Protein ubiquitination is an important posttranslational modification that regulates virtually all cellular processes. Its versatility arises from the ability of ubiquitin to form eight structurally and functionally distinct polymeric chains. It is clear that all chain types exist in cells. The two chain types that have been studied in detail have distinct, non-overlapping cellular functions. A lack of tools has hindered progress to define roles of the remaining ‘atypical’ ubiquitin chains.The objective of my laboratory is to understand specificity in the ubiquitin system, and to reveal the cellular roles of atypical ubiquitin chains. To achieve this aim, we have to define the cellular machineries that assemble atypical ubiquitin chains specifically (AIM 1). Our recent advances in enzymatic and chemical biology synthesis have made all chain types available, enabling structural and biophysical characterisation of all ubiquitin polymers (AIM 2). We will use the newly generated atypical ubiquitin chains to identify and characterise linkage-specific ubiquitin binding proteins, using mass-spectrometry. This will reveal the receptors of atypical chains in cells, as well as novel ubiquitin binding domains (AIM 3). Access to all linkage types further allows comprehensive analysis of specificity in the ubiquitin system for the first time. We recently discovered that the Ovarian Tumour (OTU) family of deubiquitinating enzymes (DUBs) comprises specific members for each linkage-type. OTU domain DUBs hence represent a great starting point to understand mechanism of linkage specificity and to define substrates modified with atypical chains in cells (AIM 4). Finally, studying cellular functions of linkage-specific OTU DUBs will uncover novel physiological roles of atypical chains (AIM 5).We are only beginning to understand the full potential of ubiquitin in cellular regulation. Our studies on atypical ubiquitination will contribute to these exciting emerging areas of research.