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

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Yeast Functional Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2477))

Abstract

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.

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Acknowledgments

C.T is supported by funding awarded to M.O. from the Canadian Institutes for Health Research (PJT153313).

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Authors and Affiliations

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

    Christian Trahan & Marlene Oeffinger

  2. Département de biochimie et médicine moléculaire, Faculté de médecine, Université de Montréal, Montréal, QC, Canada

    Marlene Oeffinger

  3. Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, QC, Canada

    Marlene Oeffinger

Authors
  1. Christian Trahan
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  2. Marlene Oeffinger
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Corresponding author

Correspondence to Marlene Oeffinger .

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Editors and Affiliations

  1. Institut de Biologie Paris Seine, Sorbonne Université, Paris, France

    Frédéric Devaux

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Trahan, C., Oeffinger, M. (2022). Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae. In: Devaux, F. (eds) Yeast Functional Genomics. Methods in Molecular Biology, vol 2477. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2257-5_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2257-5_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2256-8

  • Online ISBN: 978-1-0716-2257-5

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