New procedure for the use of high-performance liquid chromatography–electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides

Apffel, Alex; Chakel, John A.; Fischer, Steven M.; Lichtenwalter, Kay; Hancock, William S.

doi:10.1016/s0021-9673(97)00256-2

Cited by 122 publications

(123 citation statements)

References 22 publications

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“…Eluents of low electric conductivity and high content of volatile organic solvent are most suitable for nucleic acid analysis by ESI-MS [27,28,34,40,41]. 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].…”

Section: Optimization Of Ion-pair Reagent Concentrationmentioning

confidence: 99%

“…Commonly applied off-line and on-line sample purification protocols for nucleic acids prior to mass spectrometric analysis include multiple ethanol precipitation [19], membrane filtration [20], solid-phase extraction [21], microdialysis [14], affinity purification [22,23], cation-exchange [24], ligand-exchange [13], magnetic particles [25], size exclusion chromatography [26], and high-performance liquid chromatography (HPLC) [27,28]. Ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry (ICEMS) has been demonstrated to be a rapid and highly effective analytical tool for the characterization of PCR amplified sequences [15, 29 -31].…”

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confidence: 99%

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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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Section: Optimization Of Ion-pair Reagent Concentrationmentioning

confidence: 99%

mentioning

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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show abstract

“…A solution to the compatibility issue has been to use more volatile amines, such as dimethylhexylamine, which has been used in the analysis of a metabolite of clodronate [9]. There have been methods developed for the analysis of oligonucleotides and nucleotides by LC-MS using a 1,1,1,3,3,3-hexafluoro-2-isopropanoltriethylamine as the ion-pairing agent [10,11]. However, hexafluoroisopropanol is highly corrosive, relatively expensive and must be handled with care.…”

mentioning

confidence: 99%

Development of generic liquid chromatography-mass spectrometry methods using experimental design

Seto

Bateman

Gunter

2002

J. Am. Soc. Mass Spectrom.

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“…95,96 This method allows the separation of RNase digested RNAs, although the combination of triethylamine, as the ion pairing agent, and 1,1,1,3,3,3-hexafluoro-2-isopropanol, as a mobile phase additive to improve ESI performance, can lead to instrument contamination, which will be an issue if other analytes and/ or chromatographies are to be used on the same LC-MS system. Recently, a variety of HPLC mobile phases that are more friendly to instrumentation have been investigated, primarily within the field of therapeutic oligonucleotide analysis.…”

Section: High-performance Liquid Chromatography -Oligonucleotidesmentioning

confidence: 99%

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

Gaston

Limbach

2014

RNA Biology

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The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

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New procedure for the use of high-performance liquid chromatography–electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides

Cited by 122 publications

References 22 publications

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

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

Development of generic liquid chromatography-mass spectrometry methods using experimental design

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

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