Charge Reduced ESI Multistage MS of Bioactive Compounds

Analysis of biological systems comprised of interacting proteins (e. g. blood coagulation, allergic reaction or bacterial cell wall biosynthesis) and chemicals (e. g. endocrine disruptors, antibiotics) typically involves determining the molecular masses of the interesting molecules and elucidating their primary structures. Covalent modifications to the structure or amino acid sequence may affect the biological system and must also be determined by some means. Several complementary techniques are typically applied to obtain this kind of information (e.g. chromatographic, electrophoretic or spectroscopic techniques). However, the complexity of the investigated systems makes the sole use of these methods useless. Mass spectrometry turned out to be a complementary technique, which exhibits high sensitivity as well as high throughput and allows the acquisition of molecular mass and primary structure information. Innovations in desorption/ionization techniques and in analyzer designs have extended the applicability of this technique to the analysis of biomolecules. Especially the introduction of nano electrospray ionization (ESI) coupled to an ion storage device, in particular an quadrupole ion trap (QIT), offered new possibilities: high sensitivity and mass resolution, improved mass range and multistage fragmentation (MSn) capabilities in one single analyzer. The latter feature turned out to be the most striking and will be in the center of this research project. Up to MS10 experiments are now feasible, but a lot of investigations have to be performed to understand the fragmentation mechanisms and derived laws for peptidoglycan fragments (isolated from bacterial cell walls of e.g. H. pylori or methicillin-resistant S. aureus), steroid conjugates, phytoestrogens (surface water and fish) and lignans (from food). The charge manipulation of the multiple-charged molecular ions by means of a Po neutralizer chamber will be studied to improve the yield of single-charged peptide molecular ions out of tryptic digests of proteins as obtained in proteomics. This will be particular helpful for molecular mass and amino acid sequence determination in proteome work. Related to this topic is also the attempt to optimize the on-line coupling of isoelectric focussing of proteins performed in a capillary with a nano ESI QIT system to generate a 3-dimensional picture (pI, intensity and molecular mass of separated protein) of complex protein mixtures (e.g. blood coagulation glycoproteins), a kind of virtual 2-dimensional gelelectrophoresis. The sensitivity and performance characteristics of such an instrument make nano ESI QIT mass spectrometry an attractive technique for the near future in enviromental and bioanalysis.

The major result of the project is that we have built a prototype ion source which can be attached to commercial available mass spectrometers and this resulted in one patent submission via the Vienna University of Technology. This new nano electrospray ion source allows the improved manipulation of the charge number of biomolecules by means of the Po-210 source and the resulting mass spectrometric data allow a faster unambiguously identification of proteins in proteomics. The newly developed device should help to find faster new biomarkers for human disease diagnostics and therapy monitoring. Furthermore the on-line detection and structural characterization of environmental air pollutants (e.g. phthalates from polymers or other organic matter) will be possible with unsurpassed sensitivity. Within the project the foundations for structural database of bioactive low mass compounds were laid, which have possible applications in forensic sciences, metabolomics and illegal drug analysis.