通过减少质子转移电荷的方法对抗体进行中下分析的通用方法 - 轨道质谱质谱法
Ryan N Oates1, Linda B Lieu1, Jake T Kline2
1Department Chemistry and Biochemistry, University of Oklahoma, Norman, OK, 73109, USA.
Analytical and bioanalytical chemistry
|September 16, 2024
概括
质子转移电荷减小 (PTCR) 简化了单克隆抗体 (mAb) 子单元测序的中向下质谱学 (MD MS). 这种技术增强了碎片离子分配,并增加了对mAbs.序列覆盖的信心.
科学领域:
- 分析化学 分析化学
- 生物化学 生物化学
- 蛋白质组学是指蛋白质组学.
背景情况:
- 中向下质谱法 (MD MS) 允许在IdeS蛋白解和还原后对大型单克隆抗体 (mAb) 子单位 (~25 kDa) 进行测序.
- 大型多的双重质谱中的光谱拥堵阻碍了MD MS中精确的碎片离子分配.
- 现有的MDMS方法在简化复杂的mAb子单元的气相测序方面面临挑战.
研究的目的:
- 开发和基准测试一种新的MDMS战略,将离子碎片化技术与质子转移电荷减小 (PTCR) 结合起来.
- 为了简化mAb子单元的气相序列,并提高光谱解释性.
- 为了提高序列覆盖率和对mAb子单位碎片离子分配的信心.
主要方法:
- 在 Orbitrap Tribrid质谱仪上实施基于 PTCR 的 MD MS 策略.
- 应用液态染色学 (LC) 时间尺度分析与PTCR相结合.
- 精确估计减电量产物离子的电荷状态,以精确的m/z确定.
主要成果:
- 基于PTCR的MDMS产生了简化的质谱,离子信号重叠显著减少.
- 已证明精确检测低电荷产物离子,精确度低百万分之一 (ppm) m/z.
- 在一次运行中,单个mAb子单元的序列覆盖率达到了>80%,在组合两个实验时达到了>90%.
结论:
- 基于PTCR的MD MS是简化和增强mAb子单元分析的有效策略.
- 该方法增加了序列覆盖范围,识别碎片的数量,以及对碎片离子分配的信心.
- 这种方法可以广泛应用于各种mAb类,而无需进行广泛的优化,利用高分辨率的Orbitrap质量分析.
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