title: HPLC-UV, MALDI-TOF-MS and ESI-MS/MS analysis of the mechlorethamine DNA crosslink at a cytosine-cytosine mismatch pair.
abstract: 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.
  
  
  RESULTS
  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.
authors.affiliation: Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. pornchai.r@chula.ac.th
authors.forename: Pornchai
authors.initials: P
authors.lastname: Rojsitthisak
authors.forename: Nutthapon
authors.initials: N
authors.lastname: Jongaroonngamsang
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Romero
authors.forename: Ian S
authors.initials: IS
authors.lastname: Haworth
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry; D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003596:Cytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008466:Mechlorethamine / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D012910:Snake Venoms / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000477:Alkylating Agents; D012910:Snake Venoms; D008466:Mechlorethamine; D003596:Cytosine; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; D010727:Phosphoric Diester Hydrolases