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Proteases control a wide variety of essential physiological processes. By specific proteolytic processing, proteases participate in every biological process including cell-cycle progression, proliferation and differentiation, apoptosis, tissue remodelling, haemostasis, immunity or angiogenesis. Proteases are part of complex networks, termed protease webs, also including their substrates, activators, modulators and inhibitors. The term protease web comprises the current knowledge that proteases do not operate in isolation, but in interconnected pathways and amplification cascades where proteolytic information moves both in a unidirectional flow and in regulatory feedback loops. Inappropriate proteolysis has been found to play a major role in cancer as well as cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. Excessive or inappropriate proteolysis is seldom a result of genetic aberrations but most often results from numerous endogenous and/or exogenous factors resulting in unwanted activation of protease signalling pathways, such as the effect of atherosclerotic plaque formation or blood vessel injury on the blood coagulation cascade, which leads to the appearance of intravascular thrombi. Thus understanding the protease web will yield new diagnostic tools and validated targets for drug discovery. To identify the role of proteases in a defined biological context and to understand protease signalling in health and disease it is necessary to know the identity of the protease and protease-substrate repertoires, termed the protease degradome as well as all molecules interacting and thereby regulating and modulating the activities of individual proteases.
Our group is focused on the identification and validation of disease-associated proteases. For the determination of protease activities we developed a tool termed "Mass spectrometry assisted enzyme screening system" (MES described in detail below) [9, 14]. Mass spectrometry is a useful tool for the description of enzymatic reactions since the change of the chemical structure of the reactants is generally accompanied by a change in the molecular weight. Furthermore the chemical identity of the reaction products can be validated by tandem mass spectrometry instruments (MS/MS). Compared to a fluorescence-based assay, the MES system is about a factor of 1000 more sensitive detecting the activity of the angiotensin-converting-enzyme (ACE). It is a further benefit of the MES system that the enzymatic conversion of larger biomolecules such as peptides is possible. MES experiments are performed with MALDI or ESI mass spectrometers, depending on the scientific question.
For the purification of proteases it is mandatory to maintain their biological activities during protein purification. Liquid chromatography is well suited for protein purification since it offers the opportunity to choose parameters according to the stability window of a target protease. Because every protein shows a very individual behaviour towards chromatographic materials an empiric determination of parameters for the optimized chromatographic purification is very helpful and significantly reduces the time for protein purification. Therefore we perform a parameter screening with the protein purification parameter screening system (PPS [15] described in detail below) prior to the purification of a target protein by liquid column chromatography. HPLC-pumps designed for protein purification are used. For preparative purposes the displacement chromatography is established.
By applying MES and PPS an angiotensin-II-generating protease was identified in the porcine renal tissue [16]. A further identification of a protease is described in the patent Nr. 10 2006 049 822.
Methods & Tools: The mass spectrometry assisted enzyme screening system (MES)
For the determination of enzyme activity, usually the time dependent product formation during the enzymatic reaction is monitored. For this approach artificial labeled substrates are common. Upon turnover the labeled substrate undergoes a change in absorbance at a given wavelength. Although this is a simple means for the analysis of enzyme activity, the range of substrates that can be studied is highly restricted. As an alternative, radioactive substrates may be used because of their identical chemical nature to the natural substrates as well as their sensitivity of detection. However, radiometric assays require the separation of the radioactive products by chromatographic methods and subsequent liquid scintillation counting. Radiometric as well as optical methods both share the problem of uncertainty about the fate of the chemical structure of the substrate after the enzymatic conversion, which may yield wrong positive results.
Because enzymatic reactions in general are accompanied by a change in the molecular weight of the reactants, monitoring enzymatic activity with a mass spectrometer is an alternative, circumventing the above-mentioned disadvantages. However, mass spectra of reaction mixtures in the presence of numerous proteins and other biomolecules as well as buffer salts can result in poor signal intensities. Therefore our group developed a new automated mass spectrometry-based assay, named "mass spectrometry assisted enzyme screening (MES)" which circumvents the above-mentioned problems and yields high signal intensities of the analytes of interest, even if enzyme activities in complex protein fractions are measured.
The MES method includes 4 steps.
Figure 4: Robotics for mass spectrometry assisted enzyme screening (MES).
Methods & Tools: The protein-purification-parameter-screening system (PPS) [15].
Screening for optimum parameters for the chromatographic purification of a target protein is often laborious and time-consuming. Multiple reasons are responsible for the difficulties in the chromatography of proteins resulting from the large heterogeneity of the chemical properties of proteins. The numerous interactions between the individual functional groups of a protein with their environment - including other biomolecules, buffers, additives such as stabilizers and surfaces of chromatographic media - critically decide about the recovery. The large variety of interactions of a protein with its environment is still far away from being calculated and therefore requires a systematic empiric experimental approach to achieve optimum purification conditions. We developed a system, termed PPS (protein-purification-parameter-screening system), which enables an automated systematic empiric experimental approach (Figure 5). Up to 96 different chromatographic purification parameters can be tested in parallel within short processing times with the PPS system. The PPS procedure includes 6 steps: 1. The 96 gel aliquots in a deep well plate are equilibrated with 96 individual sample application buffers. Typically, for an ion-exchange PPS experiment, the pH of the sample application buffer is varied on the x-axis of the deep-well plate and the salt concentration on the y-axis. Thus each well displays an individual pair of chromatographic parameters. 2. Aliquots of the sample are applied to the individually prepared gels in 96 wells 3. After application of the sample aliquots to the gels the non-binding biomolecules present in the supernatant are removed and the gels are washed with the individual sample application buffers. 4. The binding proteins are eluted. 5. The total protein concentration and the concentration or the activity of the target protein is determined in the supernatant and in the eluate. 6. The resulting data are analysed for the most appropriate set of parameters for the purification of the target protein. These chromatographic parameters are used for the purification of the target protein by liquid chromatography. A special advantage is given by the possibility to apply even raw extracts directly to the PPS-system. Furthermore the possibility to vary a large number of parameters increases the chance to find secondary interaction-based separation effects, which often help to realize high specific yields.
Figure 5: Robotics for protein purification parameter screening system (PPS).
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