Rapid characterization of cross-links, mono-adducts, and non-covalent binding of psoralens to deoxyoligonucleotides by LC-UV/ESI-MS and IRMPD mass spectrometry

Abstract

SummaryUpon UV photoactivation, psoralen analogs form covalent mono-adducts and cross-links with DNA at thymine residues. Electrospray ionization mass spectrometric analysis allowed rapid and efficient determination of the reaction percentages of each psoralen analog with DNA duplexes containing different binding sites after exposure to UV irradiation. The distribution of cross-linked products and mono-adducts was monitored by both LC-UV and IRMPD-MS methods with the highest ratio of cross-linked products to mono-adducts obtained for 8-methoxypsoralen (8-MOP), psoralen (P), and 5-methoxypsoralen (5-MOP). Reactions at 5'-TA sites were favored over 5'-AT sites, and duplexes containing two and three binding sites showed extensive binding by the psoralens. 4'-aminomethyl-4,5',8-trimethylpsoralen (AMP) bound non-selectively via non-covalent interactions and was the only psoralen analog to show significant binding in the absence of UV irradiation. 8-MOP binding displayed the greatest sequence selectivity among the psoralen analogs. The sites of interstrand cross-linking were determined by fragmentation of the duplex/psoralen complexes by infrared multiphoton dissociation (IRMPD), which produced cross-linked product ions containing an intact single strand, the psoralen analog, and either a w n or a n -B portion of the complementary strand. IRMPD of DNA/AMP complexes after UV irradiation also produced high abundances of the intact single strands with the AMP ligand attached, products indicative of a significant population of mono-adducts.

Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions†

“…Table 1. Yield* of formation of bipyrimidine photoproducts in primary cultured of human keratinocytes and in human skin exposed to either UVB or UVA (data from Mouret et al [61] UV ⁄ ESI-MS or IR multiphoton dissociation mass spectrometry has recently become available (173).…”

Tandem mass spectrometry for characterization of covalent adducts of DNA with anticancer therapeutics

“…The IRMPD spectra displayed a few characteristic a − B and w ions, with the w 6 , a 8 − B, a 9 − B, and w 9 ions all retaining the platination modification. ET-IRMPD led to new products, including a, d, z, and w ions, that more specifically mapped the adduction site and confirmed that radical precursors may follow unique dissociation pathways not observed for the more conventional closed-shelled ions (Smith and Brodbelt, 2010). …”

“…The intact charge-reduced precursors produced by UVPD may be subjected to CID in a process termed electron photodetachment (EPD), and it results in formation of w, d, a • , and z • ions (Gabelica et al, 2006). UVPD creates a more diverse array of diagnostic fragment ions than any other activation method (Smith and Brodbelt, 2010). …”

“…Reprinted (adapted) with permission from Smith and Brodbelt, 2010. Copyright (2010) American Chemical Society.…”

Comparison of MS/MS Methods for Characterization of DNA/Cisplatin Adducts

“…[27, 36, 37, 39, 43-46] Infrared multiphoton dissociation (IRMPD) has also been evaluated and results in similar fragmentation pathways as CID, as demonstrated for sequencing oligonucleotides[47] and for analysis of DNA/drug complexes. [40-42, 48] Ultraviolet photodissociation (UVPD) has recently been implemented for sequencing oligonucleotides and mapping their modifications. [49] UVPD at 193 nm results in efficient charge reduction of the multiply charged oligodeoxynucleotide anions via electron detachment, in addition to extensive backbone cleavages to yield sequence ions of relatively low abundance.…”