Identifying Protein Interactomes of Target RNAs Using HyPR-MS.

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.