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Quantitative Analysis of the Endoplasmic Reticulum-Associated Proteins Using ER-Localizable Reactive Molecules
Tomonori Tamura1, Itaru Hamachi2,3
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto, Japan.
Methods in Molecular Biology (Clifton, N.J.)
|November 12, 2022
Summary
This study introduces a novel chemoproteomic method for analyzing endoplasmic reticulum (ER) proteins without cell fractionation. This technique allows for the identification and quantification of ER-associated proteins in live cells.
Area of Science:
- Cell Biology
- Proteomics
- Biochemistry
Background:
- The endoplasmic reticulum (ER) is vital for cellular functions like protein synthesis, lipid metabolism, and calcium storage.
- Analyzing the ER proteome is crucial for understanding these processes, but isolating intact ER has been a significant challenge.
- Existing methods for ER proteome analysis are often hampered by the difficulty of ER isolation or require genetic manipulation.
Purpose of the Study:
- To develop a chemoproteomic approach for global ER proteome analysis in live cells.
- To overcome the limitations of traditional ER isolation techniques.
- To enable tag-mediated enrichment and quantitative analysis of ER-associated proteins.
Main Methods:
- Utilized ER-localizable reactive molecules (ERMs) that spontaneously accumulate within the ER of live cells.
- Employed spatially limited chemical modification of ER-localized proteins with a detection/purification tag via intermolecular reactions.
- Integrated liquid chromatography-tandem mass spectrometry (LC-MS/MS) with SILAC (Stable Isotope Labeling by Amino acids in Cell culture) technology for quantitative analysis.
Main Results:
- Demonstrated a method for ER proteome analysis that does not require ER fractionation or genetic transformation.
- Successfully enriched and quantitatively analyzed ER-associated proteins using the chemoproteomic approach.
- The method leverages the high concentration of ERMs within the ER to facilitate targeted protein modification.
Conclusions:
- The described chemoproteomic approach provides a powerful tool for comprehensive ER proteome profiling in native cellular environments.
- This method facilitates a deeper understanding of ER functions by enabling accurate identification and quantification of associated proteins.
- The technique offers a significant advancement for studying cellular processes involving the endoplasmic reticulum.
Keywords:
Chemical probesChemical proteomicsER-localizable reactive moleculesEndoplasmic reticulumProtein labelingSILACUnfolded protein responseMore Related Videos
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