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Chapter 3 : Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry

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Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, Page 1 of 2

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Abstract:

This chapter reviews applications of electrospray ionization (ESI) to the analysis of RNA and its constituents. Some examples of modified nucleosides discovered by liquid chromatography (LC)/mass chromatography (MS) and characterized include four sugar-methylated nucleosides in tRNA from hyperthermophilic archaea, 5-methylcarboxymethyl-2’-O-methyluridine in rat liver tRNA, and two sugar-methylated nucleosides in the cap4 subunit of trypanosomal mRNA. A method has been implemented for mapping modified nucleosides in RNA, based on electrospray ionization mass spectrometry of RNase T-derived oligonucleotides. The ability to mass measure the small peaks suggests that oligonucleotides can be analyzed at levels lower than the 20 pmol of tRNA utilized in the present example. To examine levels of modified nucleosides in tRNA as a function of growth temperature in the thermophilic archaeon , it was grown at 70, 85, and 100°C. The strategies and examples of nucleic acid analysis using mass spectrometry presented in this chapter utilize ESI because of its successful implementation for the analysis of nucleic acids and their constituents from the monomer level in digests all the way up to intact tRNA and 5S rRNA.

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3

Key Concept Ranking

Nucleic Acid Constituents
0.78500265
Ionization Mass Spectrometry
0.5606502
High-Performance Liquid Chromatography
0.5080458
Bacteria and Archaea
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Nucleic Acids
0.46517107
0.78500265
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Figures

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Figure 1

Schematic diagram of the sample inlet and ESI for a triple quadrupole mass spectrometer.

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3
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Figure 2

Chromatogram (detection at 254 nm) from a directly combined high-performance LC/ESI-MS analysis of nucleosides from a total digest of mixed tRNA from MRE600. Major modified nucleosides are as follows: 1, Ψ; 2, D; 3, acpU; 4, sC; 5, Cm; 6, I; 7, mG; 8, mU; 9, sU; 10, UM; 11, oQ; 12, mG; 13, Gm; 14, tA; 15, mA; 16, mA. (Inset) ESI mass spectrum of the peak at centered at 16.8 min. Symbols are defined in Appendix 1.

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3
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Figure 3

Expanded UV chromatogram (A) and reconstructed ion chromatograms for the MH ion of methylguanosine (B) (/ 298) and the BH ions for methylguanine (C) (/ 166) and guanine (D) (/ 152), extracted from the analysis shown in Fig. 2 . For clarity, ion abundances have been normalized to the most abundant peak in each panel.

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3
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Figure 4

Product ion spectra from collision-induced dissociation of the BH ions (/ 166) characteristic for base-methylated guanosines, acquired from an LC/MS/MS analysis of a digest of wheat germ tRNA. (A) 1-Methylguanosine; (B) -methylguanosine; (C) 7-methylguanosine.

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3
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Figure 5

Scheme for mapping modified nucleosides in RNA by mass spectrometric analysis of RNase Τ-generated oligonucleotides.

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3
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Figure 6

Chromatogram from directly combined high-performance LC/ESI-MS of an RNase Tdigest of 20 pmol of tRNA .

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3
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Figure 7

ESI mass spectrum of ACUoQUAms AA4ψCUGp (peak 10 in Fig. 6 ). (Inset) Oligonucleotide masses derived from the mass spectrum.

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3
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Tables

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Table 1

Assignments of oligonucleotides from RNase T digestion of tRNA

Citation: Crain P. 1998. Detection and Structure Analysis of Modified Nucleosides in RNA by Mass Spectrometry, p 47-57. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch3

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