Mass and sequence verification of modified oligonucleotides using electrospray tandem mass spectrometry

Abstract

Electrospray coupled with tandem mass spectrometry is shown to have utility in determining the molecular mass and the sequence of short modified oligonucleotides. The product ion spectrum of a triply deprotonated molecule precursor is dominated by ions formed via two fragmentation pathways: first, fragmentation across the SP-0 phosphate bond, and second, loss of a non-terminal base with concomitant cleavage of the ribose 3'C-O bond. If either of these two pathways is complete with fragmentation occurring at each equivalent position along the phosphadiester backbone, then the oligomer may be sequenced. Alternatively, if both these pathways are only partially complete, their complementarity may be used for more extensive sequencing. The inductive effect of the modification on the nucleobase is shown to influence the latter fragmentation process. The greater the electron affinity of the base modification, the more facile is the loss of that base when the molecule is collisionally activated. In fact, this latter fragmentation dominates the product ion spectrum when a nucleobase contains a substituent that is highly inductively withdrawing. This domination can be such that few other first-generation fragment ions are present in the product ion spectrum. Sequence information may then require utilization of second-generation product ions.

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

“…They observed a base loss preference of AϾTӷGϾC. Similar to the McLuckey group, Barry and coworkers proposed a base-catalyzed 2-step, 1,2 elimination scheme where the negatively charged oxygen of the 3Јphosphodiester group abstracts a proton from the 2Ј position of the sugar resulting in base loss [13]. The observed preference for nucleobase loss and subsequent formation of a n -B fragment ions is AϾTϾGϷC.…”

“…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].…”

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

“…Consequently, MSMS of oligodeoxynucleotides has been successfully applied to characterize or confirm structures [11], to detect chemical modifications [2,10,18], and to identify sequence variations [14, 19 -21]. However, the deduction of sequence information is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS.…”