| Autor: |
Alexandre, Désert, Karine, Guitot, Audrey, Michaud, Daniel, Holoch, Raphaël, Margueron, Fabienne, Burlina, Dominique, Guianvarc'h |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
Methods in molecular biology (Clifton, N.J.). 2529 |
| ISSN: |
1940-6029 |
| Popis: |
Identification of histone lysine methyltransferase (HKMT) substrates has recently benefited from chemical-biology-based strategies in which artificial S-adenosyl-L-methionine (SAM) cofactors are engineered to allow substrate labeling using either the wild-type target enzyme or designed mutants. Once labeled, substrates can be selectively functionalized with an affinity tag, using a bioorthogonal ligation reaction, to allow their recovery from cell extracts and subsequent identification. In this chapter, we describe steps on how to proceed to set up such an approach to characterize substrates of specific HKMTs of the SET domain superfamily, from the characterization of the HKMT able to accommodate a SAM surrogate containing a bioorthogonal moiety, to the proteomic analysis conducted on a cell extract. We focus in particular on the controls that are necessary to ensure reliable proteomic data analysis. The example of PR-Set7 on which we have implemented this approach is shown. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
