Ultraviolet/matrix-assisted laser desorption/ionization mass spectrometric characterization of 2,5-dihydroxybenzoic acid-induced reductive hydrogenation of oligonucleotides on cytosine residues

Abstract

The changes in the ion signals in the isotope cluster, mass resolution, signal-to-noise ratio and mass accuracy for matrix-assisted laser desorption/ionization (MALDI) of DNA oligonucleotides (dGGATC, dCAGCt, and dAACCGTT) and their fragment ions were evaluated, and these data were compared with those obtained using 3-hydroxypicolinic acid. Mass spectra obtained by using 2,5-dihydroxybenzoic acid (2,5-DHB) appear to have differences from the theoretical isotopic clusters, which arise by reductive hydrogenation producing a second peak at the M + 2 isotope of the native oligonucleotide. Based on the patterns of the isotopic envelope observed in the in-source decay fragments, we propose that cytosine is the site of reduction. We do not find evidence of reduction of oligonucleotides, viz. dTGGGGTT, that do not contain cytosine; however, 2'-deoxycytidine and 2'-deoxycytidine-5'-monophosphate undergo reductive hydrogenation. Several experiments were carried out in an effort to determine whether the reductive hydrogenation occurs during sample preparation or as a result of laser irradiation. The results of these experiments suggest that it occurs during sample preparation. The relative intensities of ion signals corresponding to the reduced base can be altered by using different matrix additives (aminonaphthalenes) or a different substrate (copper). Also, the oxidized form of 2,5-DHB is trapped by reaction with the side chain of cysteine in glutathione, providing evidence that the reaction occurs in solution as the matrix crystallizes.

Ionic-liquid matrices for quantitative analysis by MALDI-TOF mass spectrometry

“…Nevertheless, with carefully designed sample preparation methods (e.g., employing a thin-layer matrix [12] or using a matrix/comatrix to increase sample spot homogeneity [13]), some groups have achieved satisfactory quantitative results for ODNs over a dynamic range of at least one order of magnitude. Other sample preparations aimed at providing more homogeneous crystallization between analyte and matrix include "seed-layer" [14 -16], two-layer (underlayer/overlayer) [17,18], fast evaporation of matrix solvent to produce a thin-layer matrix film [19], and electrospray deposition of the matrix/sample [20]. Although improvements in quantification were achieved [12,13], these sample preparation methods were strongly dependent on the nature of the sample and the expertise of the spectrometrist.…”