红外多光子解离使膜蛋白复合体和G蛋白合受体的自上而下的表征成为可能
Corinne A Lutomski1,2, Tarick J El-Baba1,2, Joshua D Hinkle3
1Physical and Theoretical Chemistry Laboratory, Department of Chemistry University of Oxford Oxford OX1 3QU UK.
Angewandte Chemie (Weinheim an der Bergstrasse, Germany)
|March 22, 2024
概括
这项研究引入了一种使用红外激光和质谱学的新方法,以有效地从膜蛋白中去除洗剂. 这种技术允许进行详细的自上而下的分析,改善了膜蛋白及其复合物的识别.
科学领域:
- 生物化学 生物化学
- 分析化学 分析化学
- 结构生物学 结构生物学
背景情况:
- 由于它们的疏水性和洗剂稳定性,很难使用原生质谱分析膜蛋白.
- 传统的洗剂去除方法限制了能源的应用,阻碍了上下质谱分析.
- 顶向下质谱对于详细描述和识别蛋白质及其复合体至关重要.
研究的目的:
- 开发一种有效地从膜蛋白中去除洗剂的方法,以改进质谱分析.
- 为了使膜蛋白及其复合体的详细上下质谱学.
- 为了阐明膜蛋白在红外多光子解离过程中的碎片化机制.
主要方法:
- 在高压线性离子陷中使用了经过修改的Orbitrap Eclipse Tribrid质谱仪和红外激光.
- 应用可调节的红外激光强度和时间,用于控制的洗剂微粒解离.
- 使用红外多光子解离 (IRMPD) 用于自上而下的质谱.
- 进行了气相分子动力学模拟,以分析碎片化模式.
主要成果:
- 通过调整红外激光参数,成功释放膜蛋白从洗剂囊中.
- 确定了洗剂红外吸收和容易去除菌之间的相关性.
- 通过自上而下的MS实现了良好的序列覆盖,以通过自上而下的MS来明确识别膜蛋白和复合体.
- 在GPCRs的跨膜域内确定了特定的分裂模式,并提出了碎片化机制.
结论:
- 开发的红外激光辅助质谱技术有效地从膜蛋白中去除洗剂.
- 这种方法显著提高了用于膜蛋白分析的自上而下的质谱学的能力.
- 该研究提供了有关膜蛋白分析的碎片化模式和结构方面的见解.
相关概念视频
01:21G-protein Coupled Receptors
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...