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, makes analysis of linkage type-specific ubiquitin signaling a challenging and complex task. Here we review the strategies and molecular tools available for enrichment, detection, and characterization of linkage type-specific ubiquitin signaling. 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 molecues 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.