代谢物添加物的蛋白质学研究提供了强大的工具.
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