Gold(I) Cationization Promotes Ring Opening in Lysine-Containing Cyclic Peptides

  • David J. Foreman
  • John T. Lawler
  • Mary L. Niedrauer
  • Matthew A. Hostetler
  • Scott A. McLuckeyEmail author
Focus: Honoring Helmut Schwarzʻs Election to the National Academy of Sciences: Research Article


A strategy to sequence lysine-containing cyclic peptides by MSn is presented. Doubly protonated cyclic peptides ions are transformed into gold (I) cationized peptide ions via cation switching ion/ion reaction. Gold(I) cationization facilitates the oxidation of neutral lysine residues in the gas phase, weakening the adjacent amide bond. Upon activation, facile cleavage N-terminal to the oxidized lysine residue provides a site-specific ring opening pathway that converts cyclic peptides into acyclic analogs. The ensuing ion contains a cyclic imine as the new N-terminus and an oxazolone, or structural equivalent, as the new C-terminus. Product ions are formed from subsequent fragmentation events of the linearized peptide ion. Such an approach simplifies MS/MS data interpretation as a series of fragment ions with common N- and C-termini are generated. Results are presented for two cyclic peptides, sunflower trypsin inhibitor and the model cyclic peptide, β-Loop. The power of this strategy lies in the ability to generate the oxidized peptide, which is easily identified via the loss of HAuNH3 from [M + Au]+. While some competitive processes are observed, the site of ring opening can be pinpointed to the lysine residue upon MS4 enabling the unambiguous sequencing of cyclic peptides.


Gold cationization Cyclic peptides Ion/ion reactions Sunflower trypsin inhibitor 



This work was supported by the National Institutes of Health (NIH) under Grant GM R37-45372. Vanessa M. Kung of the Gellman Lab at the University of Wisconsin is acknowledged for synthesis of the β-Loop cyclic peptide and Samuel H. Gellman is acknowledged for providing the peptide to our laboratory.

Supplementary material

13361_2019_2247_MOESM1_ESM.docx (1.4 mb)
ESM 1 (DOCX 1385 kb)


  1. 1.
    Craik, D.J., Daly, N.L., Bond, T., Waine, C.: Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol. 294, 1327–1336 (1999)CrossRefGoogle Scholar
  2. 2.
    Craik, D.J.: Discovery and applications of the plant cyclotides. Toxicon. 56, 1092–1102 (2010)CrossRefGoogle Scholar
  3. 3.
    Colgrave, M.L., Craik, D.J.: Thermal, chemical, and enzymatic stability of the cyclotide Kalata B1: the importance of the cyclic cystine knot. Biochemistry. 43, 5965–5975 (2004)CrossRefGoogle Scholar
  4. 4.
    Craik, D.J., Conibear, A.C.: The chemistry of cyclotides. J. Org. Chem. 76, 4805–4817 (2011)CrossRefGoogle Scholar
  5. 5.
    Pränting, M., Lööv, C., Burman, R., Göransson, U., Andersson, D.I.: The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against gram-negative bacteria. J. Antimicrob. Chemother. 65, 1964–1971 (2010)CrossRefGoogle Scholar
  6. 6.
    Gründemann, C., Koehbach, J., Huber, R., Gruber, C.W.: Do Plant Cyclotides Have Potential As Immunosuppressant Peptides? J. Nat. Prod. 75, 167–174 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Tang, J., Wang, C.K., Pan, X., Yan, H., Zeng, G., Xu, W., He, W., Daly, N.L., Craik, D.J., Tan, N.: Isolation and characterization of cytotoxic cyclotides from Viola tricolor. Peptides. 31, 1434–1440 (2010)CrossRefGoogle Scholar
  8. 8.
    Colgrave, M.L., Huang, Y.-H., Craik, D.J., Kotze, A.C.: Cyclotide Interactions with the Nematode External Surface. Antimicrob. Agents Chemother. 54, 2160 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Colgrave, M.L., Kotze, A.C., Kopp, S., McCarthy, J.S., Coleman, G.T., Craik, D.J.: Anthelmintic activity of cyclotides: In vitro studies with canine and human hookworms. Acta Tropica. 109, 163–166 (2009)CrossRefGoogle Scholar
  10. 10.
    Daly, N.L., Koltay, A., Gustafson, K.R., Boyd, M.R., Casas-Finet, J.R., Craik, D.J.: Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity. J. Mol. Biol. 285, 333–345 (1999)CrossRefGoogle Scholar
  11. 11.
    Hallock, Y.F., Sowder, R.C., Pannell, L.K., Hughes, C.B., Johnson, D.G., Gulakowski, R., Cardellina, J.H., Boyd, M.R.: Cycloviolins A−D, Anti-HIV Macrocyclic Peptides from Leonia cymosa1. J. Org. Chem. 65, 124–128 (2000)CrossRefGoogle Scholar
  12. 12.
    Jennings, C., West, J., Waine, C., Craik, D., Anderson, M.: Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis. Proc. Natl. Acad. Sci. 98, 10614 (2001)CrossRefGoogle Scholar
  13. 13.
    Jennings, C.V., Rosengren, K.J., Daly, N.L., Plan, M., Stevens, J., Scanlon, M.J., Waine, C., Norman, D.G., Anderson, M.A., Craik, D.J.: Isolation, Solution Structure, and Insecticidal Activity of Kalata B2, a Circular Protein with a Twist: Do Möbius Strips Exist in Nature? Biochemistry. 44, 851–860 (2005)CrossRefGoogle Scholar
  14. 14.
    Barbeta, B.L., Marshall, A.T., Gillon, A.D., Craik, D.J., Anderson, M.A.: Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc. Natl. Acad. Sci. 105, 1221 (2008)CrossRefGoogle Scholar
  15. 15.
    Caceres, C.C., Bansal, P.S., Navarro, S., Wilson, D., Don, L., Giacomin, P., Loukas, A., Daly, N.L.: An engineered cyclic peptide alleviates symptoms of inflammation in a murine model of inflammatory bowel disease. J. Biol. Chem. 292, 10288–10294 (2017)CrossRefGoogle Scholar
  16. 16.
    Durek, T., Cromm, P.M., White, A.M., Schroeder, C.I., Kaas, Q., Weidmann, J., Ahmad Fuaad, A., Cheneval, O., Harvey, P.J., Daly, N.L., Zhou, Y., Dellsén, A., Österlund, T., Larsson, N., Knerr, L., Bauer, U., Kessler, H., Cai, M., Hruby, V.J., Plowright, A.T., Craik, D.J.: Development of novel melanocortin receptor agonists based on the cyclic peptide framework of sunflower trypsin Inhibitor-1. J. Med. Chem. 61, 3674–3684 (2018)CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gunasekera, S., Fernandes-Cerqueira, C., Wennmalm, S., Wähämaa, H., Sommarin, Y., Catrina, A.I., Jakobsson, P.-J., Göransson, U.: Stabilized cyclic peptides as scavengers of autoantibodies: neutralization of anticitrullinated protein/peptide antibodies in rheumatoid arthritis. ACS Chem. Biol. 13, 1525–1535 (2018)CrossRefGoogle Scholar
  18. 18.
    de Veer, S.J., Li, C.Y., Swedberg, J.E., Schroeder, C.I., Craik, D.J.: Engineering potent mesotrypsin inhibitors based on the plant-derived cyclic peptide, sunflower trypsin inhibitor-1. Eur. J. Med. Chem. 155, 695–704 (2018)CrossRefGoogle Scholar
  19. 19.
    Swedberg, J.E., Wu, G., Mahatmanto, T., Durek, T., Caradoc-Davies, T.T., Whisstock, J.C., Law, R.H.P., Craik, D.J.: Highly potent and selective plasmin inhibitors based on the sunflower trypsin Inhibitor-1 scaffold attenuate fibrinolysis in plasma. J. Med. Chem. 62, 552–560 (2019)CrossRefGoogle Scholar
  20. 20.
    Fesik, S.W., Bolis, G., Sham, H.L., Olejniczak, E.T.: Structure refinement of a cyclic peptide from two-dimensional NMR data and molecular modeling. Biochemistry. 26, 1851–1859 (1987)CrossRefGoogle Scholar
  21. 21.
    Coles, M., Sowemimo, V., Scanlon, D., Munro, S.L.A., Craik, D.J.: A conformational study by proton NMR of a cyclic pentapeptide antagonist of endothelin. J. Med. Chem. 36, 2658–2665 (1993)CrossRefGoogle Scholar
  22. 22.
    Mazzeo, M., Isernia, C., Rossi, F., Saviano, M., Pedone, C., Paolillo, L., Benedetti, E., Pavone, V.: Conformational behaviour of a cyclolinopeptide a analogue: two-dimensional NMR study of cyclo(Pro1-Pro-Phe-Phe-Ac6c-IIe-ala-Val8). J. Pept. Sci. 1, 330–340 (1995)CrossRefGoogle Scholar
  23. 23.
    Porter, C., Wilce, J.: NMR analysis of G7-18NATE, a nonphosphorylated cyclic peptide inhibitor of the Grb7 adapter protein. Pept. Sci. 88, 174–181 (2007)CrossRefGoogle Scholar
  24. 24.
    Johnson, M., Liu, M., Struble, E., Hettiarachchi, K.: Characterization of cyclic peptides containing disulfide bonds. J. Pharm. Biomed. Anal. 109, 112–120 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Northfield, S., Wielens, J., Headey, S., Williams-Noonan, B., Mulcair, M., Scanlon, M., Parker, M., Thompson, P., Chalmers, D.: Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV‐1 Integrase. ChemMedChem. (2018)Google Scholar
  26. 26.
    Laurencin, M., Simon, M., Fleury, Y., Baudy-Floc'h, M., Bondon, A., Legrand, B.: Selectivity modulation and structure of α/aza-β3 cyclic antimicrobial peptides. Chem. Eur. J. 24, 6191–6201 (2018)CrossRefGoogle Scholar
  27. 27.
    Gross, M.L., McCrery, D., Crow, F., Tomer, K.B., Pope, M.R., Ciuffetti, L.M., Knoche, H.W., Daly, J.M., Dunkle, L.D.: The structure of the toxin from helminthosporium carbonum. Tetrahedron Lett. 23, 5381–5384 (1982)CrossRefGoogle Scholar
  28. 28.
    Tilvi, S., Naik, C.: Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia. J. Mass Spectrom. 42, 70–80 (2007)CrossRefGoogle Scholar
  29. 29.
    Mohimani, H., Yang, Y.L., Liu, W.T., Hsieh, P.W., Dorrestein, P.C., Pevzner, P.A.: Sequencing cyclic peptides by multistage mass spectrometry. Proteomics. 11, 3642–3650 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Attard, T.J., Carter, M.D., Fang, M., Johnson, R.C., Reid, G.E.: Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry. J. Am. Soc. Mass. Spectrom. 1–14 (2018)Google Scholar
  31. 31.
    Parsley, N.C., Kirkpatrick, C.L., Crittenden, C.M., Rad, J.G., Hoskin, D.W., Brodbelt, J.S., Hicks, L.M.: PepSAVI-MS reveals anticancer and antifungal cycloviolacins in Viola odorata. Phytochemistry. 152, 61–70 (2018)CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Paizs, B., Suhai, S.: Fragmentation pathways of protonated peptides. Mass Spectrom. Rev. 24, 508–548 (2005)CrossRefGoogle Scholar
  33. 33.
    Tomer, K.B., Crow, F.W., Gross, M.L., Kopple, K.D.: Fast-atom bombardment combined with tandem mass spectrometry for the determination of cyclic peptides. Anal. Chem. 56, 880–886 (1984)CrossRefGoogle Scholar
  34. 34.
    Hitzeroth, G., Vater, J., Franke, P., Gebhardt, K., Fiedler, H.-P.: Whole cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and in situ structure analysis of streptocidins, a family of tyrocidine-like cyclic peptides. Rapid Commun. Mass Spectrom. 19, 2935–2942 (2005)CrossRefGoogle Scholar
  35. 35.
    Poth, A.G., Colgrave, M.L., Philip, R., Kerenga, B., Daly, N.L., Anderson, M.A., Craik, D.J.: Discovery of cyclotides in the Fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins. ACS Chem. Biol. 6, 345–355 (2011)CrossRefGoogle Scholar
  36. 36.
    Chan, L.Y., He, W., Tan, N., Zeng, G., Craik, D.J., Daly, N.L.: A new family of cystine knot peptides from the seeds of Momordica cochinchinensis. Peptides. 39, 29–35 (2013)CrossRefGoogle Scholar
  37. 37.
    Narayani, M., Chadha, A., Srivastava, S.: Cyclotides from the indian medicinal plant Viola odorata (Banafsha): identification and characterization. J. Nat. Prod. 80, 1972–1980 (2017)CrossRefGoogle Scholar
  38. 38.
    Crittenden, C.M., Parker, W.R., Jenner, Z.B., Bruns, K.A., Akin, L.D., McGee, W.M., Ciccimaro, E., Brodbelt, J.S.: Exploitation of the ornithine effect enhances characterization of stapled and cyclic peptides. J. Am. Soc. Mass Spectrom. 27, 856–863 (2016)CrossRefGoogle Scholar
  39. 39.
    McGee, W.M., McLuckey, S.A.: The ornithine effect in peptide cation dissociation. J. Mass Spectrom. 48, 856–861 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Eller, K., Schwarz, H.: Organometallic in the gas phase. Chem. Rev. 91, 1121–1177 (1991)CrossRefGoogle Scholar
  41. 41.
    Schwarz, H.: Relativistic effects in gas-phase ion chemistry: an experimentalist’s view. Angew. Chem. Int. Ed. 42, 4442–4454 (2003)CrossRefGoogle Scholar
  42. 42.
    Schröder, D., Schwarz, H., Hrušák, J., Pyykkö, P.: Cationic gold (I) complexes of xenon and of ligands containing the donor atoms oxygen, nitrogen, phosphorous, and sulfur. Inorg. Chem. 37, 624–632 (1998)CrossRefGoogle Scholar
  43. 43.
    O’Hair, R.A.J., “Mass spectrometry of organogold compounds” in The Chemistry of Organogold Compounds, Rappoport, Z.; Liebman, J.F.; Marek, I. (eds.) John Wiley & Sons, Ltd: Chichester, UK, Chapter 4, 2014, 57–105Google Scholar
  44. 44.
    Foreman, D.J., Betancourt, S.K., Pilo, A.L., McLuckey, S.A.: Novel peptide ion chemistry associated with gold (I) cationization: preferential cleavage at lysine residues. Int. J. Mass Spectrom. 427, 114–122 (2018)CrossRefGoogle Scholar
  45. 45.
    Espinosa, J.F., Gellman, S.H.: A designed β-hairpin containing a natural hydrophobic cluster. Angew. Chem. Int. Ed. 39, 2330–2333 (2000)CrossRefGoogle Scholar
  46. 46.
    Xia, Y., Wu, J., McLuckey, S.A., Londry, F.A., Hager, J.W.: Mutual storage mode ion/ion reactions in a hybrid linear ion trap. J. Am. Soc. Mass Spectrom. 16, 71–81 (2005)CrossRefGoogle Scholar
  47. 47.
    Liang, X., Xia, Y., McLuckey, S.A.: Alternately pulsed nanoelectrospray ionization/atmospheric pressure chemical ionization for ion/ion reactions in an electrodynamic ion trap. Anal. Chem. 78, 3208–3212 (2006)CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Gunawardena, H.P., O'Hair, R.A.J., McLuckey, S.A.: Selective disulfide Bond cleavage in gold(I) cationized polypeptide ions formed via gas-phase ion/ion cation switching. J. Proteome Res. 5, 2087–2092 (2006)CrossRefGoogle Scholar
  49. 49.
    Mentinova, M., McLuckey, S.A.: Cleavage of multiple disulfide bonds in insulin via gold cationization and collision-induced dissociation. Int. J. Mass Spectrom. 308, 133–136 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Londry, F.A., Hager, J.W.: Mass selective axial ion ejection from a linear quadrupole ion trap. J. Am. Soc. Mass Spectrom. 14, 1130–1147 (2003)CrossRefGoogle Scholar
  51. 51.
    Novák, J., Lemr, K., Schug, K.A., Havlíček, V.: CycloBranch: de novo sequencing of nonribosomal peptides from accurate product ion mass spectra. J. Am. Soc. Mass Spectrom. 26, 1780–1786 (2015)CrossRefGoogle Scholar
  52. 52.
    Ngoka, L.C., Gross, M.L.: A nomenclature system for labeling cyclic peptide fragments. J. Am. Soc. Mass Spectrom. 10, 360–363 (1999)CrossRefGoogle Scholar
  53. 53.
    Yu, W., Vath, J.E., Huberty, M.C., Martin, S.A.: Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry. Anal. Chem. 65, 3015–3023 (1993)CrossRefGoogle Scholar
  54. 54.
    Sullivan, A.G., Brancia, F.L., Tyldesley, R., Bateman, R., Sidhu, K., Hubbard, S.J., Oliver, S.G., Gaskell, S.J.: The exploitation of selective cleavage of singly protonated peptide ions adjacent to aspartic acid residues using a quadrupole orthogonal time-of-flight mass spectrometer equipped with a matrix-assisted laser desorption/ionization source. Int. J. Mass Spectrom. 210-211, 665–676 (2001)CrossRefGoogle Scholar
  55. 55.
    Bleiholder, C., Suhai, S., Harrison, A.G., Paizs, B.: Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). J. Am. Soc. Mass. Spectrom. 22, 1032–1039 (2011)CrossRefGoogle Scholar
  56. 56.
    Schwartz, B.L., Bursey, M.M.: Some proline substituent effects in the tandem mass spectrum of protonated pentaalanine. Biol. Mass Spectrom. 21, 92–96 (1992)CrossRefGoogle Scholar
  57. 57.
    Vaisar, T., Urban, J.: Probing proline effect in CID of protonated peptides. J. Mass Spectrom. 31, 1185–1187 (1996)CrossRefGoogle Scholar
  58. 58.
    Pilo, A.L., Peng, Z., McLuckey, S.A.: The dehydroalanine effect in the fragmentation of ions derived from polypeptides. J. Mass Spectrom. 51, 857–866 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Peng, Z., Bu, J., McLuckey, S.A.: The generation of dehydroalanine residues in protonated polypeptides: ion/ion reactions for introducing selective cleavages. J. Am. Soc. Mass Spectrom. 28, 1765–1774 (2017)CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryPurdue UniversityWest LafayetteUSA

Personalised recommendations