MATRIX-ASSISTED LASER DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY OF CHEMICALLY-MODIFIED OLIGONUCLEOTIDES/

A variety of chemically modified oligonucleotides have been studied by matrix-assisted laser desorption/ionization time-of-flight mass spect rometry (MALDI-TOFMS) in the negative ion mode. These include oligonuc leotides containing modified bases, such as uracil glycol, bromoguanin e, O-6-butylguanine, as well as oligonucleotides in which the phosphod iester groups had been replaced by other functional groups, such as ph osphorothioates. With the linear TOF mass spectrometer, there is no or very Little fragmentation observed, and the determination of the mole cular weight by MALDI-TOFMS offers a convenient way for identifying/co nfirming the presence of the modification. With internal calibration, a mass accuracy of 0.01% can be achieved. Such mass accuracy makes it possible to directly differentiate a small uridine-containing oligonuc leotide from its cytidine-containing analogue. Because of factors such as sample inhomogeneity, laser output fluctuation, and the dynamic ra nge of the detector, quantitation by MALDI-TOFMS has been difficult. N evertheless, semiquantitative information can be obtained for those an alytes that are closely related in structure. Monitoring the products of the synthesis of monophosphorothioated oligoribonucleotide 16-mers by MALDI-TOFMS revealed that the sulfur atom in the phosphorothioate g roup can be replaced by an oxygen atom during the succeeding introduct ion of phosphodiester groups. The earlier the phosphorothioate group i s introduced during the synthesis of the 16-mer, the greater is the ex tent of sulfur to oxygen replacement.