Skip to main content
SpringerLink
Log in

Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine

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

Abstract

We describe the use of the 13C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein–peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a 13C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein–protein interaction studies.

ᅟ

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
Figure 4
Figure 5
Figure 6
Figure 7
Scheme 1

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. 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, 5802–5806 (2000)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. 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, 1348–1352 (2012)

    Article  CAS  Google Scholar 

  5. Kalisman, N., Adams, C.M., Levitt, M.: Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling. Proc. Natl. Acad. Sci. U. S. A. 109, 2884–2889 (2012)

    Article  CAS  Google Scholar 

  6. Leitner, A., Joachimiak, L.A., Bracher, A., Mönkemeyer, L., Walzthoeni, T., Chen, B., Pechmann, S., Holmes, S., Cong, Y., Ma, B., Ludtke, S., Chiu, W., Hartl, F.U., Aebersold, R., Frydman, J.: The molecular architecture of the eukaryotic chaperonin TRiC/CCT. Structure 20, 814–825 (2012)

    Article  CAS  Google Scholar 

  7. 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, 717–726 (2010)

    Article  CAS  Google Scholar 

  8. Krauth, F., Ihling, C.H., Rüttinger, H.H., Sinz, A.: Heterobifunctional isotope-labeled amine-reactive photo-cross-linker for structural investigation of proteins by matrix-assisted laser desorption/ionization tandem time-of-flight and electrospray ionization LTQ-Orbitrap mass spectrometry. Rapid Commun. Mass Spectrom. 23, 2811–2818 (2009)

    Article  CAS  Google Scholar 

  9. Gomes, A.F., Gozzo, F.C.: Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS. J. Mass Spectrom. 45, 892–899 (2010)

    Article  CAS  Google Scholar 

  10. Suchanek, M., Radzikowska, A., Thiele, C.: Photo-leucine and photo-methionine allow identification of protein–protein interactions in living cells. Nat. Methods 2, 261–267 (2005)

    Article  CAS  Google Scholar 

  11. Xie, J., Schultz, P.G.: A chemical toolkit for proteins - an expanded genetic code. Nat. Rev. Mol. Cell Biol. 7, 775–782 (2006)

    Article  CAS  Google Scholar 

  12. Dimova, K., Kalkhof, S., Pottratz, I., Ihling, C., Rodriguez-Castaneda, F., Liepold, T., Griesinger, C., Brose, N., Sinz, A., Jahn, O.: Structural insights into the calmodulin-munc13 interaction obtained by cross-linking and mass spectrometry. Biochemistry 48, 5908–5921 (2009)

    Article  CAS  Google Scholar 

  13. Pettelkau, J., Schröder, T., Ihling, C.H., Olausson, B.E.S., Kölbel, K., Lange, C., Sinz, A.: Structural insights into retinal guanylylcyclase-GCAP-2 interaction determined by cross-linking and mass spectrometry. Biochemistry 51, 4932–4949 (2012)

    Article  CAS  Google Scholar 

  14. Jahn, O., Eckart, K., Brauns, O., Tezval, H., Spiess, J.: The binding protein of corticotropin-releasing factor: ligand-binding site and subunit structure. Proc. Natl. Acad. Sci. U. S. A. 99, 12055–12060 (2002)

    Article  CAS  Google Scholar 

  15. O'Neil, K.T., Ericksonviitanen, S., DeGrado, W.F.: Photolabeling of calmodulin with basic, amphiphilic alpha-helical peptides containing para-benzoylphenylalanine. J. Biol. Chem. 264, 14571–14578 (1989)

    Google Scholar 

  16. Schwarz, R., Tänzler, D., Ihling, C.H., Müller, M.Q., Kolbel, K., Sinz, A.: Monitoring conformational changes in peroxisome proliferator-activated receptor alpha by a genetically encoded photoamino acid, cross-linking, and mass spectrometry. J. Med. Chem. 56, 4252–4263 (2013)

    Article  CAS  Google Scholar 

  17. Schulz, C., Lytovchenko, O., Melin, J., Chacinska, A., Guiard, B., Neumann, P., Ficner, R., Jahn, O., Schmidt, B., Rehling, P.: Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex. J. Cell Biol. 195, 643–656 (2011)

    Article  CAS  Google Scholar 

  18. Dorman, G., Prestwich, G.D.: Benzophenone photophores in biochemistry. Biochemistry 33, 5661–5673 (1994)

    Article  CAS  Google Scholar 

  19. Kage, R., Leeman, S.E., Krause, J.E., Costello, C.E., Boyd, N.D.: Identification of methionine as the site of covalent attachment of a p-benzoyl-phenylalanine-containing analogue of Substance P on the Substance P (NK-1) receptor. J. Biol. Chem. 271, 25797–25800 (1996)

    Article  CAS  Google Scholar 

  20. Sachon, E., Bolbach, G., Lavielle, S., Karoyan, P., Sagan, S.: Met174 side chain is the site of photoinsertion of a Substance P competitive peptide antagonist photoreactive in position 8. FEBS Lett. 544, 45–49 (2003)

    Article  CAS  Google Scholar 

  21. Wittelsberger, A., Thomas, B.E., Mierke, D.F., Rosenblatt, M.: Methionine acts as a “magnet” in photoaffinity crosslinking experiments. FEBS Lett. 580, 1872–1876 (2006)

    Article  CAS  Google Scholar 

  22. Götze, M., Pettelkau, J., Schaks, S., Bosse, K., Ihling, C., Krauth, F., Fritzsche, R., Kühn, U., Sinz, A.: StavroX–A software for analyzing crosslinked products in protein interaction studies. J. Am. Soc. Mass Spectrom. 23, 76–87 (2012)

    Article  Google Scholar 

  23. Peri, S., Steen, H., Pandey, A.: GPMAW—a software tool for analyzing proteins and peptides. Trends Biochem. Sci. 26, 687–689 (2001)

    Article  CAS  Google Scholar 

  24. de Koning, L.J., Kasper, P.T., Back, J.W., Nessen, M.A., Vanrobaeys, F., Van Beeumen, J., Gherardi, E., de Koster, C.G., de Jong, L.: Computer-assisted mass spectrometric analysis of naturally occurring and artificially introduced cross-links in proteins and protein complexes. FEBS J. 273, 281–291 (2006)

    Article  Google Scholar 

  25. Rinner, O., Seebacher, J., Walzthoeni, T., Mueller, L., Beck, M., Schmidt, A., Mueller, M., Aebersold, R.: Identification of cross-linked peptides from large sequence databases. Nat. Methods 5, 315–318 (2008)

    Article  CAS  Google Scholar 

  26. Du, X., Chowdhury, S.M., Manes, N.P., Wu, S., Mayer, M.U., Adkins, J.N., Anderson, G.A., Smith, R.D.: Xlink-Identifier: an automated data analysis platform for confident identifications of chemically cross-linked peptides using tandem mass spectrometry. J. Proteome Res. 10, 923–931 (2011)

    Article  CAS  Google Scholar 

  27. Müller, M.Q., Dreiocker, F., Ihling, C.H., Schäfer, 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)

    Article  Google Scholar 

  28. 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)

    Article  CAS  Google Scholar 

  29. Müller, D.R., Schindler, P., Towbin, H., Wirth, U., Voshol, H., Hoving, S., Steinmetz, M.O.: Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927–1934 (2001)

    Article  Google Scholar 

  30. Seebacher, J., Mallick, P., Zhang, N., Eddes, J.S., Aebersold, R., Gelb, M.H.: Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing. J. Proteome Res. 5, 2270–2282 (2006)

    Article  CAS  Google Scholar 

  31. Wilkins, B.J., Daggett, K.A., Cropp, T.A.: Peptide mass fingerprinting using isotopically encoded photo-crosslinking amino acids. Mol. Biosyst. 4, 934–936 (2008)

    Article  CAS  Google Scholar 

  32. 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 

  33. Blumenthal, D.K., Takio, K., Edelman, A.M., Charbonneau, H., Titani, K., Walsh, K.A., Krebs, E.G.: Identification of the calmodulin-binding domain of skeletal-muscle myosin light chain kinase. Proc. Natl. Acad. Sci. U. S. A. 82, 3187–3191 (1985)

    Article  CAS  Google Scholar 

  34. Ikura, M., Clore, G.M., Gronenborn, A.M., Zhu, G., Klee, C.B., Bax, A.: Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–638 (1992)

    Article  CAS  Google Scholar 

  35. Krueger, J.K., Bishop, N.A., Blumenthal, D.K., Zhi, G., Beckingham, K., Stull, J.T., Trewhella, J.: Calmodulin binding to myosin light chain kinase begins at substoichiometric Ca2+ concentrations: a small-angle scattering study of binding and conformational transitions. Biochemistry 37, 17810–17817 (1998)

    Article  CAS  Google Scholar 

  36. Kalkhof, S., Ihling, C., Mechtler, K., Sinz, A.: Chemical cross-linking and high-performance Fourier transform ion cyclotron resonance mass spectrometry for protein interaction analysis: application to a calmodulin/target peptide complex. Anal. Chem. 77, 495–503 (2004)

    Article  Google Scholar 

  37. Duda, T., Fik-Rymarkiewicz, E., Venkataraman, V., Krishnan, R., Koch, K.W., Sharma, R.K.: The calcium-sensor guanylate cyclase activating protein type 2 specific site in rod outer segment membrane guanylate cyclase type 1. Biochemistry 44, 7336–7345 (2005)

    Article  CAS  Google Scholar 

  38. Olshevskaya, E.V., Hughes, R.E., Hurley, J.B., Dizhoor, A.M.: Calcium binding, but not a calcium-myristoyl switch, controls the ability of guanylyl cyclase-activating protein GCAP-2 to regulate photoreceptor guanylyl cyclase. J. Biol. Chem. 272, 14327–14333 (1997)

    Article  CAS  Google Scholar 

  39. Dizhoor, A.M., Hurley, J.B.: Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins. Methods 19, 521–531 (1999)

    Article  CAS  Google Scholar 

  40. Koch, K.W., Dell'Orco, D.: A calcium-relay mechanism in vertebrate phototransduction. ACS Chem. Neurosci. 4, 90–917 (2013)

    Article  Google Scholar 

  41. Ames, J.B., Dizhoor, A.M., Ikura, M., Palczewski, K., Stryer, L.: Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases. J. Biol. Chem. 274, 19329–19337 (1999)

    Article  CAS  Google Scholar 

  42. Schröder, T., Lilie, H., Lange, C.: The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca2+. Protein Sci. 20, 1155–1165 (2011)

    Article  Google Scholar 

  43. Schaks, S., Maucher, D., Ihling, C.H., Sinz, A.: Investigation of a calmodulin/peptide complex by chemical cross-linking and high-resolution mass spectrometry. In: König, S. (Ed.) Biomacromolecular Mass Spectrometry, ISSN: 1936-444X. Nova Science Publishers (Hauppauge, NY, USA) (2012)

  44. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V., Mann, M.: In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006)

    Article  CAS  Google Scholar 

  45. Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W.: Improved staining of proteins in polyacrylamide gels including isoelectric-focusing gels with clear background at nanogram sensitivity using coomassie brilliant blue G-250 and R-250. Electrophoresis 9, 255–262 (1988)

    Article  CAS  Google Scholar 

  46. Available at: http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct. Accessed: 21 Mar 2014

  47. Rodriguez-Castaneda, F., Maestre-Martinez, M., Coudevylle, N., Dimova, K., Junge, H., Lipstein, N., Lee, D., Becker, S., Brose, N., Jahn, O., Carlomagno, T., Griesinger, C.: Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short-term synaptic plasticity. EMBO J. 29, 680–691 (2010)

    Article  CAS  Google Scholar 

  48. 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, 834–850 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by the Deutsche Forschungsgemeinschaft (DFG, project Si 867/13-1) and the region of Sachsen-Anhalt. Dr. Thomas Schröder is acknowledged for providing GCAP-2

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, 06120, Halle (Saale), Germany

    Jens Pettelkau, Christian H. Ihling, Petra Frohberg & Andrea Sinz

  2. Proteomics Group, Max-Planck Institute of Experimental Medicine, 37075, Göttingen, Germany

    Lars van Werven & Olaf Jahn

Authors
  1. Jens Pettelkau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian H. Ihling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Petra Frohberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lars van Werven
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Olaf Jahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andrea Sinz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Olaf Jahn or Andrea Sinz.

Electronic supplementary material

Supporting information is available for this manuscript on synthesis of Fmoc-13C6-Bpa, chromatograms of peptide separation, protein and peptide sequences, SDS-PAGE, MS and MS/MS data of cross-linked products, and details of identified cross-linked products.

ESM 1

(DOC 26503 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pettelkau, J., Ihling, C.H., Frohberg, P. et al. Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine. J. Am. Soc. Mass Spectrom. 25, 1628–1641 (2014). https://doi.org/10.1007/s13361-014-0944-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13361-014-0944-6

Keywords

Search

Navigation