深度覆盖和扩展序列读取通过单个古老蛋白酶快速 de novo 通过质谱测量蛋白质测序获得
bioRxiv : the preprint server for biology
|June 12, 2025
概括
这项研究引入了新型的超热酸考古 (HTA) 蛋白酶,以实现高效的新型蛋白质测序. 这些单酶溶液提供卓越的多样性和完整的抗体测序,优于传统方法.
科学领域:
- 蛋白质组学是指蛋白质组学.
- 生物化学 生物化学
- 分析化学 分析化学
背景情况:
- 在抗体测序和抗原发现等应用中,de novo蛋白质测序至关重要.
- 使用素的传统方法在的多样性和序列覆盖方面存在局限性,特别是在抗体方面.
- 现有的多蛋白酶或混合分裂策略增加了复杂性,降低了可扩展性.
研究的目的:
- 为了评估超热酸古老 (HTA) 蛋白酶作为单酶溶液用于新型抗体测序.
- 为了比较HTA-蛋白质酶与标准蛋白质酶 (如素和化学素) 的性能.
- 评估精简的HTA-蛋白酶和EAciD碎片化工作流程的效率和可扩展性.
主要方法:
- 利用两个不同的HTA-蛋白酶进行蛋白质消化.
- 在ZenoTOF 7600质谱系统上使用EAciD碎片化.
- 使用PEAKS/DeepNovo和Stitch软件进行了新的测序分析.
主要成果:
- HTA-蛋白酶生成的独特读数大约是素或化学素的五倍.
- 在互补性确定区域 (CDR) 实现了高冗余性,以实现完整的抗体测序.
- 证明了高达四倍的对齐得分,并减少了HTA生成数据的序列错误.
结论:
- 通过HTA-EAciD方法,可以使用单个蛋白酶进行完整且明确的新型抗体测序.
- 这种精简的工作流提供了短的消化时间,最小的清洁和单次LC-MS/MS运行分析.
- HTA-EAciD方法为复杂的多酶工作流提供了一个可扩展和高效的替代方案,用于新的蛋白质测序.
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