Oligonucleotide analysis and sequencing using MALDI-TOF mass spectrometry

Marek Šebela1, Štěpán Kouřil1, Pavel Řehulka2 and Petr Tarkowski1

1Faculty of Science, Palacký University, Olomouc, Czech Republic
 2Faculty of Military Health Sciences, University of Defense, Hradec Králové, Czech Republic


Since its discovery, matrix-assisted laser desorption/ionization (MALDI), which is usually associated with a time-of-flight (TOF) mass analyzer in commercial instruments, has become a common technology in biological mass spectrometry (MS). It is useful for measurements with peptides and proteins, oligonucleotides, oligosaccharides, technical polymers, small polar compounds, viruses or intact microbial cells. The well-known possibility of fast identification or biotyping of microorganisms (the latter representing a differentiation of strains or species by distinctive biochemical characteristics – this is in principle applicable to any group of biological samples of the same type) makes MALDI-TOF MS more common and increasingly popular in biochemical, bioanalytical, medicinal or biotechnological laboratories. Both matrix choice and optimization of the sample preparation protocol are the most important steps in MALDI experiments. Matrix is indispensable in the ion formation process. The applications of MALDI-TOF MS to nucleic acid research include quality control of synthetic oligonucleotides, oligonucleotide sequencing, single-nucleotide polymorphism genotyping and microsatellite analysis. Oligonucleotides, which are largely utilized as gene probes in molecular biology, can efficiently be measured for example using 3-hydroxypicolinic acid (HPA) as a matrix but there also other matrix compounds available for this purpose: 2′,4′,6′-trihydroxyacetophenone, 6-aza-2-thiothymine, picolinic and anthranilic acids. A major obstacle is the relatively poor mass resolution, which increases in parallel with the increasing molecular mass accompanied by the decrease in signal intensity and sensitivity. Solid mixed matrices have been introduced such as HPA/picolinic acid or HPA/pyrazinecarboxylic acid to cope with these obstacles. Additives (e. g. diammonium hydrogen citrate) are then used to reduce or prevent from the formation of adducts between the phosphate groups of DNA and counter ions (Na+, K+). In this work we have used synthetic oligonucletotides up to a 60-mer to look for the most optimal sample preparation and data acquisition procedure. The ionophore antibiotics nigericin and monensin were checked as possible additives together with 1,4-bis(3-aminopropyl)piperazine. For oligonucleotide sequencing based on the fragmentation process of ion-source decay, combining HPA with different co-matrices based on aromatic carboxylic acids was evaluated to achieve excellent results with the optimized protocol.

This work was supported by grant no. 15-16888S by the Czech Science Foundation.