允许单细胞分析的技术正在获得广泛关注,这些研究大多利用基因组学的方法。虽然纳米流体技术已经实现了单细胞的质谱分析,这些测量仅限于代谢组学和脂质组学研究.单细胞蛋白质组学具有提高我们对细胞间异质性的理解的潜力。然而,这种方法面临的挑战包括样本可用性有限,以及高度敏感的样品收集方法的要求,清理,和检测。我们提出了一种克服这些限制的技术,通过将基于微量移液管(拉玻璃毛细管)的样品收集策略与离线样品制备和nanoLC-MS/MS相结合,通过自下而上的蛋白质组学策略分析蛋白质。本研究探讨了两种类型的蛋白质组学数据获取策略,即数据依赖(DDA)和数据独立获取(DIA)。研究结果表明,DIA更灵敏,能够分析来自约130μm非洲爪狼胚胎细胞中含有<6nL细胞质的>1600种蛋白质。发现该方法在从跨越1-128细胞发育阶段的单细胞获得可重复的蛋白质定量方面是稳健的。此外,我们使用微量移液管取样研究了单个胚胎细胞内的细胞间异质性,并研究了胚胎发育早期沿动物-植物轴和背-腹轴的不对称性.对动物-植物轴的研究导致发现了沿动物-植物轴的各种不对称分布的蛋白质。我们进一步将从我们的蛋白质组数据集发现的命中与其他研究进行了比较,并使用正交成像技术验证了一些命中。这项研究是非洲爪鱼种质相关蛋白DDX4/VASA植物富集的第一份报告。总的来说,这里提供的方法和数据有望在发育生物学中发挥重要作用。
Techniques that allow single cell analysis are gaining widespread attention, and most of these studies utilize genomics-based approaches. While nanofluidic technologies have enabled mass spectrometric analysis of single cells, these measurements have been limited to metabolomics and lipidomic studies. Single cell proteomics has the potential to improve our understanding of intercellular heterogeneity. However, this approach has faced challenges including limited sample availability, as well as a requirement of highly sensitive methods for sample collection, cleanup, and detection. We present a technique to overcome these limitations by combining a micropipette (pulled glass capillary) based sample collection strategy with offline sample preparation and nanoLC-MS/MS to analyze proteins through a bottom-up proteomic strategy. This
study explores two types of proteomics data acquisition strategies namely data-dependent (DDA) and data-independent acquisition (DIA). Results from the
study indicate DIA to be more sensitive enabling analysis of >1600 proteins from ∼130 μm Xenopus laevis embryonic cells containing <6 nL of cytoplasm. The method was found to be robust in obtaining reproducible protein quantifications from single cells spanning the 1-128-cell stages of development. Furthermore, we used micropipette sampling to
study intercellular heterogeneity within cells in a single embryo and investigated embryonic asymmetry along both animal-vegetal and dorsal-ventral axes during early stages of development. Investigation of the animal-vegetal axis led to discovery of various asymmetrically distributed proteins along the animal-vegetal axis. We have further compared the hits found from our proteomic data sets with other studies and validated a few hits using an orthogonal imaging technique. This
study forms the first report of vegetal enrichment of the germ plasm associated protein DDX4/VASA in Xenopus embyos. Overall, the method and data presented here holds promise to enable important leads in developmental biology.