Dissecting the spatiotemporal regulation of the cell cycle using phosphoproteomics and highlighting functional differences within proteoform families

Protein-coding genes are expressed as dynamic ensembles of unique molecular forms known as proteoforms, and differences between their amino acid sequences or post-translational modifications can imbue important functional differences. These functional differences help to drive fundamental biological processes, such as the cell cycle, a highly conserved process during which a cell grows and divides into two daughter cells. Post-translational modifications in particular can impact the localization of proteins and create important functional differences and compartmentalization of proteoform families. We have conducted a deep phosphoproteomic study of the cell cycle using bottom-up phosphoproteomics to identify over 77,000 phosphosites in U-2 OS cells following fluorescence-aided cell sorting using FUCCI cell cycle markers. These results have expanded the network of cell cycle regulation, with over 4,000 phosphosites displaying abundance changes over the cell cycle, including sites that may be linked to protein translocation events and protein abundance changes independent of RNA levels. These phosphopeptide results provide insights into the origins of cell-to-cell heterogeneity of proteoform expression, including that introduced by the cell cycle, which have relevance in studying human health and diseases such as cancer, and which provide fodder for interrogating the proteoform systems biology of the cell cycle in the future.