Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae.


Journal

Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969

Informations de publication

Date de publication:
2022
Historique:
entrez: 6 5 2022
pubmed: 7 5 2022
medline: 11 5 2022
Statut: ppublish

Résumé

Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to get a comprehensive picture of cellular behavior and to understand biological systems. In the past two decades, affinity purification coupled to mass spectrometry has become a powerful tool to comprehensively study interaction networks and their assemblies. To overcome initial limitations of the approach, in particular, the effect of protein and RNA degradation, loss of transient interactors, and poor overall yield of intact complexes from cell lysates, various modifications to affinity purification protocols have been devised over the years. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol as described here has been optimized for the yeast S. cerevisiae.

Identifiants

pubmed: 35524119
doi: 10.1007/978-1-0716-2257-5_12
doi:

Substances chimiques

Macromolecular Substances 0
Proteins 0

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

195-223

Subventions

Organisme : CIHR
ID : PJT153313
Pays : Canada

Informations de copyright

© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

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Auteurs

Christian Trahan (C)

RNP Biochemistry Laboratory, Center for Genetic and Neurological Diseases, Institut de recherches cliniques de Montréal, Montréal, QC, Canada.

Marlene Oeffinger (M)

RNP Biochemistry Laboratory, Center for Genetic and Neurological Diseases, Institut de recherches cliniques de Montréal, Montréal, QC, Canada. marlene.oeffinger@ircm.qc.ca.
Département de biochimie et médicine moléculaire, Faculté de médecine, Université de Montréal, Montréal, QC, Canada. marlene.oeffinger@ircm.qc.ca.
Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, QC, Canada. marlene.oeffinger@ircm.qc.ca.

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