Electrospray ionization tandem mass spectrometry methods for the analysis of DNA and DNA/drug complexes

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Title: Electrospray ionization tandem mass spectrometry methods for the analysis of DNA and DNA/drug complexes
Author: Smith, Suncerae I.
Abstract: Many anticancer therapies are based on the interaction of small molecule drugs with nucleic acids , particularly DNA . Electrospray ionization tandem mass spectrometry has established itself as an irreplaceable tool for the characterization of DNA adducts produced by alkylating agents , carcinogens , and antitumor drugs , in addition to the characterization of nucleic acid post -transcriptional modifications . ESI -MS was used to assess the non -covalent binding of a novel series of intercalating anthrapyrazoles to duplexes containing different sequences . Relative binding affinities paralleled the shift in melting point of the DNA duplexes measured from a previous study . Upon collisionally induced dissociation of the duplex /anthrapyrazole complexes , different binding strengths were discerned based on the fragmentation patterns . In addition , the interactions of a new series of sulfur -containing acridine ligands , some that functioned as alklyating mustards , with duplex DNA were also evaluated . Non -covalent and covalent binding of each ligand was determined , and the site of adduction (G > A ) was revealed for the covalent modifications . The distribution of cross -linked products and mono -adducts by psoralen analogs was also monitored by both LC -UV and IRMPD -MS methods . Reactions at 5’ -TA sites were favored over 5’ -AT sites . The sites of interstrand cross -linking were determined by fragmentation of the duplex /psoralen complexes by infrared multiphoton dissociation (IRMPD ) . Ultraviolet photodissociation (UVPD ) at 193 nm caused efficient charge reduction of deprotonated oligodeoxynucleotides via electron detachment . Subsequent CID of the charge -reduced oligodeoxynucleotides formed upon electron detachment , in a net process called electron photodetachment dissociation (EPD ) , resulted in a diverse array of abundant sequence ions which allowed the modification site (s ) of three modified oligodeoxynucleotides to be pinpointed to a more specific location than by conventional CID . Electron transfer dissociation (ETD ) caused efficient charge reduction of multi -protonated oligonucleotides . Subsequent CAD of the charge -reduced oligonucleotides formed upon electron transfer , in a net process termed electron transfer collision activated dissociation (ETcaD ) , resulted in rich backbone fragmentation , with a marked decrease in the abundance of base loss ions and internal fragments . ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages , with conservation of weaker non -covalent bonds . In addition , IRMPD and UVPD were used to activate charge -reduced oligonucleotides formed upon electron transfer . ET -IRMPD afforded tunable characterization of the modified DNA and RNA , allowing for modified bases to be directly analyzed . ET -UVPD promoted higher energy backbone fragmentation pathways and created the most diverse MS /MS spectra . The numerous products generated by the hybrid MS /MS techniques (ETcaD , ET -IRMPD , and ET -UVPD ) resulted in specific and extensive backbone cleavages which allowed for the modification sites of multiple oligonucleotides to be pinpointed .
URI: http : / /hdl .handle .net /2152 /ETD -UT -2010 -08 -1804
Date: 2010-12-14

Citation

Electrospray ionization tandem mass spectrometry methods for the analysis of DNA and DNA/drug complexes. Doctoral dissertation, University of Texas at Austin. Available electronically from http : / /hdl .handle .net /2152 /ETD -UT -2010 -08 -1804 .

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