Native Mass Spectrometry Analysis of Affinity-Captured Endogenous Yeast RNA Exosome Complexes
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).
Key wordsNative mass spectrometry Endogenous protein assemblies Exosome complex Affinity capture Exactive Plus EMR
This work is supported by National Institutes of Health grants P41 GM103314 and P41 GM109824. We gratefully acknowledge the members of the Chait lab and the Laboratory of Cellular and Structural Biology (headed by Professor Michael Rout) at the Rockefeller University for feedback and insightful discussions. We especially thank Andrew Krutchinsky for the design and implementation of the modifications in the nanoelectrospray setup described here. We also thank Zhanna Hakhverdyan for providing the cryomilled yeast powder.
- 4.Lössl P, van de Waterbeemd M, Heck AJ et al (2016) The diverse and expanding role of mass spectrometry in structural and molecular biology. EMBO J 16:155–166Google Scholar
- 26.LaCava J, Fernandez-Martinez J, Hakhverdyan Z, Rout MP (2016) Optimized affinity capture of yeast protein complexes. Cold Spring Harb Protoc 2016:615–619Google Scholar
- 31.Cristea IM, Chait BT (2011) Conjugation of magnetic beads for immunopurification of protein complexes. Cold Spring Harb Protoc 6:534–538Google Scholar
- 38.Reid DJ, Diesing JM, Miller MA et al (2019) MetaUniDec: high-throughput deconvolution of native mass spectra. J Am Soc Mass Spectrom 30:118–127Google Scholar