The Molecular Toolbox for Linkage Type‐Specific Analysis of Ubiquitin Signaling

Modification of proteins and other biomolecules with ubiquitin regulates virtually all aspects of eukaryotic cell biology. Ubiquitin can be attached to substrates as a monomer or as an array of polyubiquitin chains with defined linkages between the ubiquitin moieties. Each ubiquitin linkage type adopts a distinct structure, enabling the individual linkage types to mediate specific functions or outcomes in the cell. The dynamics, heterogeneity, and in some cases low abundance, make analysis of linkage type‐specific ubiquitin signaling a challenging and complex task. Herein, the strategies and molecular tools available for enrichment, detection, and characterization of linkage type‐specific ubiquitin signaling, are reviewed. The molecular “toolbox” consists of a range of molecularly different affinity reagents, including antibodies and antibody‐like molecules, affimers, engineered ubiquitin‐binding domains, catalytically inactive deubiquitinases, and macrocyclic peptides, each with their unique characteristics and binding modes. The molecular engineering of these ubiquitin‐binding molecules makes them useful tools and reagents that can be coupled to a range of analytical methods, such as immunoblotting, fluorescence microscopy, mass spectrometry‐based proteomics, or enzymatic analyses to aid in deciphering the ever‐expanding complexity of ubiquitin modifications.