Chemical proteomics of RID substrates

ABSTRACT: To globally profile the fatty-acylation substrate proteins of RID, we utilized the bioorthogonal chemical reporter Alk-16, an alkynyl-functionalized fatty acid analogue, to metabolically label fatty-acylated proteins in living cells. To identify the substrate proteins of RID, we sought to perform quantitative chemical proteomics experiments by combining the Alk-16 metabolic labeling and stable isotope labeling by amino acids in cell culture (SILAC) . In order to eliminate potential false positives due to isotope labeling and to increase identification reliability, we designed a dual SILAC workflow. In the “Forward” SILAC experiment, cells were cultured in standard SILAC heavy and light media before transfection to express RID-WT and RID-CA, respectively. In the “Reverse” SILAC experiment, the isotope labeling was switched and the heavy- and light-labeled cells were transfected with RID-CA and RID-WT, respectively. Cells were labeled with Alk-16 and the lysates were then mixed equally. The combined lysates were treated with NH2OH, followed by click reaction with azido-biotin, streptavidin enrichment, and trypsin digestion. The resulting peptide samples were analyzed by mass spectrometry for protein identification. The heavy-to-light (H/L) SILAC ratios of identified proteins were quantified to evaluate the extent of enrichment.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Tao Peng  

LAB HEAD: Tao Peng

PROVIDER: PXD046500 | Pride | 2024-06-22

REPOSITORIES: Pride