Objective
The project will explore in a pilot phase the potential of Laser Desorption/Ionization Mass Spectrometry (LDI/MS) for routine, large scale sequencing of DNA. Even in the most advanced automated instruments for the Sanger technique, separation of the polynucleotides on a gel is generally considered the limiting step, which will prohibit routine sequencing of the Human Genome in reasonable time.
An exploration was made of the potential and the limits of matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) in nucleic acids analyses.
Among the tested matrices and laser wavelengths for desorption the best results have been obtained with the combinations: succinic acid as matrix and a laser wavelength of 2.94 um and with 3-hydroxypicolinic acid as matrix and 337 nm or 355 nm laser wavelength.
The problem with heterogeneous counterions on the phosphate groups of the nucleic acids has been solved. Besides a simple purification of the matrix solutions with a ion exchange column, an in situ exchange of alkali cations with ammonium ions is performed in the sample droplet just prior to the introduction into the mass spectrometer. This leads to oligonucleotides almost exclusively associated with ammonium counterions. In the high mass range, results differ for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) samples. DNA oligonucleotides give well resolved signals up to the 30 mer level. Above this size metastable molecule ion fragmentation, dominated by single and multiple base losses, degrades the quality of the spectra. For DNA UV-MALDI with the 3-hydroxypicolinic acid matrix is superior to the infrared (IR) desorption scheme in terms of accessible mass range. For the analysis of mixtures of oligodeoxynucleotides, very promising results have been obtained for a mixture of oligothymidylic acids and of chemically degraded polyuridylic acids. Because the different oligonucleotides in a mixture have more uniform physicochemical properties than different peptides, mass analysis of mixtures of the former turned out to be easier than for peptides and proteins. Substitution of up to 30% of the matrix by urea can be tolerated for both matrices. Also in negative ion mode, addition of peptides or proteins in concentrations comparable to those of the oligonucleotides, does not disturb the analysis. Protein and peptide signals are suppressed in the presence of nucleic acids because of a higher ionization efficiency for negative ions of the latter.
One of the promising alternative approaches towards this goal is to replace separation on a gel by mass spectrometry which, in principle, has a comparable sensitivity, but is much faster, has a superior absolute accuracy in terms of the exact definition of the polynucleotide (by absolute molecular weight) and is better suited for integration into a fully automated system and data analysis. So far mass spectrometry of nucleic acids has been limited to relatively small oligonucleotides and sensitivity has been very low.
Matrix-assisted Laser Desorption/Ionization, a recently developed new ionization technique for biopolymers, has been proven to function for peptides up to a mass of several hundred thousand Dalton with unmatched sensitivity. The first results obtained on nucleic acids up to 48 000 Dalton and mixture analysis suggest that this technique might also have the potential to meet the requirements for DNA sequencing, if chemical and physical conditions and parameters are optimized.
The project will therefore explore the limits with respect to:highest mass measurable; sensitivity; and mixture analysis in nucleic acid LID mass spectrometry. Biochemical preparation and modification steps will be developed to adapt the samples to this type of analysis. Physical parameters, in particular optimal wavelength and matrix combinations in the ultraviolet and infrared, will be investigated.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences biochemistry biomolecules nucleic acids
- natural sciences biological sciences genetics DNA
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences chemical sciences analytical chemistry mass spectrometry
- natural sciences physical sciences optics laser physics
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Coordinator
48149 Münster
Germany
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