Background Identifying CO-binding proteins is vital for our knowledge of CO-regulated molecular processes. The carbamate post-translational modification is a reversible CO-mediated adduct that can form on neutral -terminal α-amino or lysine ε-amino groups. Methods We have developed triethyloxonium ion (TEO) as a chemical proteomics tool to trap the carbamate post-translational modification on protein covalently. We use C-NMR and TEO and identify ubiquitin as a plant CO-binding protein. Results We observe the carbamate post-translational modification on the ubiquitin ε-amino groups of lysines 6, 33, and 48. We show that biologically relevant near atmospheric PCO levels increase ubiquitin conjugation dependent on lysine 6. We further demonstrate that CO increases the ubiquitin E2 ligase (UBC5) charging step via the transthioesterification reaction in which Ub is transferred from the E1 ligase active site to the E2 active site. Conclusions and general significance Therefore, plant ubiquitin is a CO-binding protein, and the carbamate post-translational modification represents a potential mechanism through which plant cells can respond to fluctuating PCO.