Photodissociative Cross-Linking of Diazirine-Tagged Peptides with DNA Dinucleotides in the Gas Phase

  • Yang Liu
  • František TurečekEmail author
Focus: Honoring Helmut Schwarz’s Election to the National Academy of Sciences: Research Article


Non-covalent complexes of DNA dinucleotides dAA, dAT, dGG, dGC, and dCG with diazirine-tagged Cys-Ala-Gln-Lys peptides were generated as singly charged ions in the gas phase. Laser photodissociation at 355 nm of the diazirine ring in the gas-phase complexes created carbene intermediates that underwent covalent cross-linking to the dinucleotides. The dinucleotides differed in the cross-linking yields, ranging from 27 to 36% for dAA and dAT up to 90–98% for dGG, dGC, and dCG. Collision-induced dissociation tandem mass spectrometry (CID-MS3) of the cross-linked conjugates revealed that fragmentation occurred chiefly in the dinucleotide moieties, resulting in a loss of a nucleobase and backbone cleavages. The CID-MS3 spectra further revealed that cross-links were primarily formed in the 3′-nucleotides for the dAT, dGC, and dCG combinations. Gas-phase and solution structures of dGG complexes with S-tagged CAQK were investigated by Born-Oppenheimer molecular dynamics (BOMD) and density functional theory calculations. The low free-energy complexes had zwitterionic structures in which the peptide was protonated at the N-terminus and in the Lys residue whereas the carboxyl or dGG phosphate were deprotonated, corresponding to the respective (Cys+, Lys+, COO)+ and (Cys+, Lys+, phosphate)+ protomeric types. Both types preferred structures in which the peptide N-terminal cysteine carrying the S-photo-tag was aligned with the 3′-guanine moiety. BOMD trajectories at 310 K were analyzed for close contacts of the incipient peptide carbene with the positions in dGG that pointed to frequent contacts with the N-1, NH2, and N-7 atoms of 3′-guanine, in agreement with the cross-linking results. Carbene insertion to the guanine N-1-H and NH2 bonds was calculated by density functional and Møller-Plesset perturbational theory to be 350–380 kJ mol−1 exothermic. Based on calculations, we proposed a mechanism for the carbene reaction with guanine starting with an exothermic attack at N-7 to form a dipolar intermediate that can close an aziridine ring in another exothermic reaction, forming a stable covalent cross link.

Graphical Abstract


DNA-peptide complexes Diazirine tags Gas-phase cross-linking Sequence analysis Born-Oppenheimer molecular dynamics Close contact analysis 



This research has received funding from the National Science Foundation Division of Chemistry (Grants CHE-1661815 and CHE-1624430). F.T. thanks the Klaus and Mary Ann Saegebarth Endowment for support. Y. L. thanks Dr. Emilie Viglino for generous help with peptide synthesis.

Supplementary material

13361_2019_2189_MOESM1_ESM.pdf (2.4 mb)
ESM 1 (PDF 2459 kb)


  1. 1.
    von Hippel, P.H.: From "simple" DNA-protein interactions to the macromolecular machines of gene expression. Annu. Rev. Biophys. Biomol. Struct. 36, 79–105 (2007)CrossRefGoogle Scholar
  2. 2.
    Raindlova, V., Pohl, R., Hocek, M.: Synthesis of aldehyde-linked nucleotides and DNA and their bioconjugations with lysine and peptides through reductive amination. Chem. Eur. J. 18, 4080–4087 (2012)CrossRefGoogle Scholar
  3. 3.
    Carrette, L.L.G., Morii, T., Madder, A.: Toxicity inspired cross-linking for probing DNA−peptide interactions. Bioconjug. Chem. 24, 2008–2014 (2013)CrossRefGoogle Scholar
  4. 4.
    Flett, F.J., Walton, J.G.A., Mackay, C.G., Interthal, H.: Click chemistry generated model DNA–peptide heteroconjugates as tools for mass spectrometry. Anal. Chem. 87, 9595–9599 (2015)CrossRefGoogle Scholar
  5. 5.
    Wickramaratne, S., Boldry, E.J., Buehler, C., Wang, Y.-C., Distefano, M.D., Tretyakova, N.D.: Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine J. Biol. Chem. 290, 775–787 (2015)CrossRefGoogle Scholar
  6. 6.
    Ming, X., Groehler 4th, A., Michaelson-Richie, E.D., Villalta, P.W., Campbell, C., Tretyakova, N.Y.: Mass spectrometry based proteomics study of cisplatin-Induced DNA–protein cross-linking in human fibrosarcoma (HT1080) cells. Chem. Res. Toxicol. 30, 980–995 (2017)CrossRefGoogle Scholar
  7. 7.
    Buxton, K.E., Kennedy-Darling, J., Shortreed, M.R., Zaidan, N.Z., Olivier, M., Scalf, M., Sridharan, R., Smith, L.M.: Elucidating protein–DNA interactions in human alphoid chromatin via hybridization capture and mass spectrometry. J. Proteome Res. 16, 3433–3442 (2017)CrossRefGoogle Scholar
  8. 8.
    Weir Lipton, M.S., Fuciarelli, A.L., Springer, D.L., Hofstadler, S.A., Edmonds, C.G.: Analysis of radiation induced nucleobase–peptide cross-links by electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 11, 1673–1676 (1997)CrossRefGoogle Scholar
  9. 9.
    Golden, M.C., Resing, K.A., Collins, B.D., Willis, M.C., Koch, T.H.: Mass spectral characterization of a protein-nucleic acid photocross-link. Protein Sci. 8, 2806–2812 (1999)CrossRefGoogle Scholar
  10. 10.
    Wong, D.L., Reich, N.O.: Identification of tyrosine 204 as the photo-cross-linking site in the DNA−EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry. Biochemistry. 39, 15410–15417 (2000)CrossRefGoogle Scholar
  11. 11.
    Rieger, R.A., McTigue, M.M., Kycia, J.H., Gerchman, S.E., Grollman, A.P., Iden, C.R.: Characterization of a cross-linked DNA-endonuclease VIII repair complex by electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 11, 505–515 (2000)CrossRefGoogle Scholar
  12. 12.
    Steen, H., Petersen, J., Mann, M., Jensen, O.: Mass spectrometric analysis of a UV-cross-linked protein–DNA complex: Tryptophans 54 and 88 of E. coli SSB cross-link to DNA. Protein Sci. 10, 1989–2001 (2001)CrossRefGoogle Scholar
  13. 13.
    Wagenknecht, H.-A., Rajski, S.R., Pascaly, M., Stemp, E.D.A., Barton, J.K.: Direct observation of radical intermediates in protein-dependent DNA charge transport. J. Am. Chem. Soc. 123(19), 4400–4407 (2001)CrossRefGoogle Scholar
  14. 14.
    Doneanu, C.E., Gafken, P.R., Bennett, S.E., Barofsky, D.F.: Mass spectrometry of UV-cross-linked protein-nucleic acid complexes: identification of amino acid residues in the single-stranded DNA-binding domain of human replication protein A. Anal. Chem. 76, 5667–5676 (2004)CrossRefGoogle Scholar
  15. 15.
    Pourshahian, S., Limbach, P.A.: Application of fractional mass for the identification of peptide-oligonucleotide cross-links by mass spectrometry. J. Mass Spectrom. 43, 1081–1088 (2008)CrossRefGoogle Scholar
  16. 16.
    Rosenfeld, K.K., Ziv, T., Goldin, S., Glaser, F., Manor, H.: Mapping of DNA binding sites in the tetrahymena telomerase holoenzyme proteins by UV cross-linking and mass spectrometry. J. Mol. Biol. 410, 77–92 (2011)CrossRefGoogle Scholar
  17. 17.
    Steen, H., Jensen, O.N.: Analysis of protein–nucleic acid interactions by photochemical cross-linking and mass spectrometry. Mass Spectrom. Rev. 21, 163–182 (2002)CrossRefGoogle Scholar
  18. 18.
    Tacheny, A., Dieu, M., Arnould, T., Renard, P.: Mass spectrometry-based identification of proteins interacting with nucleic acids. J. Proteome. 94, 89–109 (2013)CrossRefGoogle Scholar
  19. 19.
    Laughlin, S., Wilson, W.D.: May the best molecule win: competition ESI mass spectrometry. Int. J. Mol. Sci. 16, 24506–24531 (2015)CrossRefGoogle Scholar
  20. 20.
    Groehler, A., Degner, A., Tretyakova, N.Y.: Mass spectrometry-based tools to characterize DNA-protein cross-linking by bis-electrophiles. Basic Clin. Pharmacol. Toxicol. 121(S3), 63–77 (2017)CrossRefGoogle Scholar
  21. 21.
    Lin, S., Cotter, R.J., Woods, A.S.: Detection of non-covalent interaction of single and double stranded DNA with peptides by MALDI-TOF. Proteins Struct. Funct. Genet. Suppl. 2, 12–21 (1998)CrossRefGoogle Scholar
  22. 22.
    Veenstra, T.D.: Electrospray ionization mass spectrometry: a promising new technique in the study of protein/DNA noncovalent complexes. Biochem. Biophys. Res. Commun. 257, 1–5 (1999)CrossRefGoogle Scholar
  23. 23.
    Beck, J.L., Colgrave, M.L., Ralph, S.F., Sheil, M.M.: Electrospray ionization mass spectrometry of oligonucleotide complexes with drugs, metals, and proteins. Mass Spectrom. Rev. 20, 61–87 (2001)CrossRefGoogle Scholar
  24. 24.
    Alves, S., Woods, A., Tabet, J.C.: Charge state effect on the zwitterion influence on stability of non-covalent interaction of single-stranded DNA with peptides. J. Mass Spectrom. 42, 1613–1622 (2007)CrossRefGoogle Scholar
  25. 25.
    Alves, S., Woods, A., Devolve, A., Tabet, J.C.: Influence of salt bridge interactions on the gas-phase stability of DNA/peptide complexes. Int. J. Mass Spectrom. 278, 122–128 (2008)CrossRefGoogle Scholar
  26. 26.
    Brahim, B., Tabet, J.C., Alves, S.: Positive and negative ion mode comparison for the determination of DNA/peptide noncovalent binding sites through the formation of "three-body" noncovalent fragment ions. Eur. J. Mass Spectrom. 24, 168–177 (2018)CrossRefGoogle Scholar
  27. 27.
    Liu, Y., Ramey, Z., Tureček, F.: Non-covalent interactions of a neuroprotective peptide revealed by photodissociative cross-linking in the gas phase. Chem. Eur. J. 24, 9259–9263 (2018)CrossRefGoogle Scholar
  28. 28.
    Mann, A.P., Scodeller, P., Hussain, S., Joo, J., Kwon, E., Braun, G.B., Molder, T., She, Z.-G., Kotamraju, V.R., Ranscht, B., Krajewski, S., Teesalu, T., Bhatia, S., Sailor, M.J., Ruoslahti, E.: A peptide for targeted, systemic delivery of imaging and therapeutic compounds into acute brain injuries. Nat. Commun. 7, 11980 (2016)CrossRefGoogle Scholar
  29. 29.
    Huang, R.C.C., Bonner, J.: Histone, a suppressor of chromosomal RNA synthesis. Proc. Natl. Acad. Sci. U. S. A. 48, 1216–1222 (1962)CrossRefGoogle Scholar
  30. 30.
    Bannister, A.J., Kouzarides, T.: Regulation of chromatin by histone modifications. Cell Res. 21, 381–395 (2011)CrossRefGoogle Scholar
  31. 31.
    Strahl, B.D., Allis, C.D.: The language of covalent histone modifications. Nature. 403(6765), 41–45 (2000)CrossRefGoogle Scholar
  32. 32.
    Jenuwein, T., Allis, C.D.: Translating the histone code. Science. 293(5532), 1074–1080 (2001)CrossRefGoogle Scholar
  33. 33.
    Pepin, R., Shaffer, C.J., Tureček, F.: Position-tunable diazirine tags for peptide-peptide ion cross-linking in the gas-phase. J. Mass Spectrom. 52, 557–560 (2017)CrossRefGoogle Scholar
  34. 34.
    Das, J.: Aliphatic diazirines as photoaffinity probes for proteins: recent developments. Chem. Rev. 111, 4405–4417 (2011)CrossRefGoogle Scholar
  35. 35.
    Nguyen, H.T.H., Andrikopoulos, P.C., Rulíšek, L., Shaffer, C.J., Tureček, F.: Photodissociative cross linking of noncovalent peptide-peptide ion complexes in the gas phase. J. Am. Soc. Mass Spectrom. 29, 1706–1720 (2018)CrossRefGoogle Scholar
  36. 36.
    Shaffer, C.J., Andrikopoulos, P.C., Řezáč, J., Rulíšek, L., Tureček, F.: Efficient covalent bond formation in gas-phase peptide-peptide ion complexes with the photoleucine stapler. J. Am. Soc. Mass Spectrom. 27, 633–645 (2016)CrossRefGoogle Scholar
  37. 37.
    Berendsen, H.J., Postma, J.V., van Gunsteren, W.F., DiNola, A.R.H.J., Haak, J.R.: Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690 (1984)CrossRefGoogle Scholar
  38. 38.
    Stewart, J.J.P.: Optimization of parameters for semi-empirical methods V: modification of NDDO approximations and applications to 70 elements. J. Mol. Model. 13, 1173–1213 (2007)CrossRefGoogle Scholar
  39. 39.
    Řezáč, J., Fanfrlík, J., Salahub, D., Hobza, P.: Semiempirical quantum chemical PM6 method augmented by dispersion and H-bonding correction terms reliably describes various types of noncovalent complexes. J. Chem. Theor. Comput. 5, 1749–1760 (2009)CrossRefGoogle Scholar
  40. 40.
    Stewart, J.J.P.: MOPAC 16. Stewart Computational Chemistry, Colorado Springs, CO (2016)Google Scholar
  41. 41.
    Řezáč, J.: Cuby: an integrative framework for computational chemistry. J. Comput. Chem. 37, 1230–1237 (2016)CrossRefGoogle Scholar
  42. 42.
    Řezáč, J.: Cuby—ruby framework for computational chemistry, version 4,
  43. 43.
    Becke, A.D.: Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A. 38, 3098–3100 (1988)CrossRefGoogle Scholar
  44. 44.
    Chai, J.D., Head-Gordon, M.: Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections. Phys. Chem. Chem. Phys. 10, 6615–6620 (2008)CrossRefGoogle Scholar
  45. 45.
    Tomasi, J., Mennucci, B., Cammi, R.: Quantum mechanical continuum solvation models. Chem. Rev. 105, 2999–3093 (2005)CrossRefGoogle Scholar
  46. 46.
    Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Petersson, G.A., Nakatsuji, H., Caricato, M., Marenich, A.V., Bloino, J., Janesko, B.G., Gomperts, R., Mennucci, B., Hratchian, H.P., Ortiz, J.V., Izmaylov, A.F., Sonnenberg, J.L., Williams-Young, D., Ding, F., Lipparini, F., Egidi, F., Goings, J., Peng, B., Petrone, A., Henderson, T., Ranasinghe, D., Zakrzewski, V.G., Gao, J., Rega, N., Zheng, G., Liang, W., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Throssell, K., Montgomery Jr., J.A., Peralta, J.E., Ogliaro, F., Bearpark, M.J., Heyd, J.J., Brothers, E.N., Kudin, K.N., Staroverov, V.N., Keith, T.A., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A.P., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Millam, J.M., Klene, M., Adamo, C., Cammi, R., Ochterski, J.W., Martin, R.L., Morokuma, K., Farkas, O., Foresman, J.B., Fox, D.J.: Gaussian 16, revision A01. Gaussian, Inc., Wallingford CT (2016)Google Scholar
  47. 47.
    Marek, A., Tureček, F.: Collision-induced dissociation of diazirine-labeled peptide ions. Evidence for Brønsted-acid assisted elimination of nitrogen. J. Am. Soc. Mass Spectrom. 25, 778–789 (2014)CrossRefGoogle Scholar
  48. 48.
    Seburg, R.A., McMahon, R.J.: Photochemistry of matrix-isolated diazoethane and methyldiazirine: ethylidene trapping? J. Am. Chem. Soc. 114, 7183–7189 (1992)CrossRefGoogle Scholar
  49. 49.
    Jackson, J.E., Soundararajan, N., White, W., Liu, M.T.H., Bonneau, R., Platz, M.S.: Measurement of the absolute rate of 1,2-hydrogen migration in benzylchlorocarbene. J. Am. Chem. Soc. 111, 6874–6875 (1989)CrossRefGoogle Scholar
  50. 50.
    Pezacki, J.P., Couture, P., Dunn, J.A., Warkentin, J.: Rate constant for 1,2-hydrogen migration in cyclohexylidene and in substituted cyclohexylidenes. J. Org. Chem. 64, 4456–4464 (1999)CrossRefGoogle Scholar
  51. 51.
    Stevens, I.D.R., Liu, M.T.H., Soundararajan, N., Paike, N.: The barrier for 1,2-hydrogen shift in dialkyl carbenes. Tetrahedron Lett. 30, 481–484 (1989)CrossRefGoogle Scholar
  52. 52.
    Murray, K.K.: DNA sequencing by mass spectrometry. J. Mass Spectrom. 31, 1203–1215 (1996)CrossRefGoogle Scholar
  53. 53.
    McLuckey, S.A., Van Berkel, G.J., Glish, G.L.: Tandem mass spectrometry of small, multiply charged oligonucleotides. J. Am. Soc. Mass Spectrom. 3, 60–70 (1992)CrossRefGoogle Scholar
  54. 54.
    Roepstorff, P., Fohlman, J.: Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Mass. Spectrom. 11, 601 (1984)CrossRefGoogle Scholar
  55. 55.
    Biemann, K.: Nomenclature for peptide fragment ions (positive ions). Methods Enzymol. 193, 886–887 (1990)CrossRefGoogle Scholar
  56. 56.
    Wu, J., McLuckey, S.A.: Gas-phase fragmentation of oligonucleotide ions. Int. J. Mass Spectrom. 237, 197–241 (2004)CrossRefGoogle Scholar
  57. 57.
    Schurch, S.: Characterization of nucleic acids by tandem mass spectrometry—the second decade (2004–2013): from DNA to RNA and modified sequences. Mass Spectrom. Rev. 35, 483–523 (2016)CrossRefGoogle Scholar
  58. 58.
    Liu, M.T.H.: Chemistry of Diazirines, Vol. II, CRC Press, Boca Raton, FL (1987)Google Scholar
  59. 59.
    Tippmann, E.M., Platz, M.S., Svir, I.B., Klymenko, O.V.: Evidence for specific solvation of two halocarbene amides. J. Am. Chem. Soc. 126, 5750–5762 (2004)CrossRefGoogle Scholar
  60. 60.
    Mohan, U., Burai, R., McNaughton, B.R.: Reactivity between acetone and single-stranded DNA containing a 5′-capped 2′-fluoro-N7-methyl guanine. Tetrahedron Lett. 55, 3358–3360 (2014)CrossRefGoogle Scholar
  61. 61.
    Nowak, I., Robins, M.J.: Synthesis of 3′-deoxynucleosides with 2-oxabicyclo[3.1.0]hexane sugar moieties: addition of difluorocarbene to a 3′,4′-unsaturated uridine derivative and 1,2-dihydrofurans derived from D- and L-xylose. J. Org. Chem. 72, 3319–3325 (2007)CrossRefGoogle Scholar
  62. 62.
    Jerbi, J., Springborg, M.: Reactivity descriptors for DNA bases and the methylation of cytosine. Int. J. Quantum Chem. 118, e25538 (2018)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of WashingtonSeattleUSA

Personalised recommendations