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Post-translational modification of proteins with the small polypeptide ubiquitin plays a pivotal role in many cellular processes, altering protein lifespan, location, and function and regulating protein-protein interactions. Ubiquitination exerts its diverse functions through complex mechanisms by formation of different polymeric chains and subsequent recognition of the ubiquitin signal by specific protein interaction domains. Despite some recent advances in the analytical tools for the analysis of ubiquitination by mass spectrometry, there is still a need for additional strategies suitable for investigation of cellular ubiquitination at the proteome level. Here, we present a stable tagged ubiquitin exchange (StUbEx) cellular system in which endogenous ubiquitin is replaced with an epitope-tagged version, thereby allowing specific and efficient affinity purification of ubiquitinated proteins for global analyses of protein ubiquitination. Importantly, the overall level of ubiquitin in the cell remains virtually unchanged, thus avoiding ubiquitination artifacts associated with overexpression. The efficiency and reproducibility of the method were assessed through unbiased analysis of epidermal growth factor (EGF) signaling by quantitative mass spectrometry, covering over 3400 potential ubiquitinated proteins. The StUbEx system is applicable to virtually any cell line and can be readily adapted to any of the ubiquitin-like post-translational modifications.

StUbEx: Stable Tagged Ubiquitin Exchange System for the Global Investigation of Cellular Ubiquitination