We collected over 8000 mouse embryos of each developmental stage (zygote, 2-cell, 4-cell, 8-cell, morula, blastocyst) and performed tandem mass tag (TMT)-based quantitative mass spectrometry and identified nearly 5000 proteins for each stage. In-depth analysis indicates that protein expression profiles of zygote, morula and blastocyst show apparent difference from 2- to 8-cell embryos due to the maternal-totipotent-differentiation transition. We further identified novel factors and proved that they play important roles in determining pre-implantation embryo development