[Advances in the application of affinity separation for analyzing protein ubiquitination].

Protein ubiquitination is one of the most common yet complex post-translational modifications in eukaryotes that plays an important role in various biological processes including cell signal transduction, growth, and metabolism. Disorders in the ubiquitination process have been revealed to correlate with the occurrence and development of many diseases such as neurodegenerative disease, inflammation, and cancer. Investigation of protein ubiquitination is of great importance to uncover protein functions, understand the molecular mechanisms underlying biological processes, and develop novel strategies for disease treatment. Great advances have been made toward understanding protein ubiquitination; however, it remains a challenging task due to the high diversity of ubiquitination sites and structures, as well as the dynamic nature of ubiquitination in biological processes. Protein ubiquitination occurs through the formation of a covalent bond between the carboxyl terminus of ubiquitin and the ε-amino group of a lysine residue in the substrate. As a small protein, ubiquitin itself can be further modified by another ubiquitin molecule to form homotypic or heterotypic polyubiquitin chains. There are eight sites, namely seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) and one N-terminal methionine (M1), in one ubiquitin molecule that can be used to form a ubiquitin dimer. The variations in modification sites, ubiquitin chain lengths, and conformations result in differences in protein sorting, cell signaling, and function. To resolve the high complexity of protein ubiquitination, new separation approaches are required. Affinity separation based on the specific recognition between biomolecules offers high selectivity and has been employed to study the structures and functions of ubiquitination. In addition, affinity ligands are central to the separation performance. Different affinity ligands have been developed and employed for the capture and enrichment of ubiquitylated proteins. Immunoaffinity separation based on antigen-antibody interactions has been one of the most classical separation methods. Antibodies against ubiquitin or different ubiquitin linkages have been developed and widely applied for the enrichment of ubiquitylated proteins or peptides. The specific capture allows the downstream identification of endogenous ubiquitination sites via mass spectrometry and thus facilitates understanding of the roles and dynamics of polyubiquitin signals. Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that can interact with ubiquitin or polyubiquitin chains. Ubiquitin-associated domains, ubiquitin-interacting motifs, and ubiquitin-binding zinc finger domains are the most frequently used UBDs. Due to the moderate affinity of UBDs toward ubiquitin or ubiquitin chains, tandem ubiquitin-binding entities (TUBEs) have been engineered with high affinities (