3xMBT protein pull-down - A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation

ABSTRACT: Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation is largely unknown. We have developed the naturally occurring MBT domain repeats of L3MBTL1 (3xMBT) to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. In experiments using two different instruments (experiment 1 - Orbitrap Elite, experiment 2 - Orbitrap Velos) we have used SILAC to compare proteins captured by 3xMBT to proteins captured by the D355N mutant, which does not bind methylated lysine. Data was analyzed using MaxQuant version 1.3.0.5 using default parameters except that mono and di-methylated lysine were included as variable modifications and modification-specific false discovery rate was set to 10%. Several hundred proteins are specifically enriched by 3xMBT and we have directly detected lysine methylation on about two dozen. We have also used our approach to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Three-way SILAC was used to compare methylated proteins between cells treated with UNC0638, a specific inhibitor of G9a and GLP, and cells treated with vehicle control. We find reduced capture of the known G9a substrates WIZ and ACIN1, as well as identifying DNA ligase 1 as a potential target for G9a/GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.

INSTRUMENT(S): LTQ Orbitrap Velos, LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Scott Carlson  

LAB HEAD: Scott Carlson

PROVIDER: PXD000361 | Pride | 2013-11-11

REPOSITORIES: Pride