Top-Down Interrogation of Chemically Modified Oligonucleotides by Negative Electron Transfer and Collision Induced Dissociation
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- 作者：Yang Gao ; Jiong Yang ; Mark T. Cancilla ; Fanyu Meng ; Scott A. McLuckey
- 刊名：Analytical Chemistry
- 出版年：2013
- 出版时间：May 7, 2013
- 年：2013
- 卷：85
- 期：9
- 页码：4713-4720
- 全文大小：518K
- 年卷期：v.85,no.9(May 7, 2013)
- ISSN：1520-6882
文摘
Two sets of synthetic 21鈥?3mer oligonucleotides with various types of 2鈥?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鈥?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.