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.
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Acknowledgments
We thank Dr. Alma Fujisawa and Dr. Yuki Yasueda for their great contribution to the development of ERMs and for helpful advice on the manuscript. This work was funded by a Grant-in-Aid for Scientific Research on Innovative Areas “Integrated Bio-metal Science” (19H05764) to T.T., and Japan Science and Technology Agency (JST) ERATO Grant JPMJER1802 to I.H. This work was also supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” (17H06348).
Conflicts of Interest
The authors declare that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict.
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Tamura, T., Hamachi, I. (2023). Quantitative Analysis of the Endoplasmic Reticulum-Associated Proteins Using ER-Localizable Reactive Molecules. In: Luque-Garcia, J.L. (eds) SILAC. Methods in Molecular Biology, vol 2603. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2863-8_11
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DOI: https://doi.org/10.1007/978-1-0716-2863-8_11
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