Electron detachment dissociation (EDD), recently introduced by Zubarev and co-workers for the dissociation of multiply charged biomolecular anions via a radical ion intermediate, has been shown to be analogous to electron capture dissociation (ECD) in several respects, including more random peptide fragmentation and retention of labile posttranslational modifications. We have previously demonstrated unique fragmentation behavior in ECD compared to vibrational excitation for oligodeoxynucleotide cations. However, that approach is limited by the poor sensitivity for oligonucleotide ionization in positive ion mode. Here, we show implementation of EDD on a commercial Fourier transform ion cyclotron resonance mass spectrometer utilizing two different configurations: a heated filament electron source and an indirectly heated hollow dispenser cathode electron source. The dispenser cathode configuration provides higher EDD efficiency and additional fragmentation channels for hexamer oligodeoxynucleotides. As in ECD, even-electron d/w ion series dominate the spectra, but we also detect numerous a/z (both even-electron and radical species), (a/z - B), c/x, (c/x - B), and (d/w - B) ions with minimal nucleobase loss from the precursor ions. In contrast to previous high-energy collision-activated dissociation (CAD) and ion trap CAD of radical oligonucleotide anions, we only observe minimum sugar cross-ring cleavage, possibly due to the short time scale of EDD, which limits secondary fragmentation. Thus, EDD provides fragmentation similar to ECD for oligodeoxynucleotides but at enhanced sensitivity. Finally, we show that noncovalent bonding in a DNA duplex can be preserved following EDD, illustrating another analogy with ECD. We believe the latter finding implies EDD has promise for characterization of nucleic acid structure and folding.

Two sets of synthetic 21-23mer oligonucleotides with various types of 2'-position modifications have been studied with tandem mass spectrometry using ion trap collision-induced dissociation (IT-CID) and negative electron transfer (NET)-CID. A systematic study has been conducted to define the limitations of IT-CID in sequencing such 2'-chemically modified oligonucleotides. We found that IT-CID is sufficient in characterizing oligonucleotide sequences that do not contain DNA residues, where high sequence coverage can be achieved by performing IT-CID on multiple charge states. However, oligonucleotides containing DNA residues gave limited backbone fragmentation with IT-CID, largely due to dominant fragmentation at the DNA residue sites. To overcome this limitation, we employed the negative electron transfer to strip an electron from the multiply charged oligonucleotide anion. Then, the radical anion species formed in this reaction can fragment via an alternative radical-directed dissociation mechanism. Unlike IT-CID, NET-CID mainly generates a noncomplementary d/w ion series. Furthermore, we found that NET-CID did not show preferential dissociations at the DNA residue sites and thus generated higher sequence coverage for the studied oligonucleotide. Information from NET-CID of different charge states is not fully redundant such that the examination of multiple charge states can lead to more extensive sequence confirmation. This work demonstrates that the NET-CID is a valuable tool to provide high sequence coverage for chemically modified oligonucleotides, and such detailed characterization can serve as an important assay to control the quality of therapeutic oligonucleotides that are produced under the good manufacture practice (GMP) regulations.

We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A 6 , C 6 , and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A 6 , C 6 , G 6 , U 6 , CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d-and w-type ions rather than c-and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone. (J Am Soc Mass Spectrom 2006, 17, 1369 -1375 D eoxyribonucleic acid (DNA) and ribonucleic acid (RNA) play several important roles in a variety of biological processes, including transcription and translation. RNA molecules are unique biopolymers in that they can both carry genetic information and catalytic function [1-3]. As for proteins, RNA function is structurally related and methods for sensitive and detailed structural characterization are needed. NMR spectroscopy is widely used for RNA structural analysis [4, 5] but suffers from limited sensitivity. Another powerful approach is chemical labeling followed by fluorescence resonance energy-transfer (FRET) measurements [6], which can provide single molecule sensitivity [7]. However, structural information from FRET is limited to distance constraints for the added fluorophores and does not provide a detailed picture.McCloskey and coworkers pioneered mass spectrometric analysis of nucleic acids [8,9] and have applied LC-MS to the interrogation of RNA reaction mechanisms and to the characterization of RNA modifications [10 -12]. Tandem mass spectrometry (MS/MS) is widely used to characterize oligonucleotides and PCR products [13-18]. Particularly, dissociation of even-electron oligodeoxynucleotide (DNA) anions with "slow heating" techniques, including nozzle-skimmer dissociation [19], collision activated dissociation (CAD) [20], infrared multiphoton dissociation (IRMPD) [21], and multipole storage-assisted dissociation [22] produces sequencespecific [a Ϫ B] and w-type ions (McLuckey nomenclature [23]) from cleavage of the phosphodiester backbone.With those techniques, nucleobase loss alone is also observed as a major fragmentation pathway. Such...

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