Charge State Reduction of Oligonucleotide Negative Ions from Electrospray Ionization

Cheng, Xueheng; Gale, David C.; Udseth, Harold R.; Smith, Richard D.

doi:10.1021/ac00099a016

Cited by 80 publications

(80 citation statements)

References 1 publication

(1 reference statement)

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“…For instrumentation that does not resolve adduction, mass spectral peaks are shifted to higher mass resulting in a systematic error. Microdialysis, 7,8 ethanol precipitation, 9,10 high-performance liquid chromatography, 11 spin columns, 12,13 biomagnetic bead separations, [14][15][16] and the addition of organic modifiers 17,18 are some of the approaches that have been employed to address this critical issue.…”

mentioning

confidence: 99%

Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Hannis

Muddiman

1999

Rapid Commun. Mass Spectrom.

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Accurate and precise determination of the number of repeats from a short tandem repeat (STR) sequence for a human gene locus is demonstrated for the first time by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). Specifically, the polymorphic human tyrosine hydroxylase (HUMTHO1) gene, a tetranucleotide STR forensic allele, was chosen as a model system to evaluate our approach for future characterization of both STRs and variable number of tandem repeats (VNTRs) by development of an ESI-FTICR-MS approach. The coding and noncoding strands from the HUMTHO1 9.3 allele are simultaneously resolved obtaining accurate (better than 70 ppm) average mass measurements of 25,783.23 and 24,754.55 Da for the coding and noncoding strands, respectively. The mass measurements are used to calculate the number of repeats for each strand, 'n', of 9.75169 and 9.75001 for the coding and noncoding strands, respectively. It will be shown how the value of 'n' can be used to directly determine the number of pure repeats and accurately determine the exact nature of the polymorphism within the repeat (if any). The single nucleotide deletion in the coding strand (adenine) and noncoding strand (thymine) were accurately identified using this approach. Interestingly, we observed the conversion of single-stranded to double-stranded DNA while the PCR product in the ESI buffer was being infused; the issues related to this observation will be presented. Previous results by other researchers investigating the HUMTHO1 9.3 allele using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) are directly compared with our results. Our results indicate that ESI-FTICR-MS is a powerful approach to rapidly and accurately characterize tandem repeating sequences which will ultimately lead towards the understanding of a complex class of diseases and in human identity determination.

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mentioning

confidence: 99%

Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Hannis

Muddiman

1999

Rapid Commun. Mass Spectrom.

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“…In all cases, the 4-times negatively charged ion was more affected than the 3-times negatively charged ion. The addition of ascorbic acid seems to induce "charge state reduction" (45,46), which partially compensated the negative effect of ascorbic acid addition on ionization efficiency for [PTO-3H] 3− . Both effects, charge state reduction and ion suppression, can be explained by a decrease of pH as well as an increase of ionic strength in the mobile phase due to the addition of ascorbic acid.…”

Section: Methods Developmentmentioning

confidence: 97%

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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“…The distribution and relative abundances of the charge states have been shown to depend primarily on the length and base composition of the oligonucleotide [27,28] as well as on the composition of the electrosprayed solution [29]. It is known from literature that for single-and mixed-nucleobase oligonucleotides the relative abundances of the product ions derived from a given oligomer are highly dependent upon the charge state of the precursor ion [30 -32].…”

Section: Charge State Effects On the Sequence Verification Of A 60-mementioning

confidence: 99%

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

Oberacher¹,

Parson²,

Oefner³

et al. 2004

J. Am. Soc. Mass Spectrom.

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An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably resequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product. (J Am Soc Mass Spectrom 2004, 15, 510 -522)

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Charge State Reduction of Oligonucleotide Negative Ions from Electrospray Ionization

Cited by 80 publications

References 1 publication

Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

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