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Recent reports highlight phosphorylation as a major role in the regulation of sleep-wake cycles, to a lesser extent, the phosphoproteome in Suprachiasmatic Nucleus (SCN) is not well understood. We use multiplex tandem mass tag (TMT) - labelling coupled with liquid chromatography-mass spectrometry (LC-MS) to study the rhythmic regulatory effects of electroacupuncture(EA) at different times in constant darkness and the mechanism of EA mediated SCN protein phosphorylation. We applied the mass spectrometry-based phosphoproteome to explore the molecular characteristics of EA-induced phosphorylation modification in the SCN.