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

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.