A quantitative atlas of mitotic phosphorylation was conducted to identify and quantify phosphorylation events in human cells during the G and mitotic phases of the cell cycle. Using stable isotope labeling and phosphopeptide enrichment techniques, the researchers identified over 14,000 unique phosphorylation sites on 3,682 proteins, with more than half of these sites not previously described in the literature. The majority of these sites were found in [S/T]P motifs, suggesting they may be CDK substrates. Analysis of non-proline site-containing phosphopeptides revealed two unique motifs, indicating the presence of at least two undiscovered mitotic kinases. The study compared phosphorylation levels in cells arrested in G1 and mitotic phases, revealing a significant increase in phosphorylation during mitosis. The data showed that many known cell cycle regulators were phosphorylated, and a large number of phosphorylation sites were found in [pS/pT]-P motifs, which are the minimum required sequence for CDKs. The researchers also identified several known cell cycle-regulated events and many unreported sites on known cell cycle regulators. The study used a combination of mass spectrometry and bioinformatics to analyze the data, identifying over 14,000 phosphorylation sites. The results showed that many proteins involved in critical cell cycle processes were phosphorylated, suggesting that many of the unknown targets may themselves be regulators of the cell cycle. The study also identified several new phosphorylation motifs, including those for Aurora kinase A and Polo-like kinase 1, which may be important in mitotic regulation. The researchers developed a metric called the regulated phosphorylation (RePh) score to assess the relative abundance of phosphorylation sites. This score was used to evaluate the phosphorylation levels of known mitotic proteins and to identify proteins that may be involved in cell cycle regulation. The study also identified several new kinases and substrates, including those involved in the DNA damage response and chromosome condensation. The findings of this study provide a comprehensive view of mitotic phosphorylation and highlight the importance of phosphorylation in cell cycle regulation. The data suggest that many proteins are regulated by multiple phosphorylation events, which may play a role in signal integration, creating signaling thresholds, or generating dynamic behaviors. The study also identified several new phosphorylation motifs that may be important in mitotic regulation. Overall, the study provides a valuable resource for understanding the complex regulatory mechanisms of the cell cycle.A quantitative atlas of mitotic phosphorylation was conducted to identify and quantify phosphorylation events in human cells during the G and mitotic phases of the cell cycle. Using stable isotope labeling and phosphopeptide enrichment techniques, the researchers identified over 14,000 unique phosphorylation sites on 3,682 proteins, with more than half of these sites not previously described in the literature. The majority of these sites were found in [S/T]P motifs, suggesting they may be CDK substrates. Analysis of non-proline site-containing phosphopeptides revealed two unique motifs, indicating the presence of at least two undiscovered mitotic kinases. The study compared phosphorylation levels in cells arrested in G1 and mitotic phases, revealing a significant increase in phosphorylation during mitosis. The data showed that many known cell cycle regulators were phosphorylated, and a large number of phosphorylation sites were found in [pS/pT]-P motifs, which are the minimum required sequence for CDKs. The researchers also identified several known cell cycle-regulated events and many unreported sites on known cell cycle regulators. The study used a combination of mass spectrometry and bioinformatics to analyze the data, identifying over 14,000 phosphorylation sites. The results showed that many proteins involved in critical cell cycle processes were phosphorylated, suggesting that many of the unknown targets may themselves be regulators of the cell cycle. The study also identified several new phosphorylation motifs, including those for Aurora kinase A and Polo-like kinase 1, which may be important in mitotic regulation. The researchers developed a metric called the regulated phosphorylation (RePh) score to assess the relative abundance of phosphorylation sites. This score was used to evaluate the phosphorylation levels of known mitotic proteins and to identify proteins that may be involved in cell cycle regulation. The study also identified several new kinases and substrates, including those involved in the DNA damage response and chromosome condensation. The findings of this study provide a comprehensive view of mitotic phosphorylation and highlight the importance of phosphorylation in cell cycle regulation. The data suggest that many proteins are regulated by multiple phosphorylation events, which may play a role in signal integration, creating signaling thresholds, or generating dynamic behaviors. The study also identified several new phosphorylation motifs that may be important in mitotic regulation. Overall, the study provides a valuable resource for understanding the complex regulatory mechanisms of the cell cycle.