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Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
Published on: September 28, 2017
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Identifying Protein Interactomes of Target RNAs Using HyPR-MS
Katherine B Henke1, Rachel M Miller1, Rachel A Knoener1
1Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA.
Methods in Molecular Biology (Clifton, N.J.)
|October 25, 2021
Summary
HyPR-MS (hybridization purification of RNA-protein complexes followed by mass spectrometry) is a versatile technology for discovering RNA-protein interactions. This guide details the HyPR-MS procedure for analyzing RNA-protein interactomes in various systems.
Area of Science:
- Molecular Biology
- Biochemistry
- Proteomics
Background:
- RNA-protein interactions are crucial for cellular function and homeostasis.
- Dysregulation of these interactions is implicated in numerous diseases.
- Identifying specific RNA-protein interactions is vital for understanding cellular mechanisms and disease pathology.
Purpose of the Study:
- To provide comprehensive guidance on applying HyPR-MS (hybridization purification of RNA-protein complexes followed by mass spectrometry) for RNA-protein interactome discovery.
- To detail the fundamental HyPR-MS procedure for researchers.
- To enable multiplexed discovery of specific RNA-protein interactions in various biological systems.
Main Methods:
- Experimental design including controls, capture oligonucleotides, and qPCR assays.
- Cellular formaldehyde cross-linking, lysis, and RNA solubilization.
- Isolation and purification of target RNA, followed by RT-qPCR analysis.
- Protein preparation and subsequent mass spectrometric analysis.
- Mass spectrometric data analysis for interactome identification.
Main Results:
- The chapter details the step-by-step application of HyPR-MS.
- It covers experimental design, sample preparation, and data analysis.
- Successful implementation allows for multiplexed discovery of RNA-protein interactomes.
Conclusions:
- HyPR-MS is a powerful and efficient technology for mapping RNA-protein interactions.
- The provided guidance facilitates the successful application of HyPR-MS in diverse research settings.
- This method aids in understanding the molecular basis of cellular function and disease.
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