Native Mass Spectrometry Analysis of Affinity-Captured Endogenous Yeast RNA Exosome Complexes

Methods Mol Biol. 2020;2062:357-382. doi: 10.1007/978-1-4939-9822-7_17.


Native mass spectrometry (MS) enables direct mass measurement of intact protein assemblies generating relevant subunit composition and stoichiometry information. Combined with cross-linking and structural data, native MS-derived information is crucial for elucidating the architecture of macromolecular assemblies by integrative structural methods. The exosome complex from budding yeast was among the first endogenous protein complexes to be affinity isolated and subsequently characterized by this technique, providing improved understanding of its composition and structure. We present a protocol that couples efficient affinity capture of yeast exosome complexes and sensitive native MS analysis, including rapid affinity isolation of the endogenous exosome complex from cryolysed yeast cells, elution in nondenaturing conditions by protease cleavage, depletion of the protease, buffer exchange, and native MS measurements using an Orbitrap-based instrument (Exactive Plus EMR).

Keywords: Affinity capture; Endogenous protein assemblies; Exactive Plus EMR; Exosome complex; Native mass spectrometry.

Publication types

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

MeSH terms

  • Chromatography, Affinity / methods
  • Exosome Multienzyme Ribonuclease Complex / metabolism*
  • Macromolecular Substances / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Tandem Mass Spectrometry / methods


  • Macromolecular Substances
  • Saccharomyces cerevisiae Proteins
  • Exosome Multienzyme Ribonuclease Complex