Sun Jian, Xu Xiaolu, Wei Shuo, Yu Yanbao
Department of Biological Sciences, University of Delaware, 105 The Green, Newark, Delaware, 19716 (USA).
Department of Chemistry and Biochemistry, University of Delaware, 163 The Green, Newark, Delaware, 19716 (USA).
bioRxiv. 2025 May 28:2025.05.23.655823. doi: 10.1101/2025.05.23.655823.
Early embryonic development requires tightly regulated molecular programs to coordinate cell division, fate specification, and spatial patterning. While transcriptomic profiling is widely performed, proteomic analyses of early vertebrate embryos remain limited due to technical challenges in embryonic sample preparation. Here, we propose an "in-cell proteomics" approach, which bypasses cell lysis and yolk depletion, processes individual embryos directly in functionalized filter devices, and generates liquid chromatography-mass spectrometry (LC-MS)-friendly samples in an extremely robust and streamlined manner. Combined with a single-shot data-independent acquisition (DIA) MS workflow, this approach enabled us to consistently quantify ~6,200 proteins from a single embryo, representing the deepest proteomic coverage of early development reported to date. Investigation of the temporal proteomes across five cleavage stages (1- to 16-cell) revealed a drastic proteomic shift between 2- and 4-cell stages, followed by more gradual transitions thereafter. Spatial analysis of dissected 8-cell blastomeres uncovered pronounced molecular asymmetry along the animal-vegetal axis, while dorsal-ventral differences were minimal. This study establishes a novel in-cell proteomics technology in conjunction with DIA-MS as a robust platform for high-resolution, low-input developmental proteomics analysis, and provides a comprehensive spatiotemporal protein atlas for early embryos.
早期胚胎发育需要严格调控的分子程序来协调细胞分裂、命运决定和空间模式形成。虽然转录组分析被广泛应用，但由于胚胎样本制备的技术挑战，早期脊椎动物胚胎的蛋白质组学分析仍然有限。在这里，我们提出一种“细胞内蛋白质组学”方法，该方法绕过细胞裂解和卵黄去除，直接在功能化过滤装置中处理单个胚胎，并以极其稳健和简化的方式生成适合液相色谱-质谱联用（LC-MS）分析的样本。结合单次数据非依赖采集（DIA）质谱工作流程，这种方法使我们能够从单个胚胎中持续定量约6200种蛋白质，这代表了迄今为止报道的早期发育阶段最深层次的蛋白质组覆盖范围。对五个卵裂阶段（1细胞至16细胞）的时间蛋白质组进行研究发现，在2细胞和4细胞阶段之间蛋白质组发生了剧烈变化，此后变化更为渐进。对解剖后的8细胞期卵裂球进行空间分析发现，沿动物-植物轴存在明显的分子不对称性，而背腹差异最小。本研究建立了一种结合DIA-MS的新型细胞内蛋白质组学技术，作为高分辨率、低输入量发育蛋白质组学分析的强大平台，并为早期胚胎提供了一个全面的时空蛋白质图谱。
