简单的蛋白质组学样本准备:在一个新的核心设施设置中,在8个生物矩阵中技术重复性和工作流程优化
Paraskevi Karousi1,2, Maria Voumvouraki2,3, Panagiota Efstathia Nikolaou4,5
1Section of Biochemistry and Molecular Biology, Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens, Greece.
Proteomics
|October 24, 2025
概括
这项研究优化了通过轻松提取和消化 (SPEED) 的样本制备协议,以实现可扩展的高通量蛋白质组学. 经过调整的SPEED方法使得从最少的细胞数量到各种生物样本能够准确地分析蛋白质.
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:
- "自下而上的蛋白质组学"需要有效的样本准备,以准确识别和量化蛋白质.
- 现有的协议可能很复杂,可能不适合各种生物矩阵,包括耐溶性样本.
研究的目的:
- 评估和调整通过轻松提取和消化的样品制备 (SPEED) 协议,以实现简化,无洗剂的蛋白质组学.
- 为了在各种生物样本中实现标准化,成本效益和可扩展的蛋白质组学分析.
- 为了证明协议的下降可扩展性低细胞输入和其与高通量工作流的兼容性.
主要方法:
- 精制蛋白质提取和变质化步骤为8个生物矩阵.
- 调整了SPEED协议以实现96井板兼容性,并使用低洗剂的RIPA缓冲器用于组织样本.
- 实现了对diaPASEF分析的优化数据独立采集 (DIA) 窗口,使用短时间的nanoLC-MS/MS运行.
主要成果:
- 从最少3000个用于制备的细胞和300个用于LC-MS/MS分析的细胞获得了强大的蛋白质组学测量.
- 实现了每天15-20个样本的吞吐量,使用30分钟的纳米LC-MS/MS运行.
- 证明了增强的蛋白质组覆盖范围和简化的工作流程,以挑战生物矩阵.
结论:
- 适应的SPEED协议为高通量蛋白质组学提供了强大的,可扩展的和具有成本效益的解决方案.
- 这种方法可以在各种生物环境中进行可重复的蛋白质组研究,即使采用有限的样本材料.
- 早期优化和可行性测试对于成功的蛋白质组学管道开发至关重要.
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