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). Using
this method, we performed proteomic analysis of the ER of HeLa cells
and newly assigned three proteins, namely, PAICS, TXNL1, and PPIA,
as ER-associated proteins. 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.