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使用SNOTRAP探针和基于质谱的检测方法,对S-化蛋白质进行全蛋白质样
Hongmei Yang1,2, Haitham Amal3,4,5,6, Steven R Tannenbaum3
1Departments of Biological Engineering and Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA. yanghm0327@sina.cn.
Nature protocols
|November 19, 2025
概括
研究人员开发了SNOTRAP,这是一种新方法,用于在组织中识别低丰度的S-化蛋白 (SNO蛋白). 这一进展有助于理解SNO蛋白在癌症和神经退行等疾病中的作用.
科学领域:
- 生物化学 生物化学
- 蛋白质组学是指蛋白质组学.
- 分子生物学分子生物学
背景情况:
- 蛋白S-化 (SNO) 是一种关键的翻译后修饰,调节各种细胞功能.
- 失调的SNO与各种疾病有关,包括心血管,代谢,神经退行性疾病和癌症.
- 目前用于监测SNO蛋白质的方法有限,这阻碍了对其疾病作用的全面理解.
研究的目的:
- 开发一种强大的,全蛋白质组的方法来探索人类和小鼠组织中的S-基蛋白质组.
- 为了能够系统地调查SNO蛋白在疾病发病过程中的参与.
主要方法:
- 利用一种新型的探针SNOTRAP (一种通过PEG间隔器与生物素连接的三酸 thioester),用于SNO蛋白的现场标记.
- 使用基于质谱 (MS) 的检测来识别和分析SNO蛋白质.
- 纳入化学标记,二硫化物 - 胺基酸形成,斯特雷普塔维丁捕获和N-乙烯胺胺重新标记,用于Orbitrap MS的选择性丰富和无标签量化.
主要成果:
- 成功地应用了SNOTRAP方法来探索人类和小鼠大脑组织中的S-nitrosoproteome.
- 在特定位置展示了含有SNO的选择性丰富.
- 实现了SNO蛋白质的无标签量化,克服了低丰度和短暂表达的限制.
结论:
- 该SNOTRAP方法提供了一个强大的工具,用于全面的S-nitrosoproteome分析.
- 这种方法有助于更深入地了解SNO蛋白在正常生理和疾病中的功能.
- 开发的协议对于推进针对SNO途径的治疗策略至关重要.
相关概念视频
02:57Southern Blot
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
01:15Western Blotting
Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.