Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering , Kyoto University , Katsura, Nishikyo-ku, Kyoto 615-8510 , Japan.
Institute of Transformative Bio-Molecules (ITbM) , Nagoya University , Chikusa, Nagoya 464-8602 , Japan.
J Am Chem Soc. 2018 Dec 12;140(49):17060-17070. doi: 10.1021/jacs.8b08606. Epub 2018 Nov 28.
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.
内质网（ER）是一种细胞器，通过与其他细胞器的相互作用，执行多种重要的细胞功能。尽管内质网具有重要作用，但由于这些蛋白质的不稳定性，用于描绘内质网蛋白组成和动态的化学工具仍然非常有限。在这里，我们开发了内质网定位反应性分子（称为 ERMs），作为内质网靶向化学蛋白质组学的工具。ERMs 可以自发定位于活细胞的内质网中，并选择性地用结合亲和性和成像标签标记与内质网相关的蛋白质，从而实现标签介导的内质网蛋白富集和通过液相色谱串联质谱（LC-MS/MS）鉴定。使用这种方法，我们对 HeLa 细胞的内质网进行了蛋白质组学分析，并将三种新鉴定的蛋白质，即 PAICS、TXNL1 和 PPIA，鉴定为内质网相关蛋白。ERM 探针可与我们小组之前开发的细胞核和线粒体定位反应性分子同时使用，这使得在单个生物样品中可以进行正交细胞器化学蛋白质组学研究。此外，通过将 ER 特异性标记与 SILAC（稳定同位素标记相对和绝对定量技术）相结合，对 tunicamycin 诱导的内质网应激反应中与内质网相关的蛋白质的动态变化进行了定量分析。我们的结果表明，基于 ERM 的化学蛋白质组学为标记和描绘活细胞中与内质网相关的蛋白质提供了一种强大的工具。
