Identification of Microproteins in Hep3B Cells at Different Cell Cycle Stages

Microproteins are generated from small open reading frames and turn out to play various vital biological functions. As an essential biological event of eukaryotic cells, the cell cycle is involved in cell replication and division. For such a highly regulated event, microproteins associated with cell cycle regulation remained unclarified. Utilizing a combination of bottom-up and top-down proteomics, we analyzed microproteins at specific cell cycle stages of Hep3B cells. A total of 657 microproteins were identified under three cell cycle stages, including 151 in the G0/G1 stage, 163 in the S stage, and 132 in the G2/M stage. The annotation of these microproteins showed their cell cycle-specific functions, such as translation, nuclear assembly, chromatin organization, and the G2/M transition of the mitotic cell cycle. Meanwhile, more than 50% of identified microproteins were ncRNA-encoded. These nonannotated novel microproteins contain several function domains, such as the nucleoside diphosphate kinase domain, the high mobility group domain, and the DNA-binding domain. This suggested the potential functions of these novel microproteins in specific cell cycle stages. This study presented a large-scale profile of microproteins at different cell cycle stages from Hep3B and may provide new perspectives on the regulation mechanism of the cell cycle. Liquid chromatography-mass spectrometry data were deposited to ProteomeXchange using the identifier PXD030286.