Journal
JOURNAL OF PROTEOME RESEARCH
Volume 10, Issue 6, Pages 2785-2796Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr200042u
Keywords
mass spectrometry; DNA-protein cross-links; nitrogen mustards; western blot; DNA repair
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Funding
- National Cancer Institute [R01-CA-1006700]
- University of Minnesota Academic Health Center
- NIH [T32-GM08700, ES010056, ES013125]
- University of Minnesota Masonic Cancer Center
- University of Minnesota Graduate School
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Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI+-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.
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