HPLC-UV, MALDI-TOF-MS and ESI-MS/MS analysis of the mechlorethamine DNA crosslink at a cytosine-cytosine mismatch pair

• Published in: PloS one 2011-06, Vol.6 (6), p.e20745
• Main Authors: Rojsitthisak, Pornchai, Jongaroonngamsang, Nutthapon, Romero, Rebecca M, Haworth, Ian S
• Format: Article
• Language: English
• Subjects: Alkylating Agents - chemistry; Alkylation; Ammonia; Ammonium; Animals; Base Pair Mismatch; Base Sequence; Biology; Carbon-carbon composites; Cattle; Chemistry; Chromatography, High Pressure Liquid - methods; Connectivity; Crosslinking; Cytosine; Cytosine - chemistry; Deoxyribonucleic acid; Desorption; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA adducts; DNA damage; Enzymes; Gel electrophoresis; Genetic research; High performance liquid chromatography; Intestine; Ions; Lasers; Liquid chromatography; Mass Spectrometry; Mechlorethamine; Mechlorethamine - chemistry; Molecular weight; Mustard; Mutation; Pharmaceutical sciences; Phosphatases; Phosphodiesterase; Phosphoric Diester Hydrolases - metabolism; Phosphoric Monoester Hydrolases - metabolism; Polymer crosslinking; Pyrimidines; Quaternary; Scientific imaging; Snake Venoms - enzymology; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrophotometry, Ultraviolet - methods; Tandem Mass Spectrometry; Venom
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0020745

Mechlorethamine [ClCH(2)CH(2)N(CH(3))CH(2)CH(2)Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown. HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair) indicated formation of an interstrand crosslink at m/z 9222.088 [M-2H+Na](+). Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH(2)CH(2)N(CH(3))CH(2)CH(2)] at m/z 269.2 [M](2+) (expected m/z 269.6, exact mass 539.27) and its hydrolytic product dC-mech-OH at m/z 329.6 [M](+) (expected m/z 329.2). Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M](+), which are both due to loss of the 4-amino group of cytosine (as ammonia), in addition to dC and dC+HN(CH(3))CH = CH(2), respectively. The presence of m/z 269.2 [M](2+) and loss of ammonia exclude crosslink formation at cytosine N(4) or O(2) and indicate crosslinking through cytosine N(3) with formation of two quaternary ammonium ions. Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N(3) position of cytosine.