Composition and Dynamics of Protein Complexes Measured by Quantitative Mass Spectrometry of Affinity-Purified Samples - Institut Pasteur Access content directly
Book Sections Year : 2022

Composition and Dynamics of Protein Complexes Measured by Quantitative Mass Spectrometry of Affinity-Purified Samples

Abstract

Multiple protein complexes are fundamental parts of living systems. Identification of the components of these complexes and characterization of the molecular mechanisms that allow their formation, function and regulation can be done by affinity purification of proteins and associated factors followed by mass spectrometry of peptides. Speed and specificity for the isolation of complexes from whole cell extracts improved over time together with the reliable identification and quantification of proteins by mass spectrometry. Relative quantification of proteins in such samples can now be done to characterize even relatively non-abundant complexes. We describe here our experience with proteins fused with the Z domain, derived from staphylococcal protein A, and IgG affinity purification for the analysis of protein complexes involved in RNA metabolism in the budding yeast Saccharomyces cerevisiae. We illustrate the use of enrichment calculations for proteins in purified samples as a way to robust identification of protein partners. While the protocols presented here are specific for yeast, their principles can be applied to the study of protein complexes in any other organism.
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pasteur-04042946 , version 1 (23-03-2023)

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Abdelkader Namane, Cosmin Saveanu. Composition and Dynamics of Protein Complexes Measured by Quantitative Mass Spectrometry of Affinity-Purified Samples. Frédéric Devaux. Yeast Functional Genomics, 2477, Springer US, pp.225-236, 2022, Methods in Molecular Biology, 978-1-0716-2257-5 (ebook). ⟨10.1007/978-1-0716-2257-5_13⟩. ⟨pasteur-04042946⟩
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