电泳-相对离子流动性加深单细胞蛋白质组学在毛细血管电泳质谱学质谱学
bioRxiv : the preprint server for biology
|September 24, 2024
概括
我们开发了电泳相对应 (Eco) 数据独立采集,以改善单细胞质谱 (MS) 蛋白质组学. 这种方法增强了离子流动性的分辨率,使复杂的生物样本中能够更深入地识别和量化蛋白质组.
科学领域:
- 蛋白质组学是指蛋白质组学.
- 分析化学 分析化学
- 生物技术是生物技术.
背景情况:
- 在单细胞质谱 (MS) 蛋白质组学中检测微量敏感信号仍然是一个重大挑战.
- 在检测之前的分离技术对于提高蛋白质组识别和量化准确度至关重要.
- 毛细管电泳 (CE) 电喷射电离 (ESI) 之前被认为是按质量与电荷的比率对进行排序.
研究的目的:
- 为了证明电泳运动相关性转移到气相,使离子的时间排序.
- 引入和验证电泳相对应 (Eco) 数据独立获取,以提高成员国的离子移动性 (IM) 解析.
- 应用生态数据独立获取 (ddaPASEF) 来对单个胚胎细胞进行深度蛋白质组分析.
主要方法:
- 使用毛细管电泳 (CE) 与电喷射电离 (ESI) 结合,以订购.
- 通过利用电荷依赖离子移动 (IM) 趋势,开发了生态数据独立采集 (Eco-ddaPASEF).
- 在被困离子移动性质谱仪 (timsTOF PRO) 上应用生态框架来缩小离子移动性获取窗口.
主要成果:
- 在液相电泳流动性和气相离子流动性 (ρ > 0.97) 之间显示出强烈的相关性.
- 与经典的 ddaPASEF 相比,实现了显著增强的 IM 解析.
- 从HeLa细胞消化中鉴定出大约50%的更多蛋白质 (∼962个蛋白质从≤200 pg在<20分钟内).
- 在单个 *Xenopus laevis* 胚胎干细胞中成功分析了 1,157 种蛋白质,分析了 <4% 的总蛋白质组.
结论:
- 生态框架增强了使用 ddaPASEF 的离子移动性光谱学 (IMS) 中的蛋白质体灵敏度.
- 该方法可以对单细胞,包括胚胎干细胞进行深度蛋白质组分析.
- 这种方法有可能用于胚胎细胞分化的蛋白质驱动分类.
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