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Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry

Nature Protocols volume 9, pages 1301–1319 (2014)Cite this article

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Abstract

An understanding of the mechanism accompanying functional conformational changes associated with protein activation has important implications for drug design. Here we describe a powerful method, conformational changes and dynamics using stable-isotope labeling and mass spectrometry (CDSiL-MS), which involves chemical labeling by isotope-coded forms of N-ethylmaleimide or succinic anhydride to site-specifically label the side chains of cysteines or lysines, respectively, in native proteins. Subsequent MS analysis allows the quantitative monitoring of reactivity of residues as a function of time, providing a measurement of the labeling kinetics and thereby enabling elucidation of conformational changes of proteins. We demonstrate the utility of this method using a model G protein–coupled receptor, the β2-adrenergic receptor, including experiments that characterize the functional conformational changes associated with activation of distinct signaling pathways induced by different β-adrenoceptor ligands. The procedure requires 5 d, and it can easily be adapted to systems in which soluble and detergent-solubilized membrane protein targets, which undergo function-dependent conformational changes, can be interrogated structurally to allow drug screening.

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Figure 1: Reaction scheme for stable-isotope labeling of cysteines and lysines in proteins.
Figure 2: Overview of CDSiL-MS strategy designed to monitor conformational changes in proteins.
Figure 3: Different functions that can be used to fit the behavior of labeling kinetics.
Figure 4: Quantification of stable-isotope–labeled peptides for CDSiL-MS strategy.
Figure 5: CDSiL-MS–based monitoring of conformational rearrangements at crucial structural elements of the β2AR.
Figure 6: CDSiL-MS–based strategy for measuring functional residue-specific conformational rearrangements in β2AR.
Figure 7: Multiple β2AR residues from different structural elements that can be monitored with the CDSiL-MS strategy.

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Acknowledgements

We thank R.J. Lefkowitz and B.K. Kobilka for invaluable guidance; T.G. Oas for enthusiastic support; A.K. Shukla and S. Ahn for stimulating ideas; and R.T. Strachan and A. Blanc for critically reading the manuscript. We gratefully acknowledge T. Haystead (Duke University) and D. Loiselle for valuable assistance with the mass spectrometry experiments; we are also grateful to C.H. Borchers (University of Victoria, Canada) and I.A. Kaltashov (University of Massachusetts) for helpful discussions; we also thank X. Jiang for excellent technical assistance. This work was supported, in whole or in part, by US National Institutes of Health grant no. HL-075443 Proteomics Core support to K.X.

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Authors and Affiliations

  1. Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA

    Alem W Kahsai, Sudarshan Rajagopal & Kunhong Xiao

  2. Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA

    Alem W Kahsai

  3. Department of Biochemistry and Molecular Biology, Shandong University, School of Medicine, Jinan, China

    Jinpeng Sun

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Contributions

A.W.K. and K.X. designed and conducted the experiments; A.W.K., K.X., S.R. and J.S. analyzed data; A.W.K., S.R. and K.X. wrote the paper; all authors read, edited and discussed the paper.

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Correspondence to Kunhong Xiao.

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Kahsai, A., Rajagopal, S., Sun, J. et al. Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry. Nat Protoc 9, 1301–1319 (2014). https://doi.org/10.1038/nprot.2014.075

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