INTERPRETATION OF OLIGONUCLEOTIDE MASS-SPECTRA FOR DETERMINATION OF SEQUENCE USING ELECTROSPRAY-IONIZATION AND TANDEM MASS-SPECTROMETRY

Procedures are described for interpretation of mass spectra from colli sion-induced dissociation of polycharged oligonucleotides produced by electrospray ionization. The method is intended for rapid sequencing o f oligonucleotides of completely unknown structure at approximately th e 15-mer level and below, front DNA or RNA. Identification of sequence -relevant ions that are produced from extensive fragmentation in the q uadrupole -collision cell are based primarily on (1) recognition of 3' - and 5'- terminal residues as initial steps in mass ladder propagatio n, (2) alignment of overlapping nucleotide chains that have been const ructed independently from each terminus, and (3) use of experimentally measured molecular mass in rejection of incorrect sequence candidates . Algorithms for sequence derivation are embodied in a computer progra m that requires < 2 s for execution. The interpretation procedures are demonstrated for sequence location of simple forms of modification in the base and sugar. The potential for direct sequencing of components of mixtures is shown using an unresolved fraction of unknown oligonuc leotides from ribosomal RNA.