Application of Molecular Mass Spectrometry for The Structural Characterization of a DNA-Protein Cross-Links

Jiawei Gong, Morwena Jane Solivio, Edward J Merino, Julio Alberto Landero Figueroa


To date, many different analytical methods have been used to investigate the cross-linking reaction mechanism and to obtain the chemical structure of DNA-Protein Cross-links (DPCs). Direct MS analysis of DPCs is challenging because of the ionization properties of DNA and the protein. However, peptide sequencing and mass spectrometry (MS) as analytical techniques are playing increasingly important roles for the structure determination of DPCs model. In our previous study, a novel approach was presented for purification, detection and quantification of DPCs by newly developed inductively coupled plasma mass spectrometry (ICPMS/MS), which allows sub-ppb detection of S and P, key heteroelements in DNA and proteins.

In this study, we enhanced our previously developed method and it was complemented by the use of molecular MS to allow complete characterization of a DNA-protein cross-link.  First, a small molecule model is utilized to identify the adduct structure that will likely occur in an intact DNA-protein cross-link.  We investigate the thermal stability of DNA-protein cross-links, both in an intact DPC and a small molecule adduct to determine feasibility of digestion/thermal degradationof DNA without the cross-link information being lost.  Thermal degradation was conducted to reduce the cross-linked DNA into a single nucleoside. The remaining protein-nucleoside adduct then was proteolytically digested, generating a peptide-nucleoside adduct. The absence of the phosphate moiety allows for facile structural characterization via electrospray ionization mass spectrometry (ESI-MS).  Additional calculations were done for peptide matching allowing us to determine the cross-link location in the protein, made possible via MS/MS analysis.  Additionally, we show that steric effects play an important role in DPC formation.


DNA-Protein Crosslink; ICP-MS/MS; LC-MS

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Journal of the Mexican Chemical Society (J. Mex. Chem. Soc.) Vol 62, No 2 (2018). Quarterly publication (April-June). Edited and distributed by Sociedad Química de México, A.C. Barranca del Muerto 26, Col. Crédito Constructor, Del. Benito Juárez, C.P. 03940, Mexico City. Phone: +5255 56626837; +5255 56626823 Contact: Editor-in-Chief: Ignacio González-Martínez. Indexed Journal. Certificate of reserved rights for recurrent publications under digital distribution granted by the Instituto Nacional del Derecho de Autor (INDAUTOR): 04-2018-091118040000-203. Certificate of lawful title and content: Under procedure. ISSN-e granted by the Instituto Nacional del Derecho de Autor (INDAUTOR): 2594-0317. ISSN granted by the Instituto Nacional del Derecho de Autor (INDAUTOR): 1870-249X. Postal registration of printed matter deposited by editors or agents granted by SEPOMEX: IM09-0312 Copyright © Sociedad Química de México, A.C. Total or partial reproduction is prohibited without written permission of the right holder. The Figures/schemes quality and the general contents of this publication are full responsibility of the authors. Updated December 12th, 2018 by Adriana Vázquez (editorial assistant, e-mail:, J. Mex. Chem. Soc., Sociedad Química de México, A.C.