The length of a ubiquitin chain is a general determinant for selective recognition by ubiquitin-binding proteins.

The attachment of differently linked ubiquitin (Ub) chains of varying length to proteins is a prevalent posttranslational modification in eukaryotic cells. The fate of a modified protein is determined by Ub-binding proteins (UBPs) that interact with Ub chains in a linkage-selective manner. Therefore, proteome-wide interaction studies using differently linked Ub chains have become a focus of research activities. However, the impact and functional consequences of chain length on the binding selectivity of UBPs remain mostly elusive, due to a lack of available tools and sufficient amounts of pure, length-defined Ub chains. Here we generated linkage- and length-defined Ub chains using click-chemistry and gel-free fractionation and employed such defined polymers in affinity-based enrichment assays to identify length- and linkage-selective interactors on a proteome-wide scale. For the first time, this revealed that the length of a Ub chain has generally a major impact on its ability to be selectively recognized by UBPs.