Based on the data of the human genome project, today it is known that the human body is equipped with approximately 600 proteases that contribute to the regulation of important physiological processes. Our knowledge, however, of many key proteases is still limited. In this context, our research is focused on the development of novel diagnostic tools for proteolytic activity based on synthetic protease inhibitors as recognition devices.
For the different application areas including fluorescence microscopic imaging, affinity blotting, and flow cytometry, these inhibitors are functionalized with the appropriate reporter groups. We apply for the design of these inhibitors peptidic and peptidomimetic concepts coupled with structure-based approaches. Additionally, natural products with protease-inhibiting properties play as lead structures also an important role. Currently, eleven human cysteine cathepsins are known that belong to the clan CA (family C1) of cysteine proteases. Beside their role in the lysosomal protein breakdown, these enzymes are involved in the regulation of variety of important physiological processes like bone resorption, proenzyme activation and hormone maturation. Moreover, it is well accepted that these proteases play a crucial role in a variety of diseases among them osteoporosis and cancer. To better dissect partially overlapping functions of these proteases or even to identify new functions, affinity probes are powerful tools. In this context, the thiol-reactive group (2S,3S)-oxirane-2,3-dicarboxylic acid represents a privileged platform for the development of such affinity probes. By functionalizing both carboxylic acid groups with peptide sequences it is possible to address simultaneously S and S’ sites along the entire active-site cleft. The structure-based selection of these peptide sequences then allows for a selective inhibition of individual cysteine cathepsins.
(A) Chemical structure of inhibitor 1. (B) X-ray crystal structure of inhibitor 1 in complex with cathepsin B (pdb code: 1SP4).
This concept has been already successfully applied for cathepsin B. By addressing the residues His-110 and His-111, which are responsible for the dipeptidyl carboxypeptidase activity of this protease, we could develop the selective inhibitor 1 (Fig. 1), whose binding mode was confirmed by X-ray crystal structure analysis (cooperation with Dr. D. Turk, Josef Stefan Institute, Ljubljana, Slovenia). Using the biotin-functionalized inhibitor, we could already identify two novel physiological functions of cathepsin B: (i) the protease protects activated cytotoxic T-lymphocytes from self-destruction (cooperation with Dr. P. Henkart, NIH Bethesda, USA) and (ii) is involved in the migration of keratinocytes (cooperation with Prof. K. Brix, Jacobs University, Bremen, Germany).
The group of Prof. Dr. Christian Neusüß is performing research and development on modern instrumental techniques and methods for the analysis of organic molecules. The main focus of the research is on the combining separation techniques with mass spectrometry. Especially in the field of capillary electrophoresis-mass spectrometry (CE-MS) an extensive know-how is established covering both instrumental developments (e.g. interfaces) and method development. Among many applications the characterization of biotechnologically derived (glycol)proteins can be distinguished. A method for the separation and mass spectrometric characterization of human erythropoietin (EPO) enables the differentiation and determination of similarity of biosimilar products. As an example, the separation and mass spectral characterization of EPO is shown in
Figure 1. High resolution mass spectrometry allows the differentiation of closely related molecules. Furthermore, the accurate mass enables the identification of small organic molecules based on the determination of the elemental composition. These routines have been applied to the analysis of natural products and glycans derived from glycoproteins and antibodies.
Current projects deal with the development of two-dimensional electromigration techniques on-line coupled to mass spectrometry. This is especially of interest for the coupling of existing CE-methods which are not compatible with electrospray-mass spectrometry. The characterization of antibodies is a main focus of many research projects with the biopharmaceutical industry. The research laboratory is well equipped with i) several modern mass spectrometers (i.e. two quadrupole-time-of-flight mass spectrometers - QTOF MS -, an ion trap mass spectrometer and a quadrupole mass spectrometer) ii) more than half a dozen of capillary electrophoresis instruments from various supplier, and iii) two High Performance Liquid chromatography.
The group of Prof. Junker is interested in several research areas combining organic chemistry and analytical chemistry methods. The main interest of the group is to understand the mode of drug action and delivery in biological systems. Organic synthesis is used to optimize mass spectrometry (MALDI-MS) methods and to modify small molecules for improved drug delivery and to use them as marker systems.
In this context new methods for matrix assisted laser desorption & ionization mass spectrometry (MALDI-MS) are developed. In MALDI-MS the analyte is mixed with a matrix, which is usually a small organic compound. The matrix is used in a large excess and cocrystallizes with the analyte forming a compound cluster. The next step is the energy transfer from the laser beam to the matrix compound leading to desorption and ionization of the matrix-analyte aggregate. In the gas phase this cluster disintegrates and releases the ionized analyte. This process is very gentle for the analyte, the energy is mainly transferred to the matrix. Therefore, this method is exceptional suited for the analysis of fragile and large biomolecules like proteins or DNA, which stay intact. This soft ionization process has the great advantage that it can be used for the two-dimensional scanning of surfaces of e.g. human tissues. But unfortunately, this process relies on several complicated parameters making it difficult to predict.
One important parameter for the optimization of this process is the choice of the right matrix. Until today only a handful of matrices are in use. These common matrices were selected by analytical chemists who had no access to organic chemistry resources. Therefore, not much is known about the relationships between matrix structure and their performance as a MALDI matrix. The goal of the Many projects in Aalen is to resolve this issue by a combination of organic synthesis of structurally related matrix compounds and their evaluation by screening methods. During the course of these projects several hundreds of compounds were evaluated against a large number of different analyte groups like peptides, glycopeptides, proteins, natural products and lipids. This led to the discovery of several new matrices with improved properties and performance in comparison to standard matrices. The final goal is to establish structure activity relationships for the different compound classes and to define general rules for the design of MALDI matrix compounds.
Different natural environments are screened for microorganisms that exhibit interesting bioactivities, e.g. soil, water, leaves, biofilms, food. Microbes were isolated and the production of unusual antimicrobial activities were tested by antimicrobial growth assays. Sequently we purify the antimicrobial active agents by multistep IEC and rpHPLC and elucidate their structure by Mass spectrometry and NMR.
An important group of antimicrobial active compounds are peptide antibiotics which are found among all classes of life. Peptide antibiotics are characterized by unusual (nonproteinogenic) structural elements and unusual chemical linkages. They fall into two classes, nonribosomally synthesised peptides generated by nonribosomal peptide synthetases (NRPSs) and ribosomally formed peptides which mature in a series of posttranslational modifications and proteolytic processing (e.g. lantibiotics). Both pathways allow the integration of rare constituents into the peptide structure as well as the generation of unusual linkages, as for example cyclisation and branching. We were involved in the structural elucidation of a series of the nonribosomal cyclic Bacillus subtilis peptide antibiotics surfactin, mycosubtilin, and fengycin (which is furthermore branched). Furthermore the following lantibiotic were characterized: Subtilin and entianin, both exist also in a succinylated isoform, ericin A and ericin S, as well as the cyclic structures sublancin and subtilosin. Reviews are given in Stein (2005 & 2008); Fuchs et al (2011), Arnison et al (2013).
Biosynthesis of peptide antibiotics
Furthermore, we are interested in the biosynthetic gene cluster involved in the production of unusual peptide antibiotics. Our research addresses the characterization of the biosynthetic machinery involved in the posttranslational modifications of pre-propeptides, self-protecting proteins of the microbes against its own product (immunity system), as well as the regulation of lantibiotic biosythesis. In B. subtilis A1/3 the production of two lanthionine-containing peptides has been shown, ericin A und S. A gene cluster was identified and sequenced (for a summary see Stein, 2005; Halami, 2011, Arnison et al., 2013).
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of intact bacterial cells
We are intensively involved in extending the potential of MALDI-TOF MS of the analysis of intact bacterial cells. This emerging efficient analytical technique is applied for the discovery of microorganisms that produce lantibiotics and/or bacteriocins (Stein 2008, Fuchs et al, 2011). In cooperation with Dr. H.-D. Junker (HTW, Aalen, Germany) a series of organic molecules with MALDI-matrix properties are studied in order to improve both, the biotyping of microorganisms by MALDI MS of intact cells directly from agar plates or liquid cultures as well as the identification of unusual secondary metabolites.
MALDI-TOF MS of wood compounds
In cooperation with Dr. Kantlehner and Dr. Neusüß (HTW, Aalen, Germany) the potential of mass spectrometry is analyzed to characterize the highly complex mixture of hemi-cellulose and lignin extracs form different kind of wood which were isolated by a novel procedure using ionic liquids.
Inorganic Trace Analytics, mainly in Micro grade and Solar grade Polysilicon- and Silicon Products and its Upstream and Downstream Manufacturing
Since the 1950s, silicon, the second most abundant element, has been widely used to produce integrated circuits. Silicon is not found free in the nature, but it occurs as oxide or silicate. Silicon´s oxidation properties make it the primary semiconductor material for the integrated circuit manufacturing and for the solar cell fabrication. Oxidation and diffusion are the basic processes mainly in semiconductor manufacturing but also in solar cell fabrication these processes keep an important role. Due to increased electronic density, finer circuit patterns and the production of more efficient solar cells the performances of these semiconductors and solar wafers are greatly influenced by the mobility of metallic contaminants within the devices. The drive towards the reduction of these contaminants has led to the need of higher
sensitivity analytical methods to be used for the cleanliness evaluation. The importance of thoroughly cleaning silicon surfaces prior to any temperature process, oxidation or diffusion, has long been recognized. The purity of wafer surfaces is an essential requisite for the successful fabrication of very large scale integrated (VLSI) and ultra large scale integrated (ULSI) silicon circuits. Metal contaminants such as Fe, Ni, Cu and Zn, are detrimental for present semiconductor surfaces and nowadays even for solar grade material, because they spread and diffuse into the silicon bulk.
Our research interest lies in the analytical method development, Quality and data evaluation of various Polysilicon-Production steps such as TCS/STC-Distillation, tank farm management concept and Solid Post-processing steps. The most important steps here are: Surface-, Subsurface- and Bulk analysis of Polysilicon Chunks by ICP-MS/INAA (Method Development, Benchmarking, Correlation, Interferences, Sample Preparation Methods, Standardization, Limit of Detection, Sensitivity, UPW Analysis) Method Development and Routine Analysis of Etching Solutions by ICP-OES and Online Titration Method Development and Routine Analysis of TCS/STC Samples by ICP-OES and ICP-MS for Metals and Boron/Phosphorous in different Production Steps (distillation, tank farm) GC and GC-MS Analysis for TCS / STC / DCS / MDCS, Offline and Online
Applied Chemometrics and Design of Experiments in a wide field of industrial applications
According to different aims to which multivariate data analysis can be used diverse chemometrics techniques are necessary, such as Unsupervised pattern recognition, supervised classification and Modelling of DOE data.
In combination with spectroscopy applied in various kind of industrial areas e.g. in pharmaceutical industry, in process development in the field of biotechnology, or in archaeology we are able to handle a large amount of data with modern statistical tools. Many students are involved in such kind of applied research.