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Systems structural biology measurements by in vivo cross-linking with mass spectrometry

Nature Protocols volume 14, pages 2318–2343 (2019)Cite this article

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Abstract

This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis.

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Fig. 1: Synthesis and molecular features of PIR cross-linker.
Fig. 2: Experimental overview of in vivo XL-MS.
Fig. 3: Data analysis workflow.
Fig. 4: LC–ESI–MS analysis of the BDP–NHP cross-linker.
Fig. 5: Cross-links mapped onto BSA structure.
Fig. 6: Comparison of structural models for the mitochondrial trifunctional enzyme complex generated by XL-MS guided docking and cryo-EM.

Data availability

Data are freely available online. Raw data from Chavez, J. D. et al. Cell Syst. 6, 136–141 (2018) https://doi.org/10.1016/j.cels.2017.10.017 (used for Fig. 6) are available in the PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) with accession no. PXD007673. Cross-linking data are available in XLinkDB (http://xlinkdb.gs.washington.edu/) under network name ChavezCellSystems2017 BruceLab.

Code availability

All necessary software is freely available for the foreseeable future online at the respective sources: XLinkProphet (https://github.com/brucelab/xlinkprophet), mango (https://github.com/jpm369/mango), Comet (http://comet-ms.sourceforge.net/), ReAdW (https://sourceforge.net/projects/sashimi/files/ReAdW%20%28Xcalibur%20converter%29/), and perl (https://www.perl.org/get.html). In addition, the versions of these software used in this protocol are available in the Supplementary Software file.

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Acknowledgements

We thank members of the Bruce Lab for helpful discussions and suggestions. This work was supported by the following NIH grants: R01GM086688 to J.E.B., J.D.C., and A.K.; R01GM097112 to J.E.B. and J.P.M.; U19AI107775 to J.E.B., X.Z., and A.K.; R01HL110349 to J.E.B. and J.D.C.; and R01HL142628 to J.E.B. and J.D.C.

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  1. These authors contributed equally: Juan D. Chavez, Jared P. Mohr.

Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Juan D. Chavez, Jared P. Mohr, Martin Mathay, Xuefei Zhong, Andrew Keller & James E. Bruce

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  1. Juan D. Chavez
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Contributions

J.D.C., J.P.M., M.M., X.Z., A.K., and J.E.B. contributed to the development of this protocol. J.D.C., J.P.M., and J.E.B. wrote and edited the manuscript.

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Correspondence to James E. Bruce.

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Peer review information: Nature Protocols thanks Andrea Sinz and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Chavez, J. D., Schweppe, D. K., Eng, J. K., & Bruce, J. E. Cell Chem. Biol. 23, 716–726 (2016): https://doi.org/10.1016/j.chembiol.2016.05.012

Wu, X. et al. Nat. Commun. 7, 13414 (2016): https://doi.org/10.1038/ncomms13414

Mohr, J. P., Perumalla, P., Chavez, J. D., Eng, J. K., & Bruce, J. E. Anal. Chem. 90, 6028–6034 (2018): https://doi.org/10.1021/acs.analchem.7b04991

Keller, A., Chavez, J. D., & Bruce, J. E. Bioinformatics 35, 895–897 (2019): https://doi.org/10.1093/bioinformatics/bty720

Keller, A., Chavez, J. D., Eng, J. K., Thornton, Z., & Bruce, J. E. J. Proteome Res. 18, 753–758 (2019): https://doi.org/10.1021/acs.jproteome.8b00703

Key data used in this protocol

Chavez, J. D. et al. Cell Syst. 6, 136–141.e5 (2018): https://doi.org/10.1016/j.cels.2017.10.017

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Chavez, J.D., Mohr, J.P., Mathay, M. et al. Systems structural biology measurements by in vivo cross-linking with mass spectrometry. Nat Protoc 14, 2318–2343 (2019). https://doi.org/10.1038/s41596-019-0181-3

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