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对于低细胞输入的高通量蛋白质组积分可溶性变化试验,使用一端式.

Maico Lechner¹, Pierre Sabatier^2,3, Jesper V Olsen⁴

¹Department of Cellular and Molecular Medicine, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Communications chemistry

|September 27, 2025

概括

我们通过使用蛋白质组积溶性改变 (PISA) 简化了药物标参与分析. 这种方法显著减少了细胞需求和处理时间,提高了更广泛实验室应用的效率.

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A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

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科学领域:

生物化学生物化学
蛋白质组学是指蛋白质组学.
化学生物学化学生物学

背景情况:

细胞热转移试验 (CETSA) 和其基于质谱的变体,如蛋白质组积溶性改变 (PISA),对于监测药物向参与至关重要.
目前的PISA方法在吞吐量,可扩展性和样本量方面存在局限性,这限制了它们的广泛使用.

研究的目的:

通过整合单一提示方法,为PISA开发一个简化和可扩展的样本准备工作流程.
减少样本输入要求,提高基于CETSA的药物向药物参与概况的效率和可访问性.

主要方法:

结合PISA与一点方法用于简化样本准备.
使用的n-Dodecyl-β-D-Maltoside (DDM) 用于细胞溶解,与质谱学和一键处理兼容.
将细胞输入要求降低到每μL的200个细胞.
应用一键PISA工作流程,以96井板格式进行化酶抑制剂的分析.

主要成果:

在PISA中实现了显著较低的细胞要求 (低至200细胞/μL).
通过一点PISA方法证明了可比的蛋白质深度和改进的可重复性.
减少材料和溶剂的使用,与传统协议相比,蛋白质分解消化速度更快.
在12小时内实现了从细胞处理到质谱注射的完整工作流.

结论:

单一提示PISA方法为药物向参与研究提供了一种具有成本效益,快速和用户友好的方法.
这种集成的工作流提高了可扩展性和可访问性,使先进的蛋白质基因分析成为更多实验室可行的.
优化的工作流通过提高目标参与评估的效率来加速药物发现和开发.