HPLC-UV, MALDI-TOF-MS and ESI-MS/MS Analysis of the Mechlorethamine DNA Crosslink at a Cytosine-Cytosine Mismatch Pair

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.