Nucleic acid analysis using liquid chromatography and electrospray ionization mass spectrometry

The development of fast, reliable and sensitive methods for the analysis of biomolecules, especially nucleic acids, proteins, and peptides, is at the same time a deciding motor and has to keep pace with the steady progress of biochemical, molecular biological, and medical science. Miniaturized chromatographic separation methods are frequently the method of choice for the separation and characterization of complex biopolymer mixtures, when the amount of sample is limited. The concept of monolithic chromatographic separation media is especially favorable for the fabrication of capillary columns. Chemical immobilization of the monolith at the capillary wall eliminates the tedious preparation of retaining frits; the continuous porous polymer exhibits favorable mass transfer properties, enabling excellent separation efficiency. We prepared monolithic columns within the confines of fused silica capillaries of 200 μm i.d. by copolymerization of styrene and divinylbenzene in the presence of a suitable porogen mixture of 1-decanol and tetrahydrofuran. Different chemical and physical parameters that influence the morphology, porosity and separation efficiency of the obtained monolithic chromatographic columns were investigated, specifically the influence of the amount of cross-linking monomer, of different porogens and porogen mixtures, of polymerization temperature, and finally of the amount of radical initiator. The prepared monolithic columns were characterized by measurement of permeability with different solvents, determination of the pore size distribution by inverse size exclusion chromatography, examination of the morphology by scanning electron micrographs, and finally by chromatographic testing and determination of the separation parameters. Finally, the developed separation systems were applied to the separation of DNA ladder markers, of PCR amplified DNA fragments containing sequence-tagged sites used in the genotyping of individuals and of amplified short tandem repeat loci used in forensic science. Mass spectrometry emerged as one of the most important tools for the characterization of biological macromolecules after the introduction of soft ionization techniques like electrospray ionization. It yields information useful to determine or confirm the structure and sequence of biomolecules; it enables the detection of mutations and modifications in nucleic acids. We compared the characteristics of three different mass spectrometers, namely a quadrupole ion trap, a triple stage quadrupole and a sector-field instrument, in mass spectrometric analysis of nucleic acids. To prepare the online coupling of liquid chromatography and mass spectrometry, we optimized the conditions of the electrospray process in terms of utilized solvent, sheath liquid and concentration of ion-pair reagent. (author)