Abstract

GONG, Jiawei; SOLIVIO, Morwena J.; MERINO, Edward J.  and  LANDERO-FIGUEROA, Julio A.. Application of Molecular Mass Spectrometry for The Structural Characterization of a DNA-Protein Cross-Links. J. Mex. Chem. Soc [online]. 2018, vol.62, n.2, pp.236-246. ISSN 1870-249X.  https://doi.org/10.29356/jmcs.v62i2.405.

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 degradation of 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.

Keywords : DNA-Protein Crosslink; ICP-MS/MS; LC-MS.

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