Improvement of resolution, mass accuracy, and reproducibility in reflectedmode DE-MALDI-TOF analysis of DNA using cast evaporation-overlayer sample preparations

DNA analysis by matrix-assisted laser desorption/ionization (MALDI) mass sp ectrometry is hindered by two processes: alkali metal adduction and fragmen tation of the intact ionized molecule. The adverse effects of both processe s can be reduced by adding ammonium ion salts or compounds such as fructose to the sample preparations. Matrix additives improve sensitivity and resol ution of DNA analysis by MALDI. In addition, spot-to-spot reproducibility, resolution, and mass accuracy for DNA oligonucleotides (less than or equal to 12 mer) can be improved by the use of overlayer sample preparations with matrixes that have low aqueous solubilities, such as alpha-cyano-4-hydroci nnamic acid, ferulic acid, and 2,4,6-trihydroxyacetophenone. For example, r esolution for 5-12-mer oligonucleotides is greater than 7000 using overlaye r matrix preparations and mass accuracy values are well below 20 ppm. In ad dition to these methods, a new method for analyzing DNA in positive ion mod e is reported using acidified 3-hydroxypicolinic acid. This method does not lose sensitivity for higher mass oligonucleotides as quickly as overlayer methods, and spectra retain >6000 resolution and mass accuracies of similar to 20 ppm between different overlayer depositions.