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高通量无标签单细胞蛋白质学,通过多列纳米LC实现,周期时间为5分钟.

Chao Wang1, Hsien-Jung L Lin1, Siqi Huang1

  • 1Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States.

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概括

一个新的多列纳米LC-MS平台通过将运行时间缩短到5分钟,实现高通量单细胞蛋白质组学 (SCP). 这一突破使得每项研究可以分析成千上万的细胞,提供了深入的蛋白质组覆盖和生物洞察力.

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科学领域:

  • 蛋白质组学是指蛋白质组学.
  • 质谱测量质量谱测量
  • 细胞生物学 细胞生物学

背景情况:

  • 基于质谱 (MS) 的单细胞蛋白质组学 (SCP) 提供了高分辨率的蛋白质组分析.
  • 目前的nanoLC-MS工作流程由于长的分离梯度而具有较低的吞吐量.
  • 这限制了SCP在大规模研究中的应用.

研究的目的:

  • 为SCP开发一个高通量多列纳米LC-MS平台.
  • 为了在单个研究中分析成千上万个单细胞.
  • 为了保持深层的蛋白质组覆盖和生物解释性.

主要方法:

  • 实施一个多列纳米LC-MS系统,具有5分钟的分离窗口.
  • 运行在~100nL/分钟流速下,使用100%的工作周期.
  • 分析了4000多个样本,包括RAW264.7巨细胞.

主要成果:

  • 每天达到288个单细胞的吞吐量 (SPD).
  • 确定了每单个HeLa细胞约3,200种蛋白质,与较长梯度方法相比.
  • 在6000多种蛋白质和显著的细胞蛋白质中证明了精确的量化.
  • 揭示了LPS诱导的标记物和巨细胞激活通路.

结论:

  • 开发的平台是强大的,可扩展的,适合高吞吐量SCP.
  • 能够在一个研究中进行数千次单细胞分析.
  • 保持深层蛋白质覆盖和生物解释性用于细胞异质性研究.