TULIP2: an improved method for the identification of ubiquitin E3- specific targets
- 1Cell and Chemical Biology, Leiden University Medical Center, Netherlands
Protein modification by Ubiquitin or Ubiquitin-like modifiers is mediated by an enzyme cascade composed of E1s, E2s and E3s enzymes. E1s, or ubiquitin-activating enzymes, perform the ubiquitin activation. Next, ubiquitin is transferred to ubiquitin-conjugating enzymes or E2s. Finally, ubiquitin ligases or E3s catalyze the transfer of ubiquitin to the acceptor proteins. E3 enzymes are responsible for determining the substrate specificity. Determining which E3 enzyme maps to which substrate is a major challenge that is greatly facilitated by the TULIP2 methodology. TULIP2 methodology is fast, precise and cost-effective. Compared to previous TULIP methodology protocol, TULIP2 methodology achieves a more than 50-fold improvement in the purification yield and two orders of magnitude improvement in the signal-to-background ratio after label free quantification by mass spectrometry analysis. The method includes the generation of TULIP2 cell lines, subsequent purification of TULIP2 conjugates, preparation and analysis of samples by mass spectrometry.
Keywords: Ubiquitin, E3 enzymes, Proteomics, post-translational modifications, Mass Spectrometry
Received: 16 Sep 2019;
Accepted: 07 Nov 2019.
Copyright: © 2019 Salas-Lloret, Agabitini and González-Prieto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: PhD. Román González-Prieto, Leiden University Medical Center, Cell and Chemical Biology, Leiden, 2333, Netherlands, R.Gonzalez_Prieto@lumc.nl