化乙烯胺作为原生质谱的电子喷雾兼容中性pH缓冲剂
Bradley T V Davis1, Algirdas Velyvis1, Siavash Vahidi1
1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Analytical chemistry
|November 24, 2023
概括
新的挥发性缓冲剂2,2-二乙烯胺 (DFEA) 和2,2,2-三乙烯胺 (TFEA) 维持了本地ESI-MS的生理pH值. 这些缓冲器保留了宏分子复合体,克服了酸在研究pH敏感相互作用方面的局限性.
科学领域:
- 生物化学 生物化学
- 分析化学 分析化学
- 质谱测量质量谱测量
背景情况:
- 原生电子喷雾电离质谱法 (ESI-MS) 对于分析宏分子复合体至关重要.
- 氨酸是一种常见的缓冲剂,在生理pH (7.0-7.4) 时缺乏足够的缓冲能力.
- 由ESI引起的pH值变化会导致蛋白质展开,并破坏pH值敏感的相互作用.
研究的目的:
- 识别和评估挥发性,ESI兼容的缓冲器,以维持本地ESI-MS的生理pH.
- 在中性pH下评估2,2-二乙烯胺 (DFEA) 和2,2,2-三乙烯胺 (TFEA) 的缓冲能力.
主要方法:
- 调查了DFEA和TFEA作为潜在的缓冲器,确定了它们的pKa值.
- 使用DFEA和TFEA对模型蛋白 (细胞染色体c,肌球蛋白) 和多子单元复合体 (酒精脱酶,康卡纳瓦林A) 进行本机ESI-MS.
- 进行了蛋白质稳定性测试和碰撞诱导的展开实验.
主要成果:
- 与酸不同的是,DFEA (pKa 7.2) 和TFEA (pKa 5.5) 在生理pH下提供缓冲能力.
- 本地ESI-MS实验成功地在气相中保存了非共价蛋白-连接体复合体.
- DFEA和TFEA并没有使溶液或气相中的蛋白质不稳定.
结论:
- DFEA和TFEA是有效的,与ESI兼容的中性pH缓冲器.
- 这些缓冲器增强了本地ESI-MS的实用性,用于研究pH敏感的大分子复合体.
- DFEA和TFEA代表了分析复杂生物组件的重大进步.
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