Analysis of nucleic acids using ICEMS

Mass spectrometry of nucleic acids

Nucleic acids are polar and thermally labile macromolecules that contain acidic and basic groups. Thus, the generation of intact molecular ions of oligonucleotides turned out to be nearly impossible with classic ionization techniques like electron ionization or chemical ionization. The triggers for the widespread use of MS for nucleic acid analysis, however, were the introduction of electrospray ionization (ESI) and of matrix-assisted laser desorption/ionization (MALDI) as very soft ionization techniques for large biomolecules. These techniques enabled for the first time the sensitive mass spectrometric analysis of intact nucleic acids consisting of hundreds to thousands nts and thus opened MS the door to genetics.

ESI-MS of nucleic acids

ESI is a technique that allows the transfer of ions from solution to the gas phase, from which ions can be subjected to mass spectrometric analysis. The overall process can be divided into three major steps: (i) Production of charged droplets at the ESI capillary tip. (ii) Shrinkage of the charged droplets by solvent evaporation and repeated droplet disintegrations, leading to very small but highly charged droplets. (iii)  Production of gas-phase ions.

Solvent properties influence the electrospray characteristics in a variety of ways, notably the dependency of the minimal potential required to form the Taylor cone on surface tension, the spray current on conductivity, and the droplet size on viscosity, and their impact on the detectability of oligonucleotides has been studied. One parameter that influences the quality of mass spectra is the pH of the solution. As oligonucleotides are usually analyzed in the negative ion mode, high pH values assist ion formation. Thus, volatile bases, including triethylamine, piperidine, buthyldimethylamine, or cyclohexyldimethylamine, are often added to the oligonucleotide solution to enhance mass spectrometric detection. Low base concentrations (10-50 mM) seem to be favorable.

A characteristic of the ESI process is that multiply charged ions are produced. The mass spectrum of an oligonucleotide typically consists of a series of peaks, each of which represents an ion of the intact molecule having a specific number of protons removed from the phosphodiester groups. Mathematical procedures ('deconvolution') need to be applied to reconstruct the intact molecular mass out of the observed charge state distribution.

Why chromatography?

The success of the mass spectrometric analysis of nucleic acids largely depends on the purity of the nucleic acid molecules introduced into the mass spectrometer. Liquid chromatography (LC) represents one of the most efficient methods to purify and desalt nucleic acids prior to their mass spectrometric characterization.

The advantageous feature of ESI - the transfer of intact, double-stranded PCR products from solution into the gas phase - is disadvantageous in the context of genotyping. Base substitutions are difficult to identify in double-helical DNA by molecular mass measurements, because A-T and G-C base pairs have very similar average masses of 615.4 amu and 616.4 amu, respectively, and A/T or G/C substitutions do not cause any mass shift at all. Denaturation can be easily accomplished during the chromatographic run by using elevated column temperatures (65-75 °C).

Another reason for using chromatography as sample preparation method is its ability to fractionate nucleic acid mixtures enabling the thorough mass spectrometric characterization of samples that would have been too complex for direct analysis.

Experimental setup

Experimental setup

ICEMS as genotyping tool

genotyping tool

The most striking advantage of mass spectrometric genotyping assays is the use of the measured molecular mass information for allele calling. The molecular mass is less error-prone than other sequence-specific parameters, including migration times, retention times, or hybridization yields, as it represents an intrinsic property of an oligonucleotide that is directly related to its nucleotide composition.

Among all different mass spectrometric genotyping assays ICEMS represents one of the most powerful techniques, which offers the following advantages:

  1. Genotyping is based on measuring the molecular mass of intact PCR products with lengths up to 250bp.
  2. As only the conditions of the PCR need to be optimized, the development of single and multiplexed assays is a simple task. The highest multiplexing level can be reached with LC-MS.
  3. The allelic state derived from one single strand is usually confirmed by the result obtained from the complementary single strand. Thus, genotyping results are very accurate and reliable.
  4. ESI-MS can principally be used to scan for unknown sequence variations.
  5. SNPs, STRs and SNPSTRs can be characterized.
  6. Direct molecular haplotyping is feasible.
  7. Sample preparation efforts and reagent costs are low, especially, if LC-MS is performed.
  8. Integrated systems covering all parts of the workflow are commercially available.
  9. Semi-quantitative genotyping is possible.

Applications

ICEMS has been successfully applied in the following areas of research:

  1. Forensic genetics
  2. Association studies
  3. Pharmacogenetics
  4. Molecular diagnostics

Grants

  1. Österreichische Forschungsförderungsgesellschaft: Optimierung der Effizienz forensischer Typisierung durch massenspektrometrische Analyse von kombinierten DNA-Fragmentlängen- und Sequenzunterschieden, Brückenschlag-Programmlinie Projekt 810998, 2006-2008.
  2. Österreichische Forschungsförderungsgesellschaft: dnatox - Die Kopplung der Flüssigkeitschromatographie mit der Massenspektrometrie als Werkzeug für die Toxin- und DNA-Analytik, KIRAS PL 2 Projekt 813786, 2008-2009.
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