TULIP: Targets of Ubiquitin Ligases Identified by Proteomics
Román González-Prieto and Alfred C.O. Vertegaal
from: SUMOylation and Ubiquitination: Current and Emerging Concepts (Edited by: Van G. Wilson). Caister Academic Press, U.K. (2019) Pages: 147-160.
Abstract
Ubiquitin is a small protein that can be attached to thousands of substrates via an enzymatic cascade involving an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Over 600 different human E3 ligases have been found. The identification of specific targets for ubiquitin E3 ligases is challenging. So far, most of the approaches aimed to identify E3-specific substrates rely on indirect evidence. Here, we would like to introduce TULIP (Targets of Ubiquitin Ligases Identified by Proteomics) methodology, which enables the identification of ubiquitin E3-specific targets in a direct manner. The rationale behind this strategy is that on construction of a linear fusion between an E3 of interest and ubiquitin, this ubiquitin moiety will be employed by the linked E3 to modify its substrates. Subsequently, trapped substrates are purified in denaturing buffers to remove non-covalent interactors. Starting from the cDNA sequence of an E3 ligase of interest and finishing with the identification of the ubiquitination targets by mass spectrometry-based proteomics, the whole process takes between 4 to 6 weeks. The description of the methodology includes a discussion of potential pitfalls and specific recommendations read more ...
