Ubiquitin-mediated modulation of necroptotic effector MLKL

We aimed to identify lysine (K) residues that are endogenously ubiquitylated upon TNF-induced necroptosis. To this end, we used a quantitative mass spectrometry-based approach. Briefly, MDFs were treated with TSZ or left untreated and protein lysates were digested with trypsin into peptides. Ubiquitylated peptides bearing the di-glycine (K-GG) remnant were enriched by immunoprecipitation with anti-K-ε-GG antibody. A total of three biological replicates were prepared and the 6 samples were labelled with Tandem Mass Tags (TMT). Samples were mixed and analysing by Liquid Chromatography-Tandem Mass Spectroscopy (LC-MS). Quantification was performed to determine the relative abundance of site-specific ubiquitylation events in each condition.