Journal of The American Society for Mass Spectrometry

, Volume 30, Issue 9, pp 1643–1653 | Cite as

Identification of Cross-linked Peptides Using Isotopomeric Cross-linkers

  • Jie Luo
  • Jacob Bassett
  • Jeff RanishEmail author
Research Article


Chemical cross-linking combined with mass spectrometry (CL-MS) is a powerful method for characterizing the architecture of protein assemblies and for mapping protein–protein interactions. Despite its proven utility, confident identification of cross-linked peptides remains a formidable challenge, especially when the peptides are derived from complex mixtures. MS cleavable cross-linkers are gaining importance for CL-MS as they permit reliable identification of cross-linked peptides by whole proteome database searching using MS/MS information. Here we introduce a novel class of MS cleavable cross-linkers called isotopomeric cross-linkers (ICLs), which allow for confident and efficient identification of cross-linked peptides by whole proteome database searching. ICLs are simple, symmetrical molecules that asymmetrically incorporate heavy and light stable isotopes into the two arms of the cross-linker. As a result of this property, ICLs automatically generate pairs of isotopomeric cross-linked peptides, which differ only by the positions of the heavy and light isotopes. Upon fragmentation during MS analysis, these isotopomeric cross-linked peptides generate unique isotopic doublet ions that correspond to the individual peptides in the cross-link. The doublet ion information is used to determine the masses of the two cross-linked peptides from the same MS2 spectrum that is also used for peptide spectrum matching (PSM) by sequence database searching. Here we present the rationale for and mechanism of cross-linked peptide identification by ICL-MS. We describe the synthesis of the ICL-1 reagent, the ICL-MS workflow, and the performance characteristics of ICL-MS for identifying cross-linked peptides derived from increasingly complex mixtures by whole proteome database searching.


Isotopomeric cross-linker (ICL) Cross-linking-mass spectrometry (CL-MS) ICL-MS MS cleavable cross-linker Protein-protein interactions Isotopic doublet ion pairs Whole proteome database search 



We thank Mark Gillespie and Isil Hamdemir for comments on the manuscript and the Fred Hutchinson Cancer Research Center proteomics facility (L.A. Jones and P.R. Gafken) for MS analyses. This work was supported by NIH grants R01GM110064 and R01HL133678 to JR.

Supplementary material

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  1. 1.
    Luo, J., Cimermancic, P., Viswanath, S., Ebmeier, C.C., Kim, B., Dehecq, M., Raman, V., Greenberg, C.H., Pellarin, R., Sali, A., Taatjes, D.J., Hahn, S., Ranish, J.: Architecture of the human and yeast general transcription and DNA repair factor TFIIH. Mol. Cell. 59, 794–806 (2015)CrossRefGoogle Scholar
  2. 2.
    Schweppe, D.K., Chavez, J.D., Bruce, J.E.: XLmap: an R package to visualize and score protein structure models based on sites of protein cross-linking. Bioinformatics. 32, 306–308 (2016)CrossRefGoogle Scholar
  3. 3.
    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, 904–906 (2012)CrossRefGoogle Scholar
  4. 4.
    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, 901–903 (2012)CrossRefGoogle Scholar
  5. 5.
    Murakami, K., Elmlund, H., Kalisman, N., Bushnell, D.A., Adams, C.M., Azubel, M., Elmlund, D., Levi-Kalisman, Y., Liu, X., Gibbons, B.J., Levitt, M., Kornberg, R.D.: Architecture of an RNA polymerase II transcription pre-initiation complex. Science. 342, 1238724 (2013)CrossRefGoogle Scholar
  6. 6.
    Rinner, O., Seebacher, J., Walzthoeni, T., Mueller, L.N., Beck, M., Schmidt, A., Mueller, M., Aebersold, R.: Identification of cross-linked peptides from large sequence databases. Nat. Methods. 5, 315–318 (2008)CrossRefGoogle Scholar
  7. 7.
    Tan, D., Li, Q., Zhang, M.J., Liu, C., Ma, C., Zhang, P., Ding, Y.H., Fan, S.B., Tao, L., Yang, B., Li, X., Ma, S., Liu, J., Feng, B., Liu, X., Wang, H.W., He, S.M., Gao, N., Ye, K., Dong, M.Q., Lei, X.: Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states. Elife. 5, (2016)Google Scholar
  8. 8.
    Hoopmann, M.R., Zelter, A., Johnson, R.S., Riffle, M., MacCoss, M.J., Davis, T.N., Moritz, R.L.: Kojak: efficient analysis of chemically cross-linked protein complexes. J. Proteome Res. 14, 2190–2198 (2015)CrossRefGoogle Scholar
  9. 9.
    Combe, C.W., Fischer, L., Rappsilber, J.: xiNET: cross-link network maps with residue resolution. Mol. Cell. Proteomics. 14, 1137–1147 (2015)CrossRefGoogle Scholar
  10. 10.
    Chu, F., Baker, P.R., Burlingame, A.L., Chalkley, R.J.: Finding chimeras: a bioinformatics strategy for identification of cross-linked peptides. Mol. Cell. Proteomics. 9, 25–31 (2010)CrossRefGoogle Scholar
  11. 11.
    Soderblom, E.J., Goshe, M.B.: Collision-induced dissociative chemical cross-linking reagents and methodology: applications to protein structural characterization using tandem mass spectrometry analysis. Anal. Chem. 78, 8059–8068 (2006)CrossRefGoogle Scholar
  12. 12.
    Zhang, H., Tang, X., Munske, G.R., Tolic, N., Anderson, G.A., Bruce, J.E.: Identification of protein-protein interactions and topologies in living cells with chemical cross-linking and mass spectrometry. Mol. Cell. Proteomics. 8, 409–420 (2009)CrossRefGoogle Scholar
  13. 13.
    Lu, Y., Tanasova, M., Borhan, B., Reid, G.E.: Ionic reagent for controlling the gas-phase fragmentation reactions of cross-linked peptides. Anal. Chem. 80, 9279–9287 (2008)CrossRefGoogle Scholar
  14. 14.
    Kao, A., Chiu, C.L., Vellucci, D., Yang, Y., Patel, V.R., Guan, S., Randall, A., Baldi, P., Rychnovsky, S.D., Huang, L.: Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes. Mol. Cell. Proteomics. 10, M110 002212 (2011)CrossRefGoogle Scholar
  15. 15.
    Muller, M.Q., Dreiocker, F., Ihling, C.H., Schafer, M., Sinz, A.: Cleavable cross-linker for protein structure analysis: reliable identification of cross-linking products by tandem MS. Anal. Chem. 82, 6958–6968 (2010)CrossRefGoogle Scholar
  16. 16.
    Tang, X., Munske, G.R., Siems, W.F., Bruce, J.E.: Mass spectrometry identifiable cross-linking strategy for studying protein-protein interactions. Anal. Chem. 77, 311–318 (2005)CrossRefGoogle Scholar
  17. 17.
    Luo, J., Fishburn, J., Hahn, S., Ranish, J.: An integrated chemical cross-linking and mass spectrometry approach to study protein complex architecture and function. Mol. Cell. Proteomics. 11, M111 008318 (2012)CrossRefGoogle Scholar
  18. 18.
    Liu, F., Rijkers, D.T., Post, H., Heck, A.J.: Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat. Methods. 12, 1179–1184 (2015)CrossRefGoogle Scholar
  19. 19.
    Arlt, C., Gotze, M., Ihling, C.H., Hage, C., Schafer, M., Sinz, A.: Integrated workflow for structural proteomics studies based on cross-linking/mass spectrometry with an MS/MS cleavable cross-linker. Anal. Chem. 88, 7930–7937 (2016)CrossRefGoogle Scholar
  20. 20.
    Iacobucci, C., Gotze, M., Ihling, C.H., Piotrowski, C., Arlt, C., Schafer, M., Hage, C., Schmidt, R., Sinz, A.: A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions. Nat. Protoc. 13, 2864–2889 (2018)CrossRefGoogle Scholar
  21. 21.
    Khattab, S.N., El-Faham, A., El-Massry, A.M., Mansour, E.M.E., Abd El-Rahman, M.M.: Coupling of iminodiacetic acid with amine acid derivatives in solution and solid phase. Lett. Pept. Sci. 7, 331–345 (2001)CrossRefGoogle Scholar
  22. 22.
    He, L., Diedrich, J., Chu, Y.Y., Yates 3rd, J.R.: Extracting accurate precursor information for tandem mass spectra by RawConverter. Anal. Chem. 87, 11361–11367 (2015)CrossRefGoogle Scholar
  23. 23.
    Eng, J.K., Jahan, T.A., Hoopmann, M.R.: Comet: an open-source MS/MS sequence database search tool. Proteomics. 13, 22–24 (2013)CrossRefGoogle Scholar
  24. 24.
    Schweppe, D.K., Harding, C., Chavez, J.D., Wu, X., Ramage, E., Singh, P.K., Manoil, C., Bruce, J.E.: Host-microbe protein interactions during bacterial infection. Chem. Biol. 22, 1521–1530 (2015)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Institute for Systems BiologySeattleUSA

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