TitleDecoding the Assembly of Mixed and Branched Heterotypic Ubiquitin Chains.
Publication TypeJournal Article
Year of Publication2023
AuthorsSingh G, Kumar S, Das R
JournalAnal Chem
Date Published2023 Jul 04

Ubiquitination is a post-translational modification that regulates cell signaling, immune response, protein processing, molecular trafficking, and DNA repair. Generally, molecular trafficking and DNA repair processes need the attachment of a single ubiquitin on a substrate, known as monoubiquitination. The other functions of ubiquitin require the assembly of polymeric ubiquitin chains on the substrate, known as polyubiquitination. The chains are linked through the lysine residues of ubiquitin, and depending on which lysine is connected, the chains could be heterotypic or homotypic. Heterotypic polyubiquitin chains can be mixed, branched, or combined, generating myriad cellular signals with functions distinct from the homotypic ubiquitin chains. The molecular rules of heterotypic chain assembly are poorly understood due to the lack of techniques to monitor their assembly. New approaches are required to monitor the conjugation site of new ubiquitin molecules on a pre-existing chain. Here, we describe a new method based on isotopic labeling and mass spectrometry to study the assembly of heterotypic chains called isotopically resolved mass spectrometry of peptides (IRMSP). The technique is demonstrated using multiple ubiquitin enzymes and ubiquitin chains as substrates. It causes minimal perturbation to the enzyme/substrate and will be instrumental in studying the assembly of large polymeric ubiquitin chains. Using this technique, it is feasible to monitor how and with what rate branched ubiquitin chains grow in different directions in a single experiment.

Alternate JournalAnal Chem
PubMed ID37352502