Liquid Chromatography and Electrochromatography with Capillary Columns Coupled to Electrospray Mass Spectronomy for the Analysis of Nucleic Acids.

The central role of nucleic acids in biosciences has effectuated the rapid development of numerous techniques for their isolation, separation, characterization and quantitation. Advances in high-performance liquid chromatography and electrophoresis, particularly the development of novel microparticulate sorbents and the introduction of fused silica capillaries, have greatly extended the scope of high-resolution separation techniques for the analysis of nucleic acids in complex mixtures of biological origin. Since its invention in the early 1980s, electrospray ionization mass spectrometry has added a new dimension to the chemical characterization of biomacromolecules because the unique capability of measuring the mass of small biomolecules of a few hundred daltons and of large biopolymers as large as several megadaltons with an accuracy of better than 0.001-0.2% classifies mass spectrometry as the most accurate method for molecular mass measurement. The electrospray mass spectrometric analysis of nucleic acids is associated with difficulties arising from their polyanionic nature and tendency to form quite stable adducts with alkali cations resulting in mass spectra of poor quality. Consequently, desalting of the nucleic acid sample is very important to obtain mass spectra of high quality. Several protocols, such as ethanol precipitation, cation exchange, microdialysis, and off-line chromatography have been suggested to remove nonvolatile cations but most of them are time consuming and prone to losses of precious sample. The primary goal of this project is to develop new high-performance liquid chromatographic and capillary electrochromatographic separation methods to be used as on-line sample concentration, desalting, and separation techniques that can be readily coupled to electrospray mass spectrometry in order to allow sensitive characterization of nucleic acid samples. The proposed approach to analyze nucleic acids consists of a combination of different technologies which are 1. synthesis of polymeric stationary phase materials, 2. miniaturization of the separation process, 3. development of instrumental components, and 4. optimization of separation conditions for effective electrospray ionization. Miniaturization of the separation system through the application of capillary microcolumns is a prime prerequisite to take full advantage of the high mass sensitivity of electrospray mass spectrometry. Moreover, utilization of capillary columns of inner diameters of 50-320 m m is expected to offer significantly higher separation efficiency as compared to conventional analytical 4.6 mm columns. New column technologies such as derivatization of polymer particles and packing of fused silica capillaries with polymeric microbeads are developed to manufacture highly efficient capillary columns for high-performance liquid chromatography and capillary electrochromatography. The chromatographic mode proposed for separation of nucleic acid is ion-pair reversed- phase chromatography with a hydrophobic, polymeric stationary phase and a mobile phase containing water, acetonitrile and triethylammonium bicarbonate as volatile ion-pair reagent which guarantees efficient electrospray ionization and detection of nucleic acids. The developed and optimized analytical techniques will be applied to the analysis of oligonucleotides, DNA fragments and PCR products related to clinical diagnostics, and drug therapy. National and international co- operations are available and will be used to ensure efficient evaluation of the new analytical methods.

Highly efficient analysis systems were developed for the investigation of nucleic acids, proteins, and peptides. The systems were based on the separation of the analytes by means of capillary high-performance liquid chromatography (HPLC) and their subsequent detection by electrospray ionization mass spectrometry (ESI-MS). Using miniaturized capillary column technology developed in the course of this project, very small amounts (few 100 attomol) of mixtures of nucleic acids could be both separated and mass spectrometrically characterized. The utilized materials were shown to have a significant impact on the analytical results. Even better separation performance was obtained by the application of so called monolithic materials as stationary phase. In contrast to classical stationary phases, that are composed of tiny particles, monolithic stationary phases comprise a single piece of a porous polymer offering properties highly favourable for liquid chromatography. The developed monolithic stationary phases represent the most efficient stationary phases known today for the separation of nucleic acids. Subsequently, the monolithic columns were successfully optimized for the separation of peptides and proteins. Again, the newly developed separation systems are among the most efficient known today with regard to separation performance. The monolithic separation systems were utilized in connection with ESI-MS detection for the investigation of biochemical and proteomic problems. The nitration of proteins in the gas phase by environmentally relevant pollutants was investigated in collaboration with the Technical University of Munich. The proteins involved in Photosynthesis were analyzed in collaboration with the University of Viterbo, Italy. A new variant of the antenna protein lhcb 1 was detected and its aminoterminal sequence was determined, which is of great importance in the supramolecular organization of photosystem II. In another focus of the project, we evaluated the applicability of tandem mass spectrometry for the generation of sequence data about nucleic acids. We were able to demonstrate that sequence specific information can be obtained for oligomers as long as 80-mers. We developed a computer-based comparative sequencing algorithm for fully automated interpretation of the generated sequence information. The method was applied in collaboration with Stanford University to the detection of genetic variation and for medical diagnostics.