Identifying Protein Interactomes of Target RNAs Using HyPR-MS.

Autor:	Henke KB; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA., Miller RM; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA., Knoener RA; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA., Scalf M; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA., Spiniello M; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA.; Immuno-Hematology and Transfusion Medicine, Cardarelli Hospital, Naples, Italy., Smith LM; Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA. smith@chem.wisc.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2404, pp. 219-244.
DOI:	10.1007/978-1-0716-1851-6_12
Abstrakt:	RNA-protein interactions are integral to maintaining proper cellular function and homeostasis, and the disruption of key RNA-protein interactions is central to many disease states. HyPR-MS (hybridization purification of RNA-protein complexes followed by mass spectrometry) is a highly versatile and efficient technology which enables multiplexed discovery of specific RNA-protein interactomes. This chapter provides extensive guidance for successful application of HyPR-MS to the system and target RNA(s) of interest, as well as a detailed description of the fundamental HyPR-MS procedure, including: (1) experimental design of controls, capture oligonucleotides, and qPCR assays; (2) formaldehyde cross-linking of cell culture; (3) cell lysis and RNA solubilization; (4) isolation of target RNA(s); (5) RNA purification and RT-qPCR analysis; (6) protein preparation and mass spectrometric analysis; and (7) mass spectrometric data analysis. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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