Amity Institute of Biotechnology, Amity University, Kolkata, West Bengal, India.
Present Address: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand, India.
Curr Protoc. 2021 Sep;1(9):e262. doi: 10.1002/cpz1.262.
The dynamic and unstable nature of protein nitrosothiols (PSNOs) derived from complex biological matrices (like cell lysates) make them unsuitable for proteomic/biochemical analysis in vitro. In an attempt to increase the stability of cell-derived PSNOs, scientists have devised methods to derivatize thiols undergoing nitrosylation, with a suitable molecule, to yield a stable adduct that could easily be detected using appropriate antibodies. The Biotin Switch Assay (BTSA) is currently the most widely used method for tagging PSNOs; however, the error-prone and cumbersome nature of the BTSA protocol prompted the development of alternative mechanisms of tagging cell-derived PSNOs. One such method is the immuno-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which effectively overcomes the shortcomings of the BTSA and proves to be a promising alternative. Here we describe the protocol for DMPO-based PSNO labeling and subsequent proteomic analysis by western blotting with an anti-DMPO antibody. © 2021 Wiley Periodicals LLC. Basic Protocol: Labeling of cell-derived PSNOs by DMPO-based immuno-spin trapping and their subsequent analysis by immunostaining.
蛋白质亚硝硫醇（PSNOs）来源于复杂的生物基质（如细胞裂解物），其动态和不稳定的性质使得它们不适合用于体外的蛋白质组学/生化分析。为了提高细胞来源的 PSNOs 的稳定性，科学家们设计了将发生硝化的硫醇与合适的分子衍生化的方法，生成稳定的加合物，然后可以使用适当的抗体轻松检测到。目前，生物素开关测定法（BTSA）是标记 PSNOs 最广泛使用的方法；然而，BTSA 方案易错且繁琐，这促使人们开发出标记细胞来源的 PSNOs 的替代机制。一种这样的方法是使用 5,5-二甲基-1-吡咯啉 N-氧化物（DMPO）的免疫自旋捕获法，它有效地克服了 BTSA 的缺点，被证明是一种很有前途的替代方法。在这里，我们描述了基于 DMPO 的 PSNO 标记的方案，以及随后通过抗 DMPO 抗体进行免疫印迹的蛋白质组学分析。© 2021 年威利父子公司。基本方案：通过 DMPO 基免疫自旋捕获标记细胞来源的 PSNOs，并用抗 DMPO 抗体进行免疫染色分析。
