Utility of organic bases for improved electrospray mass spectrometry of oligonucleotides

Greig, Michael J.; Griffey, Richard H.

doi:10.1002/rcm.1290090121

Cited by 195 publications

(181 citation statements)

References 14 publications

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“…ESI mass spectrometry was performed in negative ion mode on a Q-Tof Ultima instrument using a trimer sample dissolved at 4 μM in buffer containing 5 mM each imidazole and piperidine in 1:1 isopropyl alcohol:water (68 Regrettably, similar mass spectrometry assays could not be performed on the 6J2 or 12BB8 ligation products. For 6J2, the preparative-scale yields are too low to be practical.…”

Section: Mass Spectrometry Of the 6j12 Ligation Product Trimermentioning

confidence: 99%

Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

et al. 2005

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We report Zn 2+ -dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k obs up to 0.5 min −1 at 1 mM Zn 2+ and 23 °C, pH 7.9, which is substantially faster than our previously reported Mg 2+ -dependent deoxyribozymes. Each new Zn 2+ -dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2′,3′-cyclic phosphate on a second RNA substrate. Some of the Zn 2+ -dependent deoxyribozymes create native 3′-5′ RNA linkages (with k obs up to 0.02 min −1 ), whereas all of our previous Mg 2+ -dependent deoxyribozymes that use a 2′,3′-cyclic phosphate create non-native 2′-5′ RNA linkages. On this basis, Zn 2+ -dependent deoxyribozymes have promise for synthesis of native 3′-5′-linked RNA using 2′,3′-cyclic phosphate RNA substrates, although these particular Zn 2+ -dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn 2+ -dependent deoxyribozymes instead create non-native 2′-5′ linkages, just like their Mg 2+ counterparts. Unexpectedly, other Zn 2+ -dependent deoxyribozymes synthesize one of several unnatural linkages arising from reaction of an RNA nucleophile other than a 5′-hydroxyl group. Two of these unnatural linkages are the 3′-2′ and 2′-2′ linear junctions created when the 2′-hydroxyl of the 5′-terminal guanosine of one RNA substrate attacks the 2′,3′-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA resulting from attack of a specific internal 2′-hydroxyl of one RNA substrate at the 2′,3′-cyclic phosphate. When compared with the consistent creation of 2′-5′ linkages by Mg 2+ -dependent ligation, formation of this variety of RNA ligation products by Zn 2+ -dependent deoxyribozymes highlights the versatility of transition metals like Zn 2+ for mediating nucleic acid catalysis. † This research was supported by the Burroughs Wellcome Fund (New Investigator Award in the Basic Pharmacological Sciences to S.K.S.), the March of Dimes Birth Defects Foundation Many of the Mg 2+ -dependent deoxyribozymes mediate RNA ligation via reaction of a 2′,3′-cyclic phosphate, which may be opened by an attacking nucleophile with cleavage of either the P-O 2′ bond or the P-O 3′ bond. If the nucleophilic group is a 5′-hydroxyl, then these two reaction pathways lead to the isomeric native 3′-5′ linkage or non-native 2′-5′ linkage, respectively ( Figure 1). Of course, the attacking nucleophile is not necessarily a 5′-hydroxyl, but this functional group is typically the most nucleophilic of those present in RNA. Indeed, in our previous studies, all of the Mg 2+ -dependent deoxyribozymes that use a 2′,3′-cyclic phosphate RNA substrate were found to create only non-native 2′-5′ linkages (path ii in Figure 1), although the selection strategy itself did not compel this high selectivity against 3′-5′ linkages (46-50). Therefore, understanding the distribution of ligation products upon DNAcatalyzed reaction of a 2′,3′-cyclic phosphate RNA substrate i...

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Section: Mass Spectrometry Of the 6j12 Ligation Product Trimermentioning

confidence: 99%

Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

et al. 2005

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“…In ion-pair reversed-phase HPLC of nucleic acids, an ion-pair reagent such as triethylammonium acetate or butyldimethylammonium acetate is added to the mobile phase to ensure chromatographic retention of nucleic acids on a nonpolar stationary phase [42]. Since amines have also been shown to be quite effective in the suppression of cationic adducts [43], it was evident to investigate the effect of ion-pair reagent concentration with respect to the efficiency of adduct elimination, chromatographic performance, and mass spectrometric detectability. PCR mixtures containing 100 fmol of the 61 bp PCR amplicon in 500 nl were analyzed using a gradient of 2-28% acetonitrile in 1 to 50 mmol/l butyldimethylammonium bicarbonate.…”

Section: Optimization Of Ion-pair Reagent Concentrationmentioning

confidence: 99%

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry

Oberacher

Parson

Hölzl³

et al. 2004

J. Am. Soc. Mass Spectrom.

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While electrospray ionization mass spectrometry has shown great potential for the identification of genotypes in DNA sequences amplified by polymerase chain reaction (PCR), the quantitative determination of allele frequencies remains challenging because of the presence of cationic adducts in the mass spectra which severely impairs accuracy of quantitation. We report here on the elaboration of an optimized desalting protocol for ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) of PCR amplicons which facilitates the genotyping of single nucleotide polymorphisms (SNPs). Chromatographic purification at temperatures between 50 and 70°C using monolithic reversed-phase columns and acetonitrile gradients in aqueous, 20 -30 mmol/l butyldimethylammonium bicarbonate enabled the mass spectrometric analysis of nucleic acid solutions containing up to 1.7 mol/l sodium chloride. A further improvement in removal of metal cations was achieved upon the addition of 5-10 mmol/l ethylenediaminetetraacetic acid (EDTA) to the sample solution prior to liquid chromatography. ICEMS was used for the semi-quantitative genotyping of SNPs amplified from the tetraploid genome of potato cultivars. Using a quadrupole ion trap mass spectrometer, allele frequencies were determined with an accuracy of 2-9% by measuring the relative intensities of the signals corresponding to the molecular mass of each of the alleles in the deconvoluted mass spectra. ICEMS results correlated well with those obtained by pyrosequencing, single nucleotide primer extension, and conventional dideoxy

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“…Another complexity in quantitative LC-MS is adduct formation. Generally, electrospray (ESI) or at- 4 ] ϩ are abundant in the spectra [5][6][7][8][9][10][11]. The exact mechanism of adduct formation still has to be elucidated.…”

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confidence: 99%

“…In oligonucleotide analysis, the addition of imidazole and triethylamine or piperidine has proven to be successful to reduce the number of adducted cations. Another well documented approach in this research area is the ethanol precipitation in the presence of ammonium acetate [10]. One could also try to exclude sodium from the ionization process by using ultra pure, thoroughly deionized water.…”

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confidence: 99%

Adduct formation in quantitative bioanalysis: Effect of ionization conditions on paclitaxel

Mortier

Zhang

Peteghem

et al. 2004

J. Am. Soc. Mass Spectrom.

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Quantitative analysis of target compounds with liquid chromatography atmospheric pressure ionization mass spectrometry is sometimes hampered by adduct formation. In these situations, cationization with alkali metal ions instead of proton addition is often observed in the positive ion mode. This work studies the process of adduct formation and investigates potential strategies to control this phenomenon. Paclitaxel, a pharmaceutical chemotherapeutic agent, was used as a model compound. Electrospray (ESI), atmospheric pressure chemical ionization (APCI) and sonic spray ionization (SSI) are evaluated and compared. The work was performed on two different instruments, allowing the evaluation of different ionization behavior for different source design for electrospray, if any. Different mobile phase additives were compared, including acetic acid, formic acid, ammonium formate, and a range of primary amines. Continuous infusion was used for a fast screening, to detect optimal conditions. These were then further investigated in detail by LC-MS. The results indicate that electrospray is the more sensitive interface for this compound on the investigated apparatus. Unacceptable quantitative data were acquired without additives in the mobile phase. Generally, additives increased the reproducibility significantly. A response of mainly one ion was achieved with dodecylamine/acetic acid and acetic acid/sodium acetate. The data also point out the importance of evaluating adduct formation for compounds prone to this phenomenon during method development, especially in view of accurate quantitation. (J Am Soc Mass Spectrom 2004, 15, 585-592)

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Utility of organic bases for improved electrospray mass spectrometry of oligonucleotides

Cited by 195 publications

References 14 publications

Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry

Adduct formation in quantitative bioanalysis: Effect of ionization conditions on paclitaxel

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Utility of organic bases for improved electrospray mass spectrometry of oligonucleotides

Cited by 195 publications

References 14 publications

Zn2+-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

Zn2+-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry

Adduct formation in quantitative bioanalysis: Effect of ionization conditions on paclitaxel

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Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages