Quantitative ER chemical proteomics with ER-localizable reactive molecules under ER-stress condition

ABSTRACT: The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem-mass spectrometry (LC-MS/MS). The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hela Cell

SUBMITTER: Alma Fujisawa  

LAB HEAD: Itaru Hamachi

PROVIDER: PXD011619 | Pride | 2018-11-22

REPOSITORIES: Pride