StUbEx PLUSA Modified Stable Tagged Ubiquitin Exchange System for Peptide Level Purification and In-Depth Mapping of Ubiquitination Sites

Modulation
of protein activities by reversible post-translational
modifications (PTMs) is a major molecular mechanism involved in the
control of virtually all cellular processes. One of these PTMs is
ubiquitination, which regulates key processes including protein degradation,
cell cycle, DNA damage repair, and signal transduction. Because of
its importance for numerous cellular functions, ubiquitination has
become an intense topic of research in recent years, and proteomics
tools have greatly facilitated the identification of many ubiquitination
targets. Taking advantage of the StUbEx strategy for exchanging the
endogenous ubiquitin with an epitope-tagged version, we created a
modified system, StUbEx PLUS, which allows precise mapping of ubiquitination
sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells
treated with proteasomal inhibitors resulted in the identification
of 41 589 sites on 7762 proteins, which thereby revealed the
ubiquitous nature of this PTM and demonstrated the utility of the
approach for comprehensive ubiquitination studies at site-specific
resolution.