To explore whether BALB/c-4T1 or C57BL/6-B16F10 ctEVs secreted during different circadian phases differ in proteomic composition, we analyzed ctEVs secreted over 12-hour windows (ZT0-ZT12 vs. ZT12-ZT0) using a nuclease-based aptamer release method to degrade the aptamer linkage. Released EVs were lysed and subjected to nanoscale liquid chromatography-tandem mass spectrometry.