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Liquid chromatographic and mass spectrometric determinations of mechlorethamine-DNA crosslinks |
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| รหัสดีโอไอ | |
| Title | Liquid chromatographic and mass spectrometric determinations of mechlorethamine-DNA crosslinks |
| Creator | Nutthapon Jongaroonngamsang |
| Contributor | Pornchai Rojsithisak, Haworth, Ian S. |
| Publisher | Chulalongkorn University |
| Publication Year | 2552 |
| Keyword | Liquid chromatography, DNA, Mass spectrometry |
| Abstract | The mechlorethamine crosslinking reaction with DNA at a cytosine-cytosine (C-C) mismatch pair has been used to probe the structure of DNA containing the d[GCC]•d[GCC] fragment associated with the triplet repeat expansion disease, Fragile X syndrome. The objectives of this work are to determine the structure and amount of the crosslink formed by mechlorethamine with a DNA duplex containing a C-C mismatch pair using HPLC and MALDI-TOF-MS, and to determine the target of mechlorethamine crosslinking in this duplex by enzymatic digestion, HPLC and ESI-MS/MS. HPLC analysis and molecular weight determination by MALDI-TOF-MS of the reaction products formed by mechlorethamine and the 15-mer DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases form a C-C mismatch pair) indicated formation of a mechlorethamine interstrand crosslink. HPLC and ESI-MS/MS analysis showed three products following enzymatic digestion of the crosslink by snake venom phosphodiesterase (SVPD) and calf intestinal phosphatase (CIP): dC-mechlorethamine-Cl (dC-mech-Cl), dC-mechlorethamine-OH (dC-mech-OH) and dC-mechlorethamine-dC (dC-mech-dC). Mass spectrometry also indicated that mechlorethamine forms the interstrand C-C crosslink through N3 of each cytosine base. These results are consistent with results from published gel electrophoretic data. Most importantly, they provide the first evidence of the structural connectivity of the mechlorethamine C-C crosslink, and establish this crosslink as a new chemical species. |
| URL Website | cuir.car.chula.ac.th |
