Affinity Proteomic Analysis of the Human Exosome and Its Cofactor Complexes

Methods Mol Biol. 2020;2062:291-325. doi: 10.1007/978-1-4939-9822-7_15.

Abstract

In humans, the RNA exosome consists of an enzymatically inactive nine-subunit core, with ribonucleolytic activity contributed by additional components. Several cofactor complexes also interact with the exosome-these enable the recruitment of, and specify the activity upon, diverse substrates. Affinity capture coupled with mass spectrometry has proven to be an effective means to identify the compositions of RNA exosomes and their cofactor complexes: here, we describe a general experimental strategy for proteomic characterization of macromolecular complexes, applied to the exosome and an affiliated adapter protein, ZC3H18.

Keywords: Affinity capture; Affinity proteomics; Cofactors; Exosome; Mass spectrometry.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line
  • Exosome Multienzyme Ribonuclease Complex / metabolism*
  • Exosomes / metabolism*
  • HEK293 Cells
  • Humans
  • Proteomics / methods
  • RNA / metabolism*
  • RNA-Binding Proteins / metabolism

Substances

  • RNA-Binding Proteins
  • RNA
  • Exosome Multienzyme Ribonuclease Complex