Time to rethink circadian rhythms beyond the transcriptome

The circadian clock generates ~24-hour rhythms in physiology by coordinating gene expression programs, but temporal mRNA profiles often fail to predict their protein function rhythms. This gap reflects regulatory layers beyond transcription, including rhythmic translation, protein stability, subcellular localization and post-translational modifications that collectively determine the circadian rhythms of protein abundance and activity. Here, we summarize evidence supporting a shift from RNA-level descriptions to protein-level frameworks and readouts of the circadian rhythms. Here, we highlight three topics: (i) protein abundance rhythms as informative but incomplete readouts, (ii) widespread circadian control of nuclear localization and phosphorylation that can occur without changes in total protein levels, and (iii) multi-tissue proteomic comparisons that reveal how circadian rhythms are organized differently across tissues. We then discuss how recent data-independent acquisition-based, high-throughput mass spectrometry accelerates cross-study reuse and hypothesis generation, as illustrated by a mouse circadian proteome atlas and an interactive portal enabled by Orbitrap Astral mass spectrometer. Together, these advances motivate 'functional chronobiology', linking proteome dynamics to mechanism and disease-relevant physiology.