Measurement of Intracellular Nitric Oxide with a Quantitative Mass Spectrometry Probe Approach
Measurement of Intracellular Nitric Oxide with a Quantitative Mass Spectrometry Probe Approach
复制标题
用定量质谱探针法测量细胞内一氧化氮
DOI：
10.1021/acs.analchem.1c01259
复制
发表时间：
2021
影响因子：
7.4
通讯作者：
Gui-Zhong Xin
中科院分区：
文献类型：
--
作者：
Zhu-Jun Zhong;Zhong-Ping Yao;Zi-Qi Shi;Yang-Dan Liu;Li-Fang Liu;Gui-Zhong Xin
一氧化氮 (NO) 是一种具有重要生理意义的分子，NO 的功能取决于其在生物系统，特别是细胞中的浓度。基于浓度的细胞内 NO 分析可以深入了解其在健康和疾病中的精确作用。然而，目前检测细胞内NO的方法仍然不足以进行定量分析。在这项研究中，我们报告了一种定量质谱探针方法来测量细胞中的 NO 水平。探针氨氯地平 (AML) 包含 Hantzsch 酯基，可与 NO 反应形成吡啶、脱氢氨氯地平 (DAM)。通过超高效液相色谱-串联质谱 (UPLC-MS/MS) 对 DAM 进行定量，可以特异性测量细胞内 NO 水平。值得注意的是，AML/NO反应进行得很快（1秒内），考虑到其扩散率大和半衰期短，这有利于NO检测。同时，模拟生理条件下的研究表明，AML对NO的反应是成比例的和选择性的。所提出的 UPLC-MS/MS 方法在 DAM 定量中表现出高灵敏度 (LLOQ = 0.24 nM) 和低基质干扰 (小于 15%)。此外，质谱探针方法通过测量细胞内源性和外源性 NO 得到证实。因此，以AML为探针开发的定量UPLC-MS/MS方法有望成为细胞内NO分析的新方法。
Nitric oxide (NO) is a molecule of physiological importance, and the function of NO depends on its concentration in biological systems, particularly in cells. Concentration-based analysis of intracellular NO can provide insight into its precise role in health and disease. However, current methods for detecting intracellular NO are still inadequate for quantitative analysis. In this study, we report a quantitative mass spectrometry probe approach to measure NO levels in cells. The probe, Amlodipine (AML), comprises a Hantzsch ester group that reacts with NO to form a pyridine, Dehydro Amlodipine (DAM). Quantification of DAM by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) allows specific measurement of intracellular NO levels. Notably, the AML/NO reaction proceeds rapidly (within 1 s), which is favorable for NO detection considering its large diffusivity and short half-life. Meanwhile, studies under simulated physiological conditions revealed that the AML response to NO is proportional and selective. The presented UPLC–MS/MS method showed high sensitivity (LLOQ = 0.24 nM) and low matrix interference (less than 15%) in DAM quantification. Furthermore, the mass spectrometry probe approach was demonstrated by enabling the measurement of endogenous and exogenous NO in cells. Hence, the quantitative UPLC–MS/MS method developed using AML as a probe is expected to be a new method for intracellular NO analysis.