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Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins

Nature Protocols volume 13, pages 431–458 (2018)Cite this article

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Abstract

Cross-linking coupled with mass spectrometry (XL-MS) has emerged as a powerful strategy for the identification of protein–protein interactions, characterization of interaction regions, and obtainment of structural information on proteins and protein complexes. In XL-MS, proteins or complexes are covalently stabilized with cross-linkers and digested, followed by identification of the cross-linked peptides by tandem mass spectrometry (MS/MS). This provides spatial constraints that enable modeling of protein (complex) structures and regions of interaction. However, most XL-MS approaches are not capable of differentiating intramolecular from intermolecular links in multimeric complexes, and therefore they cannot be used to study homodimer interfaces. We have recently developed an approach that overcomes this limitation by stable isotope–labeling of one of the two monomers, thereby creating a homodimer with one 'light' and one 'heavy' monomer. Here, we describe a step-by-step protocol for stable isotope–labeling, followed by controlled denaturation and refolding in the presence of the wild-type protein. The resulting light–heavy dimers are cross-linked, digested, and analyzed by mass spectrometry. We show how to quantitatively analyze the corresponding data with SIM-XL, an XL-MS software with a module tailored toward the MS/MS data from homodimers. In addition, we provide a video tutorial of the data analysis with this protocol. This protocol can be performed in ∼14 d, and requires basic biochemical and mass spectrometry skills.

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Figure 1: Overview of the homodimer analysis workflow.
Figure 2: SIM-XL's main screen.
Figure 3: Mass spectrum and reconstruction of a sample containing wild-type and 15N-labeled apoA-I.
Figure 4: Cross-linking and purification of apoA-I multimers.
Figure 5: Cross-linker (XL) library.
Figure 6: Inserting enzymes into the enzyme library.
Figure 7: Adding post-translational modifications to the library.
Figure 8: Setting up homodimer parameters.
Figure 9: Adding a new isotopically labeled residue.
Figure 10: Saving SIM-XL parameters.
Figure 11: 2D-Map of protein-interaction results.
Figure 12: Using the Circular Viewer mode to visualize cross-links from three or more proteins.
Figure 13: The Spectrum Viewer allows seamless evaluation of identifications.
Figure 14: Customizing the homodimer parameters on XL Spectrum Viewer.
Figure 15: Relative XL quantitation.
Figure 16: Enabling the Quantitation module.
Figure 17: Calculating the XICs.
Figure 18: Spatial constraints from identified residues.
Figure 19: Protein folding obtained from the identified spatial constraints.
Figure 20: Exporting the PyMOL script.
Figure 21: A poor identification.

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Acknowledgements

V.C.B. acknowledges a FAPERJ BBP grant, as well as support from CNPq and CAPES. P.C.C. acknowledges the Fundação Araucária Universal/Jovem Pesquisador Grant and support from PAPES VII and Universal CNPq. D.B.L. and J.C.-R. acknowledge the Centre National de la Recherche Scientifique (CNRS) for financial support. F.C.G. and M.F. acknowledge support from FAPESP (grants 2014/17264-3 and 2012/10862-7) and CNPq. The mass spectrometry methods were established in the UC Proteomics Laboratory on the Sciex 5600 + TripleTOF system, funded in part through an NIH-shared instrumentation grant (S10 RR027015-01; K.D. Greis, principal investigator). This work was also supported by an American Heart Association postdoctoral fellowship grant (16POST27710016 to J.T.M.) and by funding from the National Institutes of Health Heart, Lung and Blood Institute (R01 GM098458 and P01 HL128203 to W.S.D.).

Author information

Author notes

  1. Diogo B Lima and John T Melchior: These authors contributed equally to this work.

Authors and Affiliations

  1. Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France

    Diogo B Lima & Julia Chamot-Rooke

  2. Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, USA

    John T Melchior, Jamie Morris & W Sean Davidson

  3. Systems Engineering and Computer Science Program,

    Valmir C Barbosa

  4. , Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

    Valmir C Barbosa

  5. Dalton Mass Spectrometry Laboratory, University of Campinas, São Paulo, Brazil

    Mariana Fioramonte & Fabio C Gozzo

  6. Group for Computational Mass Spectrometry & Proteomics, Carlos Chagas Institute, Fiocruz Paraná, Brazil

    Tatiana A C B Souza, Juliana S G Fischer & Paulo C Carvalho

  7. Laboratory of Toxinology, Fiocruz, Rio de Janeiro, Brazil.,

    Paulo C Carvalho

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Contributions

D.B.L., P.C.C., F.C.G., and V.C.B. have participated in the development of the SIM-XL project since its beginning in 2015. D.B.L., J.T.M., W.S.D., P.C.C., and F.C.G. participated in developing the SIM-XL module tailored for homodimers. J.T.M., W.S.D., J.C.-R., J.S.G.F., J.M., and T.A.C.B.S. participated in describing the experimental methodology. D.B.L., P.C.C., and V.C.B. participated in writing the data-analysis methodology. M.F. participated in developing the SIM-XL software and in describing the computational methodology. All authors participated in writing the Introduction and Anticipated Results sections. D.B.L., J.C.-R., and P.C.C. created the supplementary video. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Paulo C Carvalho or W Sean Davidson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Wild-type contamination of a 15N-labeled peptide.

MS1 spectra for the same double charged peptide in wild-type and 15N-labeled apolipoprotein A-I1-184. Inefficient incorporation of 15N into the isotopically-labeled protein can lead to a small “phantom” peak, marked in the figure with an asterisk. The intensity of the phantom peak is proportional to the number of 14N atoms present in the peptide fragment. Based on the LC/MS/MS sensitivity and software, this can result in a false assignment of the parent ion.

Supplementary Figure 2 Vector map of apolipoproteins apoA-I and apoA-IV in pET30a(+).

The vector map has been adapted with permission from Tubb et. al. (ref. 29), © 2009 The American Society for Biochemistry and Molecular Biology. The arrow represents the transcription start site. Restriction endonucleases are shown. The “G” at the beginning of the native apolipoprotein sequence was engineered in to enhance cleavage of the tag by the TEV protease (Kapust et. al. 2002 PMID 12074568).

Supplementary information

Supplementary Figures

Supplementary Figures 1 and 2. (PDF 251 kb)

Tutorial

Video tutorial for data analysis using SIM-XL. (MP4 22951 kb)

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Lima, D., Melchior, J., Morris, J. et al. Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins. Nat Protoc 13, 431–458 (2018). https://doi.org/10.1038/nprot.2017.113

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