Ubiquitination is a unique type of protein post-translational modification in which the lysine residue on the target protein is covalently linked with ubiquitin, a 76-amino acid protein. More ubiquitin molecules could be further attached to the protein-conjugated ubiquitin, thereby assembling a ubiquitin chain. Protein ubiquitination is an enzymatic reaction cascade which requires the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin-ligating enzymes. Since its discovery four decades ago, roles of ubiquitination in protein degradation, cell cycle, autophagy, signaling transduction, and immunity have been extensively investigated. Besides the canonical biochemical and cell culture-based methods to study protein ubiquitination, other methods like targeted proteomics, cryogenic electron microscopy, and single molecule techniques have been recently employed to illustrate the global protein ubiquitination map and reveal the atomic details of this important molecular event.

This Methods Collection will include, but is not limited to, the following methods: immunoprecipitation and Ni-NTA based His pull down to examine protein ubiquitination using cell culture, cycloheximide chase assay to determine protein stability in cells, in vitro protein ubiquitination assays to analyze E3 ubiquitin ligase activity, systematic proteome-wide analysis of protein ubiquitination, targeted proteomics to profile a certain linkage of protein ubiquitination in cells or in tissue samples, crystallography and cryogenic electron microscopy to determine ubiquitin structures, and single molecule methods to assess protein ubiquitination in vitro.