Various factors influencing the signal intensity of oligonucleotides in electrospray mass spectrometry

Bleicher, Konrad H.; Bayer, Ernst

doi:10.1002/bms.1200230604

Cited by 87 publications

(76 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%

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%

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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“…The main difficulties arise due to the formation of oligonucleotide-sodium or -potassium adducts which persist after ionization and desolvation (11,12). The presence of sodium adducts becomes *To whom correspondence should be addressed l Q -s " ) 1994 …”

Section: Introductionmentioning

confidence: 99%

Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonucleotides

Potier¹,

Dorsselaer²,

et al. 1994

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We report here on the analysis of synthetic oligonucleotides by electrospray ionization mass spectrometry (ESI-MS). After intensive removal of salt ions (especially sodium cations), negative ion mass spectra, allowing mass measurement with an accuracy of 0.01%, were obtained on several oligonucleotides up to 80 nucleotides. In most cases, the resolution was sufficient to observe n-1 and n-2 forms due to internal deletions during automated synthesis, and to identify the missing nucleotides. A 132-mer, whose size is close to the limit of automated chemical synthesis, was also successfully mass measured. A quantitative study showed that ESI-MS can provide quantitative data on oligonucleotides of similar size and structure. The described methodology is used to characterize oligonucleotide analogues such as phosphorothioate oligonucleotides designed for antisense applications. Finally, analyses in the positive ion mode on a trimer TpTpT in the presence of different amine bases were performed and allowed a better understanding of the influence of these bases on the ions formation.

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“…Thus, with CycHDMAA a significant higher content of ACN in the mobile phase is necessary to elute nucleic acids from the chromatographic column. This is advantageous because the higher content of organic solvent can improve desolvation efficiency during ESI leading to improved ionization efficiency and improved detection sensitivity (39,40). Other chromatographic parameters were optimized during method development.…”

Section: Methods Developmentmentioning

confidence: 99%

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Erb

Leithner²,

Bernkop‐Schnürch³

et al. 2012

AAPS J

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Abstract. Phosporothioate oligonucleotides represent an important class of therapeutic oligonucleotides, in which none-bridging oxygen atoms of the phosphate groups are replaced by sulfur. These oligonucleotides are designed to treat disease by modulating gene expression of an affected individual. As the development and application of these therapeutical oligonucleotides require analytical support, the development, validation, and application of an assay for the quantitative analysis of a phosporothioate oligonucleotide in rat plasma is described. The method employs ion-pair reversedphase chromatography on a monolithic capillary column with acetonitrile gradients in cyclohexyldimethylammonium acetate for separation and high-resolution tandem mass spectrometry for detection of nucleic acids. Chromatographic parameters (i.e. column temperature, mobile phase composition) as well as mass spectrometric parameters (i.e. spray voltage, gas flow, and capillary position, scan mode) have been optimized for sensitive oligonucleotide quantification. Furthermore, a solid-phase extraction method was developed which enabled processing of 10 μl of plasma. The five-point calibration curve showed linearity over the range of concentrations from 100 to 1,000 nM of the oligonucleotide. The limit of detection was 50 nM. The intra-and inter-day precision and accuracies were always better than 10.2 %. Using this assay, we performed a pharmacokinetic study of the phosporothioate oligonucleotide in rat treated with a single intravenous dose of 0.39 μmol/kg. The assay sensitivity was sufficient to study the early phase elimination of the oligonucleotide. Small amounts of the oligonucleotide were detectable up to 3 h after dosing.

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Various factors influencing the signal intensity of oligonucleotides in electrospray mass spectrometry

Cited by 87 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

Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonucleotides

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

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