Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied O-18-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two O-16 atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with O-18 atoms by deubiquitination in O-18-labeled aqueous solution. By using quantitative mass spectrometry, we detected O-18 atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the O-18-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated O-18-incorporation into ubiquitin. (C) 2020 Elsevier Inc. All rights reserved.