APPLICATIONS OF DELAYED EXTRACTION MATRIX-ASSISTED LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY TO OLIGONUCLEOTIDE ANALYSIS

A delayed ion extraction technique is shown to dramatically improve ma ss resolution and the overall quality of matrix-assisted laser desorpt ion ionization (MALDI) mass spectra of oligonucleotides. Isotope limit ed mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode, This performance is as go od as that achieved to date for peptides and proteins, Applications in cluded the detection of oxidized byproducts of phosphorothioate DNA an d separation of components differing only by 15 Da at 9.5-kDa molecula r mass, In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. A n example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products, Mass accuracy wa s adequate to verify sequences of oligodeoxyribonucleotides up to 9500 -Da molecular mass. Fast fragmentation taking place between the ionizi ng pulse and the extraction pulse is demonstrated to be a sequencing t ool for small oligonucleotides, By proper selection of matrix material , wavelength, and irradiance, fast fragmentation can be promoted effic iently. Fragment ions tend to form from cleavage of phosphodiester bon ds, as previously observed in infrared MALDI.