Interpretation of Oligonucleotide Mass Spectra for Determination of Sequence Using Electrospray Ionization and Tandem Mass Spectrometry

Abstract

Procedures are described for interpretation of mass spectra from collision-induced dissociation of polycharged oligonucleotides produced by electrospray ionization. The method is intended for rapid sequencing of oligonucleotides of completely unknown structure at approximately the 15-mer level and below, from DNA or RNA. Identification of sequence-relevant ions that are produced from extensive fragmentation in the quadrupole collision cell are based primarily on (1) recognition of 3'- and 5'- terminal residues as initial steps in mass ladder propagation, (2) alignment of overlapping nucleotide chains that have been constructed 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 program that requires < 2s 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 oligonucleotides from ribosomal RNA.

Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization fourier transform ion cyclotron mass spectrometer

“…While McCloskey and coworkers did not observe the a n -5MeC fragment ion under CID conditions [12], the a n -5MeC fragment ion is observed for both oligonucleotides using the InHex IRMPD fragmentation scheme (Table 2). For both oligonucleotides, y n fragment ions account for 4-6% of the total fragment ions.…”

“…Several dissociation schemes have been used in combination with mass spectrometry to fragment DNA oligonucleotides of various lengths. These methods include nozzle-skimmer dissociation [17], collisional activated dissociation (CAD) [12,13], infrared multiple photon dissociation (IRMPD) [18], blackbody infrared radiative dissociation (BIRD) [19], and multipole storage assisted dissociation (MSAD) [20]. Several groups have shown that modified bases influence fragmentation patterns of oligonucleotides and can be used to locate modified bases that occur naturally in damaged DNA [10,[21][22][23][24].…”

“…By combining various fragmentation schemes, it was shown that complete sequence coverage could be obtained for a 50-mer DNA [7][8][9]. Other researchers demonstrated that sequence information and identification of modified bases could be obtained by combining fragmentation schemes with ion trap mass spectrometry [10] and triple quadrupole mass spectrometry [11][12][13].Based on the small amounts of sample required and the unambiguous signatures generated, mass spectrometry is emerging as the preferred platform on which to interrogate and sequence modified oligonucleotides. In order to faithfully sequence oligonucleotides containing desperate chemical modifications, an understanding of how the modifications influence the fragmentation pattern is essential.…”

“…By combining various fragmentation schemes, it was shown that complete sequence coverage could be obtained for a 50-mer DNA [7][8][9]. Other researchers demonstrated that sequence information and identification of modified bases could be obtained by combining fragmentation schemes with ion trap mass spectrometry [10] and triple quadrupole mass spectrometry [11][12][13].…”

Charge state-dependent fragmentation of oligonucleotide/metal complexes

“…Metal cations are also required for the proper folding and function of many forms of RNA, including most ribozymes [7,8]. The interactions of DNA with several natural products [9] and synthetic drugs and drug candidates [10,11] are known to be metal-mediated.Mass spectrometry (MS) is well-established as a useful tool for the structural characterization and sequencing of nucleic acids [12][13][14], and has also been employed to characterize a number of nucleic acid/metal ion interactions [15]. Several previous studies have focused on the interactions of the anticancer therapeutic cisplatin or its derivatives with DNA [16 -22].…”