Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Am Chem Soc. 2013 May 22;135(20):7693-704. doi: 10.1021/ja401565w. Epub 2013 May 8.
Nitrosothiols (RSNOs) have been proposed as important intermediates in nitric oxide (NO(•)) metabolism, storage, and transport as well as mediators in numerous NO-signaling pathways. RSNO levels are finely regulated, and dysregulation is associated with the etiology of several pathologies. Current methods for RSNO quantification depend on indirect assays that limit their overall specificity and reliability. Recent developments of phosphine-based chemical probes constitute a promising approach for the direct detection of RSNOs. We report here results from a detailed mechanistic and kinetic study for trapping RSNOs by three distinct phosphine probes, including structural identification of novel intermediates and stability studies under physiological conditions. We further show that a triarylphosphine-thiophenyl ester can be used in the absolute quantification of endogenous GSNO in several cancer cell lines, while retaining the elements of the SNO functional group, using an LC-MS-based assay. Finally, we demonstrate that a common product ion (m/z = 309.0), derived from phosphine-RSNO adducts, can be used for the detection of other low-molecular weight nitrosothiols (LMW-RSNOs) in biological samples. Collectively, these findings establish a platform for the phosphine ligation-based, specific and direct detection of RSNOs in biological samples, a powerful tool for expanding the knowledge of the biology and chemistry of NO(•)-mediated phenomena.
硝硫醇（RSNO）被认为是一氧化氮（NO（•））代谢、储存和运输的重要中间产物，也是许多 NO 信号通路的介质。RSNO 水平受到精细调节，其失调与几种病理学的病因有关。目前用于 RSNO 定量的方法依赖于间接测定，这限制了它们的整体特异性和可靠性。最近基于膦的化学探针的发展为 RSNO 的直接检测构成了一种很有前途的方法。我们在这里报告了三种不同膦探针捕获 RSNO 的详细机制和动力学研究结果，包括新型中间产物的结构鉴定和生理条件下的稳定性研究。我们进一步表明，三芳基膦-噻吩酯可以用于使用基于 LC-MS 的测定法在几种癌细胞系中绝对定量内源性 GSNO，同时保留 SNO 功能基团的元素。最后，我们证明可以使用源自膦-RSNO 加合物的共同产物离子（m/z = 309.0）来检测生物样品中的其他低分子量硝硫醇（LMW-RSNO）。总之，这些发现为基于膦键合的、特异的和直接检测生物样品中的 RSNO 建立了一个平台，这是扩展对 NO（•）介导现象的生物学和化学认识的有力工具。
