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Updated: Jul 24, 2025

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

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优化单细胞蛋白质组学使用被困离子流动性谱度进行无标签实验.

Dong-Gi Mun¹, Firdous A Bhat¹, Husheng Ding¹

¹Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First ST SW, Rochester, MN 55905, USA. pandey.akhilesh@mayo.edu.

|July 3, 2023

概括

优化受困离子移动性谱度 (TIMS) 设置可显著提高单细胞中的蛋白质组分析深度. 这一进步允许详细分析低丰度蛋白质和T细胞的翻译后修饰.

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科学领域:

蛋白质组学是指蛋白质组学
单细胞生物学单细胞生物学
质谱测量质量谱测量

背景情况:

单细胞RNA测序彻底改变了生物学研究,但不偏见的单细胞蛋白质组分析落后.
最近在小型化样本处理和质谱学方面的突破,如数据依赖采集的受困离子移动性谱学 (TIMS) 和数据依赖采集 (DDA-PASEF),已经使单细胞的蛋白质组分析成为可能.
虽然TIMS中的离子流影响蛋白质组分析,但其对低输入样本的影响需要进一步调查.

研究的目的:

优化TIMS参数,特别是离子积累/升降时间和离子移动范围,以增强低输入样本的蛋白质组概况.
通过使用优化的 TIMS 条件来评估蛋白质覆盖的深度和检测低丰度蛋白质.
证明从单细胞分析生物途径和翻译后修改的可行性.

主要方法:

优化TIMS设置,包括离子积累时间 (180毫秒) 和更窄的离子移动范围 (0.71.3 V s cm−2).
使用优化 TIMS 条件对被排序的人类初级 T 细胞进行蛋白质组分析.
分析蛋白质丰富度,划分途径,检测翻译后的修饰 (酸化,乙化).

主要成果:

优化的TIMS设置导致蛋白质覆盖率大幅增加,并从低输入样本中检测出低丰度蛋白质.
单个,五个,十个和四十个T细胞的蛋白质样本分别平均产生了365,804,1116和1651种蛋白质.
该研究成功地界定了关键的代谢和T细胞受体信号通路,并检测了单细胞的翻译后修饰.

结论:

优化的TIMS参数显著提高了单细胞蛋白质组分析的深度和灵敏度,特别是在低输入样本中.
这种方法提供了足够的蛋白质组覆盖面,用于途径分析和检测单细胞中的翻译后修饰.
开发的方法有望用于从临床样本中单细胞的无标签分析.