Mouse embryos as In Vivo models for proteomic identification and functional analysis of missing proteins

The most plausible function of human missing proteins is that they may participate in the early initiation of organogenesis. Therefore, embryonic tissues could serve as a feasible system for uncovering the existence and biological roles of these proteins. Considering ethical constraints on human embryo research, this study evaluated the feasibility of using mouse embryos as a model to investigate human missing proteins. Mouse embryos were collected from pregnant mice at 6.5, 7.0, 7.5, and 8.0 days post conception (dpc) for mass spectrometric (MS) analysis. The identified peptides, 1338 (6.5 dpc), 1292 (7 dpc), 1312 (7.5 dpc), and 1235 (8 dpc), were analyzed using a newly developed missing protein alignment software, leading to the identification of 112 human missing proteins. Among these, NKX1 was selected for antibody production and validation. Western blot analysis revealed that four missing proteins were detectable in mouse embryos. Notably, NKX1 exhibited a transient expression peak between 7 and 7.5 dpc, followed by a sharp decline at 8 dpc and minimal expression in adult tissues. Co-immunoprecipitation (Co-IP) coupled with mass spectrometry further identified NKX1-, PTCHD1-, ASXL3-, and OTOGL-associated mouse embryonic proteins, which were subsequently analyzed using Ingenuity Pathway Analysis (IPA). The predicted signaling pathways were primarily related to neural development, inflammation, mitochondrial function, and anti-aging processes. In summary, this study demonstrates that the mouse embryo serves as a valuable in vivo platform for detecting human missing proteins and elucidating their temporal expression patterns and potential physiological functions during early development.