Quantitative Analysis of the Endoplasmic Reticulum-Associated Proteins Using ER-Localizable Reactive Molecules

Abstract

The endoplasmic reticulum (ER) is an essential organelle responsible for many cellular functions, including protein synthesis and folding, lipid synthesis, membrane trafficking, and storage of Ca2+. Therefore, global profiling of ER-associated proteins should be invaluable for understanding these biological processes. However, the difficulty of isolating the intact ER hampered proteome-wide analysis of ER proteins. This chapter describes a chemoproteomic approach for ER proteome analysis using ER-localizable reactive molecules (ERMs), which need neither ER fractionation nor genetic transformation. ERMs spontaneously accumulate in the ER of live cells, and the resultant high concentration of ERMs facilitates spatially limited chemical modification of ER-localized proteins with a detection/purification tag via simple intermolecular reactions. This enables the tag-mediated enrichment and quantitative analysis of the ER-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with SILAC technology.