Simultaneous chemical proteomics of Nuclear, mitochondira and ER

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.

内质网（endoplasmic reticulum, ER）是一类通过与其他细胞器相互作用，执行多种关键细胞功能的细胞器。尽管其发挥着重要的生理作用，但由于内质网蛋白质本身具有不稳定性，用于分析内质网蛋白质组成与动态变化的化学工具仍十分有限。本研究开发了可定位于内质网的反应性分子（简称ERMs），作为聚焦内质网的化学蛋白质组学工具。ERMs可自发定位于活细胞的内质网中，并通过结合亲和标签与成像标签，选择性标记内质网相关蛋白质，从而可借助标签介导的方法，通过液相色谱-串联质谱（LC-MS/MS）对内质网蛋白质进行富集与鉴定。本研究开发的ERM探针可与本团队此前开发的定位于细胞核与线粒体的反应性分子联合使用，实现单个生物样品内的正交细胞器化学蛋白质组学分析。此外，本研究将ERMs的特异性标记与基于细胞培养氨基酸稳定同位素标记（stable isotope labeling by amino acids in cell culture, SILAC）的定量质谱技术相结合，对衣霉素诱导的内质网应激下内质网相关蛋白质的动态变化进行了定量分析。研究结果表明，基于ERM的化学蛋白质组学可为活细胞内内质网相关蛋白质的标记与分析提供强有力的工具。