In this project, we revealed that Sharpin deletion significantly inhibits tumor growth and reprograms the tumor microenvironment. To identify the potential substrates that responsible for the phenotypes, we enriched substrates with linear ubiquitin chains in WT and Sharpin KO EO771 cells and determined substrates with profound decreased level in linear ubiquitination upon Sharpin deletion. For this, cells were lysed with NP-40 lysis buffer in the presence of Protease phosphatase inhibitor cocktail, 1mM DTT and 5mM N-Ethylmaleimide. Purified GST-UBAN bound to glutathione Sepharose 4B beads were incubated with cell lysates at 4 °C overnight. Subsequently, the beads were washed four times with GST binding buffer, and the bound proteins were eluted. The eluted proteins were then subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using data-independent acquisition (DIA) mode.