Mouse embryos at the 2-cell to 8-cell stages were collected and treated with acidic Tyrode’s solution to remove the zona pellucida. Subsequently, embryos were split into single blastomeres, and nuclei were isolated from each blastomere using a micromanipulator. Proteomic samples were then prepared using the in situ digestion of alcohol-fixed cells (iSDAC) method with 50 (2-cell), 216 (4-cell), and 280 (8-cell) nuclei per sample. The nuclear proteome during mouse preimplantation development was identified by label-free quantification.