使用LC-MS/MS在细菌中的循环di-AMP, -GMP和循环GAMP的分离和定量
Silvio Uhlig1, Kristina Vevik1,2, Joke Van De Vyver1,3
1Nordic Institute of Dental Materials (NIOM), Oslo, Norway.
Journal of separation science
|May 3, 2025
概括
这项研究优化了一种检测细菌循环二核酸的方法,发现了循环二-AMP的新结构异构体. 这提高了对这些关键细菌第二信使分子的理解.
科学领域:
- 细菌学 细菌学是一门学科.
- 分析化学 分析化学
- 分子生物学分子生物学
背景情况:
- 循环二核酸是重要的细菌第二信使,调节细胞过程.
- 准确的识别至关重要,因为结构异构体可能被误认.
- 现有的方法可能难以区分各种循环二核酸异构体.
研究的目的:
- 优化UHPLC分离和MS/MS检测细菌中的循环二核酸的选择性方法.
- 要区分循环二-AMP,循环二-GMP和循环GAMP的2',2'-,2',3'-和3',3'-结合异构体.
- 在细菌物种中识别和量化未知的循环二核酸变体.
主要方法:
- 使用UHPLC与八甲基西胺列进行异构分离.
- 采用双重质谱法 (MS/MS) 与有针对性的离子监测进行量化和验证.
- 应用同位素稀释和矩阵匹配校准,以准确量化循环二-AMP和其他变体.
主要成果:
- 成功分离并检测出九种循环二核酸变体,包括结构性异构体.
- 在几种细菌物种中确定了一种未知的循环二-AMP的假定结构异构体.
- 在细菌提取物中量化时达到10%-41%的综合测量不确定性.
结论:
- 开发的方法允许选择性检测和定量细菌循环二核酸及其同位素.
- 发现一种新型的循环二-AMP同位素突显了细菌信号通路的复杂性.
- 使用总蛋白质测量的规范化提供了一个强大的方法来比较样本中的核酸度.
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