Decoding the assembly of Mixed and Branched heterotypic Ubiquitin chains

In eukaryotes, post-translational modification (PTMs) creates a proteome diversity that is essential for cellular processes. The PTM ubiquitination regulates cell signaling, immune response, protein processing, molecular trafficking, and DNA repair. While molecular trafficking typically relies on substrate monoubiquitination, the other functions require the assembly of polymeric Ubiquitin chains on the substrate. The chains are linked through lysine amino acids of Ubiquitin, and depending on which lysine is linked, the chains could be heterotypic or homotypic. The heterotypic Ubiquitin chains generate myriad cellular signals whose functions are distinct from the homotypic Ubiquitin chains. Heterotypic chains can be mixed, branched, or a combination of both. The molecular rules of heterotypic chain assembly are poorly understood. While several techniques exist to detect these chains, few exist to study their assembly. Here we describe a new technique based on isotopic labeling and mass spectrometry to study the assembly of mixed and branched heterotypic chains. The technique is demonstrated using multiple Ubiquitin enzymes and Ubiquitin chains as substrates and will be instrumental in studying the assembly of large Ubiquitin polymeric chains.