通过自上而下的质谱测量来描述重组蛋白及其碎片化
Zhen Long1, Dening Pei2, Xiang Zhu1
1Thermo Fisher Scientific Corporation, Beijing 100080, China.
Journal of pharmaceutical and biomedical analysis
|September 27, 2025
概括
本研究介绍了一种自上而下的质谱法,用于分析重组蛋白质. 该方法提供了直接测序和结构洞察力,补充了治疗性蛋白质表征的传统自下而上的方法.
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:
- 再组合蛋白质是关键的治疗方法,需要有力的表征.
- 自下而上的质谱法方法面临的挑战是重复序列和完整的蛋白质分析.
- 上下质谱法 (TDMS) 为全面的蛋白质分析提供了替代方案.
研究的目的:
- 建立和验证TDMS工作流程,用于评估重组蛋白质的结构完整性和碎片化概况.
- 为了比较基于MS2的目标和基于数据依赖获取 (DDA) 的TDMS策略.
- 通过使用TDMS,提供有关识别50kDa以下蛋白质的见解.
主要方法:
- 开发基于MS2和基于DDA的目标TDMS工作流程.
- 对三种代表性的重组蛋白质进行分析:RC1,RC2和干扰素α2b.
- 使用了高能碰撞解离 (HCD) 碎片和二硫化物减少预处理.
主要成果:
- 通过TDMS,可以通过重复序列直接测序蛋白质,克服了自下而上的限制.
- 基于DDA的TDMS实现了对12kDa以下的蛋白质的高通量识别 (RC1的片段覆盖率为50%).
- 基于目标MS2的TDMS改善了12kDa以上蛋白质的序列覆盖率 (RC2的最高49.5%).
- 对于具有二硫化物键的蛋白质来说,二硫化物键预处理是必不可少的,以提高裂变覆盖率.
结论:
- TDMS为全面的重组蛋白质表征提供了自下而上的方法的补充数据.
- 开发的TDMS策略对50kDa以下的蛋白质有效,包括具有二硫化物键的蛋白质.
- 这项工作增强了用于质量控制和治疗蛋白质开发的分析工具包.
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