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Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry

Nature Protocols volume 12, pages 23912410 (2017) | Download Citation

Abstract

Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.

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Acknowledgements

ACKNOWLEDGMENTS

We thank O. Schubert (UCLA) for insightful discussions. We also thank T. Lehmann for building the device to hold the sample tubes in the water bath and G. de Franceschi for contributing to the setup of the original LiP-MS protocol. P.P. is supported by an EU FP7-ERC Starting Grant (FP7-ERC-StG-337965), a 'Foerderungsprofessur' grant from the Swiss National Science Foundation (grant PP00P3_133670), and Promedica Stiftung (grant 2-70669-11). Y.F. is supported by an ETH Research Grant (grant no. 4412-1); I.P. is supported by an EMBO long-term fellowship (EMBO ALTF2014); and A.K. acknowledges SystemsX.ch for funding.

Author information

Author notes

    • Simone Schopper
    •  & Yuehan Feng

    Present addresses: Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark (S.S.); Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA (Y.F.).

Affiliations

  1. Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland.

    • Simone Schopper
    • , Pascal Leuenberger
    • , Yuehan Feng
    • , Ilaria Piazza
    • , Oliver Müller
    • , Paul J Boersema
    •  & Paola Picotti
  2. Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

    • Abdullah Kahraman

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Contributions

S.S., A.K. and P.P. wrote the paper. Y.F. developed and optimized the original version of the protocol. S.S., P.L., O.M. and I.P. contributed to protocol optimization. I.P. and P.L. generated figures. P.J.B. contributed to editing of the manuscript and setup of the MS pipeline. A.K. developed and optimized algorithms and tools for protein structural analyses and prepared the user guide. P.P. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Paola Picotti.

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https://doi.org/10.1038/nprot.2017.100

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