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Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry

Abstract

The growing number of applications to determine the stoichiometry, interactions and even subunit architecture of protein complexes from mass spectra suggests that some general guidelines can now be proposed. In this protocol, we describe the necessary steps required to maintain interactions between subunits in the gas phase. We begin with the preparation of suitable solutions for electrospray (ES) and then consider the transmission of complexes through the various stages of the mass spectrometer until their detection. Subsequent steps are also described, including the dissociation of these complexes into multiple subcomplexes for generation of interaction networks. Throughout we highlight the critical experimental factors that determine success. Overall, we develop a generic protocol that can be carried out using commercially available ES mass spectrometers without extensive modification.

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Figure 1: Effect of ammonium acetate concentration and added buffer salts on the spectra of alcohol dehydrogenase (ADH).
Figure 2
Figure 3: Effect of pressure on the monomer:tetramer ratio of alcohol dehydrogenase (ADH).
Figure 4: Effect of concentration on the relative intensity of nonspecific oligomers of the tetramer of pyruvate kinase (a) 30 μM (b) 10 μM (c) 1 μM (d) 200 nM.
Figure 5: Mass spectrometry (MS) and MS/MS spectra of a protein–RNA complex, human U1snRNP assembled in vitro (comprising seven Sm core proteins: D1, D2, D3, B, E, F and G, two further proteins U170k and U1A, and synthetic U1snRNA).

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Acknowledgements

We thank D. Matak-Vinkovic, A. Sandercock, M. Sharon and J. Benesch for critical reading of the manuscript and L. Lane for evaluating 'standard' protein complexes. The human U1snRNP sample was kindly donated by D. Pomeranz-Krummel in the group of K. Nagai, MRC Laboratory of Molecular Biology, Cambridge, UK. We thank the Biotechnology and Biological Sciences Research Council (BBSRC) for funding.

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Hernández, H., Robinson, C. Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat Protoc 2, 715–726 (2007). https://doi.org/10.1038/nprot.2007.73

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