This mini review provides insights into the synergistic power of chemical biology techniques for generating defined ubiquitin (Ub) variants and the use of affinity enrichment mass spectrometry to decipher the Ub code. It focuses on unconjugated Ub variants, which have emerged as additional signaling molecules in the Ub lexicon. Furthermore, it outlines the use of this approach with Ub-like proteins. (Figure created in BioRender. (2025) https://BioRender.com/7vby989).

The covalent attachment of ubiquitin (Ub) to target proteins (ubiquitylation) represents one of the most versatile post-translational modifications (PTM) in eukaryotic cells. Substrate modifications range from a single Ub moiety being attached to a target protein to complex Ub chains that can also contain Ubls (Ub-like proteins) or chemical modifications like acetylation or phosphorylation. The entirety of this complex system is entitled as “the Ub code”. To regulate the Ub code, cells have an arsenal of enzymes to install, translate, and reverse these modifications. However, deciphering the Ub code is challenging due to the difficulty of generating defined Ub/Ubl−protein conjugates. In this mini review, an overview of chemical biology techniques for the generation of defined Ub variants and their subsequent application in affinity enrichment experiments to identify interacting proteins by mass spectrometry is provided. The main focus is on unconjugated Ub variants since they are not well understood even though a “second messenger”-like function of those have been found. Finally, the opportunities to expand this approach to Ubl proteins are briefly discussed.