TMIMS creates mouse circadian proteome atlas covering 32 organs — Foundation for elucidating protein functions

A research group led by Researcher Yuta Otobe and Project Leader Hikari Yoshitane (also Associate Professor, Graduate School of Science, the University of Tokyo) from the Circadian Clock Project at Tokyo Metropolitan Institute of Medical Science (TMIMS) has used the next-generation mass spectrometer Orbitrap Astral to perform high-precision analysis of 24-hour variations in protein abundance and phosphorylation states in 32 organs and brain regions across the entire mouse body, and constructed a mouse circadian proteome atlas. As a result of the analysis, they identified a total of 18,956 types of proteins from 32 tissues and 584 samples.

The group also succeeded in creating a comprehensive circadian clock map that covers approximately 74% of all mouse proteins registered in UniProt (a database providing amino acid sequence and functional information for proteins, operated by the Swiss Institute of Bioinformatics and the European Bioinformatics Institute). This data is publicly available as the Mouse Circadian Proteome Atlas (https://chronoproteinology.org/circadian_atlas) and is expected to be widely utilized as a foundational resource for investigating "when, where, and which proteins" are functioning, contributing to future circadian clock and drug discovery research. The work was published in the online version of Molecular Cell.

In recent years, the development of next-generation sequencers has enabled comprehensive analysis of daily variations in mRNA in many organs. However, it is proteins that actually perform cellular functions, and their amounts and activities cannot be explained by mRNA variations alone. Because proteins undergo major functional changes—not only through their expression levels but also through intracellular localization and post-translational modifications such as phosphorylation—it has been technically difficult to capture temporal variations at the whole-body level.

The mouse circadian proteome atlas constructed by the research team has made it possible to compare "what time" each protein reaches its peak in "which organ." For example, the clock transcription factor NR1D1 was found to peak in many tissues during the rest phase of mice but showed different amplitudes in the brain versus peripheral tissues.

Furthermore, the researchers conducted an integrated analysis of mRNA, whole-cell proteome, nuclear fraction proteome, and phospho-proteome for the liver, a major organ of the circadian clock. The results revealed that among mRNAs showing rhythmicity, only about 20% showed rhythms at the protein level, indicating that mRNA information alone cannot explain daily variations in organ function. Additionally, the group found many cases where proteins that appeared in constant numbers in whole cells showed strong rhythms within the nucleus, demonstrating that it is "location" rather than "quantity" that is temporally controlled.

In the phosphorylation analysis, 26,000 phosphorylated peptides were identified in the whole liver and 73,000 were identified in the nuclear fraction, with 10-20% of these showing 24-hour rhythms. Many phosphorylation sites had independent variations of protein abundance, suggesting that the time-dependent activities of kinases and phosphatases are important for rhythm formation in liver function.

In small brain regions that have been difficult to analyze, approximately 12,000 proteins were identified from the central clock (the suprachiasmatic nucleus, SCN), and it was discovered that many phosphorylations peak in the subjective morning. This suggests that pathways involved in neural activity may be activated during this time of day.

Furthermore, when mice carrying the hPER2-S662G mutation, which causes familial advanced sleep phase syndrome, were analyzed, the peak times of multiple clock proteins advanced by approximately three hours, and molecular changes consistent with behavioral rhythm changes were confirmed. It became clear that a single amino acid mutation has widespread effects on protein abundance and phosphorylation states throughout the body, and this is expected to contribute to understanding the mechanisms by which circadian rhythm abnormalities are linked to disease risk. In the future, this is expected to be applied to medical practice as a foundation for chronotherapy that optimizes medication timing.

Yoshitane commented: "In this study, we targeted 32 brain regions and organs, but there are still areas we have not fully investigated, such as the skin, bladder, and inner ear, so we would like to continue our research. Furthermore, we found that rhythmicity differs greatly between whole cells and nuclear extracts. In the future, we would like to examine more detailed subcellular compartments such as mitochondria, Golgi apparatus, and synapses. We also hope to study how these change with aging and lifestyle factors to develop applications of the study results."

Journal Information
Publication: Molecular Cell
Title: A mouse circadian proteome atlas
DOI: 10.1016/j.molcel.2025.12.020