Getting to the root of branched ubiquitin chains: a review of current methods and functions

Nearly 20 years since the first branched ubiquitin (Ub) chains were identified by mass spectrometry, our understanding of these chains and their function is still evolving. This is due to the limitations of classical Ub research techniques in identifying these chains and the vast complexity of potential branched chains. Considering only lysine or N-terminal methionine attachment sites, there are already 28 different possible branch points. Taking into account recently discovered ester-linked ubiquitination, branch points of more than two linkage types, and the higher-order chain structures within which branch points exist, the diversity of branched chains is nearly infinite. This review breaks down the complexity of these chains into their general functions, what we know so far about the different linkage combinations, branched chain-optimized methodologies, and the future perspectives of branched chain research.