Proteomic strategies for characterizing ubiquitin-like modifications

The modification of proteins by the addition of ubiquitin and other ubiquitin-like proteins (UBLs) is involved in a wide range of cellular processes including cell cycle progression, the DNA damage response, endocytosis, cell signalling, autophagy and protein quality control. The UBL family comprises more than a dozen structurally related members, with ubiquitin, small ubiquitin-like modifier (SUMO) proteins, NEDD8, ISG15 and FAT10 being the most commonly known. Each UBL is associated with a distinct set of enzymes that alter the architecture and fate of their cognate proteins. UBL-conjugating enzymes add one or more UBLs to lysine and non-lysine acceptor sites on their target proteins, forming a complex distribution of monomeric and polymeric modifications. Different approaches and strategies are available to identify the sites of UBL modification, the types of modification and their dynamics upon various cellular stimuli; these techniques can decipher the complex architecture of UBL substrates and expand our understanding of UBL functions and their importance in cellular homeostasis and human diseases. This Primer covers the current methods for identifying UBL substrates, their modification sites and UBL chain linkages, and describes where the application of these methods can be used to gain biological insights into UBL functions. This Primer discusses approaches for characterizing ubiquitin-like proteins (UBLs), their targets and modification sites, and dynamic changes in modifications. The authors cover techniques for enriching modified proteins and mass spectrometry analysis strategies and workflows, describe how these techniques have been applied and outline how limitations are being addressed by new techniques.