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

Abstract

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.

Data on mass spectrometry-based proteomics for studying the involvement of CYLD in the ubiquitination events downstream of EGFR activation

“…Peptides were separated by reversed-phase in an EASY-nLC 1000 (Thermo Scientific) coupled to a Q Exactive mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source. Chromatographic separation of the peptides was performed as previously described [9] using 0.5% acetic acid as solvent A, 80% acetonitrile in 0.5% acetic acid as solvent B and an analytical in-house packed column of ReproSil Pur C 18 -AQ, 3 μm resin (Dr Maisch GmbH). The mass spectrometers were operated in positive ionization mode, in a top 12 data-dependent manner, at a resolution of 70,000 (at m / z 400) and AGC target of 1e6 for the MS survey, scanning from 300 to 1750 m / z .…”

Resolving the Complexity of Ubiquitin Networks

“…Blagoy Blagoev's group has developed the StUbEx PLUS technique, which overcomes two drawbacks of Ub remnant profiling: recognition of UBL proteins and remnant peptide amino acid sequence preference (Akimov et al, 2018b). To detect ubiquitination sites, the method utilizes internally 6xHIS-tagged Ub in the endogenous Ub knockdown background (Akimov et al, 2018b). Insertion of 6xHIS tag near the C terminus of Ub enables enrichment of HIS-Ub-modified substrates (Figure 5D).…”

“…Subsequent proteolytic cleavage after Lys residues generates ubiquitinated peptides, which can be detected by MS. In a proof of concept experiment, StUbEx PLUS identified over 41,000 unique diGly-Ub remnant peptides in nearly 7,800 Ub targets in U2OS cells upon proteasome inhibition (Akimov et al, 2018b). However, StUbEx PLUS is more laborious technique than Ub remnant profiling.…”

Instant Clue: A Software Suite for Interactive Data Visualization and Analysis

“…For example, with the latest generation of mass spectrometry instruments, it is possible to quantify a global proteome of more than 12,000 proteins within a day. In addition, the enrichment of post-translational modifications, including phosphorylations and ubiquitylations results in more than 40,000 modification sites, that can be achieved with moderate laboratory effort 1 – 4 . Such experiments demand cooperative work between biologists with specialized knowledge in their field and computational researchers to unravel meaningful findings.…”