零度以下温度染色学和自上而下的质谱学用于蛋白质高阶结构的表征:方法验证和应用于治疗性抗体
Jingxi Pan1, Suping Zhang, Carol E Parker
1University of Victoria-Genome British Columbia Proteomics Centre , Vancouver Island Technology Park, 3101-4464 Markham St., Victoria, British Columbia V8Z 7X8, Canada.
Journal of the American Chemical Society
|August 26, 2014
概括
一种新的自上而下的/交换质谱法 (HDX-MS) 方法结合了电子捕获解离与零下温度HPLC. 该技术允许在生理条件下进行蛋白质结构分析,克服了以前方法的局限性.
科学领域:
- 生物化学和结构生物学.
- 质谱测量质量谱测量
- 蛋白质动力学 蛋白质动力学
背景情况:
- 了解蛋白质高阶结构和动态是功能关键.
- 通过质谱法 (MS) 监测的胺/交换 (HDX) 是评估蛋白质结构变化的流行的技术.
- 目前的自上而下的HDX-MS方法仅限于挥发性缓冲区,并与具有二硫化物键的复杂蛋白质作斗争.
研究的目的:
- 开发一种新的自上而下的HDX-MS方法,适用于所有蛋白质类型和样本条件.
- 为了使蛋白质结构研究在生理条件下,保持本地结构和功能.
- 克服现有的直接输注上下策略的局限性.
主要方法:
- 顶向下电子捕获解离 (ECD) 与零度以下温度高性能液态染色学 (HPLC) 的组合.
- 使用ECD进行完整蛋白质的碎片化.
- 采用零度以下温度的HPLC来最大限度地减少分析期间的反交换.
主要成果:
- 实现了仅为2%的低回交水平.
- 对包括治疗性抗体在内的各种蛋白质类型的证明适用性.
- 生成高分辨率的结构信息,接近单个残留水平.
- 使用calmodulin对NMR数据进行验证.
结论:
- 开发的自上而下的HDX-MS方法克服了以前的局限性,使得在生理条件下进行研究成为可能.
- 这种技术为在各种样本类型中表征蛋白质结构和动态提供了一种多功能方法.
- 该方法提供了接近单一残留的分辨率,增强了我们对蛋白质行为的理解.
更多相关视频
05:45Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry
Published on: June 8, 2021
2.6K
09:26Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis
Published on: May 23, 2021
2.8K
相关概念视频
01:33Peptide Identification Using Tandem Mass Spectrometry
6.2K
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
6.2K
01:21Mass Spectrometry: Complex Analysis
2.1K
Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
2.1K