Skip to main content
SpringerLink
Log in

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

  • Research Article
  • Published:
Journal of The American Society for Mass Spectrometry

Abstract

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of cross-linked and non-cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3

Similar content being viewed by others

References

  1. Rappsilber, J.: The beginning of a beautiful friendship: Cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes. J. Struct. Biol. 173(3), 530–540 (2011)

    Article  CAS  Google Scholar 

  2. Sinz, A.: Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. Mass Spectrom. Rev. 25(4), 663–682 (2006)

    Article  CAS  Google Scholar 

  3. Leitner, A., Walzthoeni, T., Kahraman, A., Herzog, F., Rinner, O., Beck, M., Aebersold, R.: Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics. Mol. Cell. Proteomics. 9(8), 1634–1649 (2010)

    Article  CAS  Google Scholar 

  4. Schilling, B., Row, R.H., Gibson, B.W., Guo, X., Young, M.M.: MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides. J. Am. Soc. Mass Spectrom. 14(8), 834–850 (2003)

    Article  CAS  Google Scholar 

  5. Young, M.M., Tang, N., Hempel, J.C., Oshiro, C.M., Taylor, E.W., Kuntz, I.D., Gibson, B.W., Dollinger, G.: High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 97(11), 5802–5806 (2000)

    Article  CAS  Google Scholar 

  6. Walzthoeni, T., Claassen, M., Leitner, A., Herzog, F., Bohn, S., Forster, F., Beck, M., Aebersold, R.: False discovery rate estimation for cross-linked peptides identified by mass spectrometry. Nat. Methods 9(9), 901–903 (2012)

    Article  CAS  Google Scholar 

  7. Yang, B., Wu, Y.-J., Zhu, M., Fan, S.-B., Lin, J., Zhang, K., Li, S., Chi, H., Li, Y.-X., Chen, H.-F., Luo, S.-K., Ding, Y.-H., Wang, L.-H., Hao, Z., Xiu, L.-Y., Chen, S., Ye, K., He, S.-M., Dong, M.-Q.: Identification of cross-linked peptides from complex samples. Nat. Methods 9(9), 904–906 (2012)

    Article  CAS  Google Scholar 

  8. Herzog, F., Kahraman, A., Boehringer, D., Mak, R., Bracher, A., Walzthoeni, T., Leitner, A., Beck, M., Hartl, F.-U., Ban, N., Malmström, L., Aebersold, R.: Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry. Science 337(6100), 1348–1352 (2012)

    Article  CAS  Google Scholar 

  9. Taverner, T., Hall, N.E., O'Hair, R.A.J., Simpson, R.J.: Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry. J. Biol. Chem. 277(48), 46487–46492 (2002)

    Article  CAS  Google Scholar 

  10. Santos, L.F.A., Iglesias, A.H., Pilau, E.J., Gomes, A.F., Gozzo, F.C.: Traveling-Wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking. J. Am. Soc. Mass Spectrom. 21(12), 2062–2069 (2010)

    Article  CAS  Google Scholar 

  11. Smith, D.P., Anderson, J., Plante, J., Ashcroft, A.E., Radford, S.E., Wilson, A.J., Parker, M.J.: Trifluoromethyldiazirine: an effective photo-induced cross-linking probe for exploring amyloid formation. Chem. Commun. (44), 5728–5730 (2008)

    Google Scholar 

  12. Kindy, J.M., Taraszka, J.A., Regnier, F.E., Clemmer, D.E.: Quantifying peptides in isotopically labeled protease digests by ion mobility/time-of-flight mass spectrometry. Anal. Chem. 74(5), 950–958 (2002)

    Google Scholar 

  13. Soderberg, C.A.G., Lambert, W., Kjellstrom, S., Wiegandt, A., Wulff, R.P., Mansson, C., Rutsdottir, G., Emanuelsson, C.: Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation. PLoS One 7(6), e38927 (2012)

  14. Acton, T.B., Xiao, R., Anderson, S., Aramini, J., Buchwald, W.A., Ciccosanti, C., Conover, K., Everett, J., Hamilton, K., Huang, Y.J., Janjua, H., Kornhaber, G., Lau, J., Lee, D.Y., Liu, G.H., Maglaqui, M., Ma, L.C., Mao, L., Patel, D., Rossi, P., Sahdev, S., Shastry, R., Swapna, G.V.T., Tang, Y.F., Tong, S.C., Wang, D.Y., Wang, H., Zhao, L., Montelione, G.T.: Preparation of protein samples for NMR structure, function, and small-molecule screening studies. Methods Enzymol. 493, 21–60 (2011)

    Google Scholar 

  15. Cort, J.R., Selan, U., Schulte, A., Grimm, F., Kennedy, M.A., Dahl, C.: Allochromatium vinosum DsrC: solution-state NMR structure, redox properties, and interaction with DsrEFH, a protein essential for purple sulfur bacterial sulfur oxidation. J. Mol. Biol. 382(3), 692–707 (2008)

    Article  CAS  Google Scholar 

  16. Livesay, E.A., Tang, K., Taylor, B.K., Buschbach, M.A., Hopkins, D.F., LaMarche, B.L., Zhao, R., Shen, Y., Orton, D.J., Moore, R.J., Kelly, R.T., Udseth, H.R., Smith, R.D.: Fully Automated four-column capillary LC-MS system for maximizing throughput in proteomic analyses. Anal. Chem. 80(1), 294–302 (2007)

    Article  Google Scholar 

  17. Ibrahim, Y., Belov, M.E., Tolmachev, A.V., Prior, D.C., Smith, R.D.: Ion funnel trap interface for orthogonal time-of-flight mass spectrometry. Anal. Chem. 79(20), 7845–7852 (2007)

    Article  CAS  Google Scholar 

  18. Ibrahim, Y.M., Prior, D.C., Baker, E.S., Smith, R.D., Belov, M.E.: Characterization of an ion mobility-multiplexed collision-induced dissociation-tandem time-of-flight mass spectrometry approach. Int. J. Mass spectrom. 293(1/3), 34–44 (2010)

    CAS  Google Scholar 

  19. Jaitly, N., Mayampurath, A., Littlefield, K., Adkins, J. N., Anderson, G. A., Smith, R. D.: Decon2LS: an open-source software package for automated processing and visualization of high resolution mass spectrometry data. BMC Bioinforma. 10(87), (2009)

  20. Crowell K.L., Shah, A.R., Slysz, G., Lamarche, B.L., Meng, D., Baker, E.S., Monroe, M.E., Anderson, G.A., Smith, R.D.: LC-IMS-MS feature finder: detecting multidimensional features in IMS-TOF MS data. Proceedings of the American Society for Mass Spectrometry Conference, Denver, CO (June 2011).

  21. Mädler, S., Bich, C., Touboul, D., Zenobi, R.: Chemical cross-linking with NHS esters: a systematic study on amino acid reactivities. J. Mass Spectrom. 44(5), 694–706 (2009)

    Article  Google Scholar 

  22. Kalkhof, S., Sinz, A.: Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters. Anal. Bioanal. Chem. 392, 305–312 (2008)

    Article  CAS  Google Scholar 

  23. Kahraman, A., Malmström, L., Aebersold, R.: Xwalk: computing and visualizing distances in cross-linking experiments. Bioinformatics 27(15), 2163–2164 (2011)

    Article  CAS  Google Scholar 

  24. Leitner, A., Reischl, R., Walzthoeni, T., Herzog, F., Bohn, S., Förster, F., Aebersold, R.: Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography. Mol. Cell. Proteom. 11(3) (2012)

  25. Chen, Z.A., Jawhari, A., Fischer, L., Buchen, C., Tahir, S., Kamenski, T., Rasmussen, M., Lariviere, L., Bukowski-Wills, J.-C., Nilges, M., Cramer, P., Rappsilber, J.: Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 29(4), 717–726 (2010)

    Article  CAS  Google Scholar 

  26. Fabris, D., Yu, E.T.: Elucidating the higher-order structure of biopolymers by structural probing and mass spectrometry: MS3D. J. Mass Spectrom. 45(8), 841–860 (2010)

    Article  CAS  Google Scholar 

  27. Li, H., Wells, S.A., Jimenez-Roldan, J.E., Römer, R.A., Zhao, Y., Sadler, P.J., O'Connor, P.B.: Protein flexibility is key to cisplatin crosslinking in calmodulin. Protein Sci. 21(9), 1269–1279 (2012)

    Article  CAS  Google Scholar 

  28. Valentine, S.J., Counterman, A.E., Hoaglund, C.S., Reilly, J.P., Clemmer, D.E.: Gas-phase separations of protease digests. J. Am. Soc. Mass Spectrom. 9(11), 1213–1216 (1998)

    Article  CAS  Google Scholar 

  29. Woods, A., Koomen, J., Ruotolo, B., Gillig, K., Russel, D., Fuhrer, K., Gonin, M., Egan, T., Schultz, J.: A study of peptide–peptide interactions using MALDI ion mobility o-TOF and ESI mass spectrometry. J. Am. Soc. Mass Spectrom. 13(2), 166–169 (2002)

    Article  CAS  Google Scholar 

  30. Baker, E.S., Livesay, E.A., Orton, D.J., Moore, R.J., Danielson, W.F., Prior, D.C., Ibrahim, Y.M., LaMarche, B.L., Mayampurath, A.M., Schepmoes, A.A., Hopkins, D.F., Tang, K., Smith, R.D., Belov, M.E.: An LC-IMS-MS platform providing increased dynamic range for high-throughput proteomic studies. J. Proteome Res. 9(2), 997–1006 (2009)

    Article  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Gaetano Montelione and the NESG for providing the plasmid construct for expressing SrfN/STM0082 and the mixture of unlabeled and 13C,15N-labeled SO_2176, Dr. Sam Payne and Dr. Gordon Slysz for helpful discussions, and Dr. Abdullah Kahraman for assistance with the Xwalk program. This research was supported by the National Institute of General Medical Sciences (NIGMS grant GM094623). SrfN protein was produced in a project funded by the National Institute of Allergy and Infectious Diseases (IAA Y1-AI-8401-01). The work used instrumentation and capabilities developed with support from the NIGMS grant 8 P41 GM103493-10, and the US Department of Energy/Office of Biological and Environmental Research (DOE/BER). This work was performed in EMSL, a DOE/BER National Scientific User Facility located at PNNL in Richland, Washington.

Author information

Author notes

  1. Thomas Taverner

    Present address: Mango Solutions, 2 Methuen Park, Chippenham, SN14 0 GB, UK

Authors and Affiliations

  1. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA

    Eric D. Merkley, Erin S. Baker, Kevin L. Crowell, Daniel J. Orton, Charles Ansong, Yehia M. Ibrahim, Meagan C. Burnet, John R. Cort, Gordon A. Anderson, Richard D. Smith & Joshua N. Adkins

Authors
  1. Eric D. Merkley
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erin S. Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kevin L. Crowell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel J. Orton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas Taverner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles Ansong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yehia M. Ibrahim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Meagan C. Burnet
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. John R. Cort
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gordon A. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Richard D. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Joshua N. Adkins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joshua N. Adkins.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 2423 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Merkley, E.D., Baker, E.S., Crowell, K.L. et al. Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking. J. Am. Soc. Mass Spectrom. 24, 444–449 (2013). https://doi.org/10.1007/s13361-012-0565-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13361-012-0565-x

Key words

Search

Navigation