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WO2004029286A2 - Procede pour identifier des activites enzymatiques dans des extraits de proteine quelconques - Google Patents

Procede pour identifier des activites enzymatiques dans des extraits de proteine quelconques Download PDF

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Publication number
WO2004029286A2
WO2004029286A2 PCT/DE2003/003109 DE0303109W WO2004029286A2 WO 2004029286 A2 WO2004029286 A2 WO 2004029286A2 DE 0303109 W DE0303109 W DE 0303109W WO 2004029286 A2 WO2004029286 A2 WO 2004029286A2
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protein
fractions
immobilized
proteins
substrates
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German (de)
English (en)
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WO2004029286A3 (fr
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Hartmut SCHLÜTER
Joachim Jankowski
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Charite Universitaetsmedizin Berlin
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Charite Universitaetsmedizin Berlin
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Priority to AU2003273738A priority patent/AU2003273738A1/en
Publication of WO2004029286A2 publication Critical patent/WO2004029286A2/fr
Publication of WO2004029286A3 publication Critical patent/WO2004029286A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions

Definitions

  • the invention relates to a method for recognizing enzyme activities of any protein extracts.
  • proteome proteins expressed by the genome
  • Proteome analysis is expected to provide important new insights into understanding cellular functions and their disorders. Proteins include enzymes that are involved in all life processes. Due to their catalytic properties, enzymes can be found in the cell in much lower concentrations than many structural proteins, so that this important group of proteins often eludes 2D electrophoresis analysis. So far, only a fraction of all the enzymes expressed in the human organism over a lifetime are known. In the past, some of these enzymes have already described the integration into the development of disease processes and thus contributed to the detection and treatment of diseases. Functional proteome research should multiply knowledge in the future.
  • Proteome research to date has been largely based on the 2D electrophoresis technique, which is used to compare protein patterns of two different cell states.
  • the great advantage of 2D electrophoresis can be seen in the high resolution in protein separation and the rapid identification possibility of the proteins of interest. Up to 10,000 proteins can be separated with a 2D gel.
  • the 2D electrophoresis technique is accompanied by some disadvantages which make this technique unsuitable for functional questions in proteome research.
  • membrane proteins or proteins> 100 kDa must be separated using other separation techniques (e.g. using 1 D SDS). Proteins that only occur in the lowest concentrations can be made visible, but are often difficult to identify.
  • ICAT isotope coded affinity tags
  • WO 01/94924 A2 and DE 100 27 794 A1 describe a method for analyzing enzyme-catalyzed reactions using MALDI-TOF mass spectrometry. However, this method is only aimed at the analysis of enzyme-catalyzed reactions, e.g. for the determination of the enzyme kinetics.
  • DE 100 27 794 A1 defines even enzyme-catalyzed reactions as “enzymatic reactions with whole cells”. A similar process is also described in DE 100 44 132 A1, in which whole cells are also used therefore gain no knowledge of individual proteins.
  • Enzyme activity in the sense of the invention is understood to mean that such biomolecules such as proteins or peptides are able to form, respectively, biomolecule-enzyme complexes. have epitopes on their surface that react with enzymes. Furthermore, that after the complex has been dissolved, the original substrate is chemically modified as the reaction product (s), the formation of the product being based on a defined unit of time. Furthermore, it is also understood to mean that the biomolecules are in a position (generally) to form biomolecule-substrate complexes in which the substrate not an enzyme, but, for example, antibodies, synthetic substances, medicines, pharmaceuticals, etc.
  • proteins are extracted from biological sources such as human or animal tissue or from plant cells.
  • fractionation of the protein extract The protein extract is purified over several chromatographic steps until individual proteins are largely homogeneous.
  • samples that can originate from different purification steps are collected separately to create a protein library.
  • each individual sample is divided into 2 or more parts after fractionation. Part of the sample is covalently bound to particles.
  • Another part of the sample is frozen for later identification of proteins of interest.
  • the proteins are covalently bound to particles. After binding, the particles are freed of non-binding substances.
  • the immobilized particles are stored in the refrigerator or freezer. Suitable agents can be added to the particles for stabilization.
  • Aliquots of the immobilized protein fractions from the protein library are incubated with a substrate. The substrate is so chooses that it (in the presence of the desired protein) is chemically changed by the desired catalytic activity of the protein (enzyme) and the chemical reaction can be recognized via the reaction product formed.
  • enzyme activities of the immobilized proteins of the fractions are ultimately measured using mass spectrometric methods.
  • process steps b) to e) can be repeated cyclically for more effective fractionation.
  • substrates for the reaction with the immobilized protein fractions are understood to mean biomolecules of any kind occurring in nature, but also synthetic organic molecules or hybrids of biomolecules occurring in nature, for example — but not exclusively — enzymes, proteins, Peptides etc.
  • the substrates can be combined to form substrate libraries, such substrate libraries consisting of a mixture of several substrates or of a substrate (molecule) with multifunctional groups in the sense of reaction-specific groups.
  • the aim of the invention is therefore to develop an automated platform system which produces protein libraries from protein extracts, searches them for enzyme activities and identifies the enzymes on which the activities are based.
  • the protein library is created by immobilizing protein fractions. The immobilization stabilizes the enzyme activities and the ability for long-term storage and enables mass spectrometric analysis of the reaction products.
  • the immobilized proteins are incubated with the substrates specific for the enzymes sought. The presence of a sought enzyme can be assumed to be probable if the expected reaction products are detected.
  • the reaction products are detected using mass spectrometry, which provides a high degree of flexibility with regard to the substrates that can be used, the detection of unexpected reaction products and the rapid screening of a protein library.
  • a given protein extract is characterized by means of preliminary tests ("scouting" tests) with regard to its composition, in order to develop a fractionation strategy from the data obtained, which strategy is based on the combination of several orthogonal chromatographic separation techniques ( (separation techniques which differ significantly in their separation mechanisms) enables fractions to be obtained with as small a number of proteins as possible, and an aliquot of each protein fraction is frozen for possible later identification and stored. This measure also makes it possible to subject a fraction of interest to further cleaning steps if necessary.
  • the protein library of the immobilized protein fractions is designed in such a way that a single fraction can be tested against a large number of different substrates independently of time.
  • the choice of substrates depends on the screening goal.
  • the substrates are naturally defined for the search for synthesizing or metabolizing enzymes.
  • the reaction products are usually detected by mass spectrometry, but other detection systems such as bio-assays can also be used.
  • bio-assays can also be used.
  • the special feature of the method according to the invention consists in the method step of immobilizing the proteins after their purification. Immobilization has several indispensable advantages for the system:
  • the immobilization additionally enables long-term storage, which is necessary for the construction and use of protein libraries.
  • the novelty of the system is the approach to detect the reaction products created by direct mass spectrometric analysis (ESI or MALDI-MS) of the incubation solutions. This direct detection is only possible through the immobilization of the proteins.
  • the immobilization of the proteins prevents loss of signal intensity in the mass spectrometric analyzes due to the absence of the proteins.
  • the gel particles on which the proteins are immobilized are therefore of central importance in the context of the invention.
  • the development of magnetic gel particles is intended to simplify the automated production and handling of the protein library.
  • the particular advantage of the invention lies in its broad applicability with respect to the substrates, since there is no definition of fluorescent substrates, substrates with UV-absorbing groups or isotope-labeled substrates, but with underivatized, not -radioactive original substrates can be worked. This prevents the risk that the substrates will no longer be recognized by their enzymes due to steric changes caused by derivatization, or that false positive results may result from the model substrates derivatized with chromophores being converted to chemical bonds other than those expected (e.g.
  • Another particular advantage over any previously developed methodology is the ability not only to detect the presence of a sought protein with known reaction products, but also to detect proteins whose catalyzed reactions tion was previously unknown.
  • This advantage is of particular interest for the study of the metabolism of new active substances and the identification of the associated enzymes as well as for the exploration of new metabolic pathways.
  • a protein library is produced from a complex protein extract by fractionation and subsequent immobilization of the purified proteins, which is then searched for suitable enzyme activities using suitable substrates.
  • the protein extracts to be separated are selected to be large enough that even after multiple fractionation steps a sufficient amount of protein is retained for immobilization and identification; an absolute amount of the protein in the extract of 10 - 100 g has proven to be useful.
  • the protein libraries are created by gentle fractionation of protein extracts. For this purpose, larger protein extracts in the gram range are separated with a few separation steps to such an extent that the smallest possible number of proteins can be found in the individual protein fractions. This is achieved by using a special form of displacement chromatography (sample self-displacement).
  • the purified proteins are immobilized on affinity chromatography gel particles.
  • magnetic gel particles are also used for the immobilization, which have a suitable gel surface, which ensure the highest possible enzyme activity.
  • the gel particles are moved, preferably automated, through immobilization, washing, dosing and incubation steps. This auto At the same time, matatisation advantageously ensures that MALDI sample carriers or an electrospray injection system are loaded for the subsequent mass specrometric measurements; this provides an automated interface to the mass spectrometer.
  • reaction products with mass spectrometry eg MALDI or ESI-MS
  • the analysis of the reaction products with mass spectrometry (eg MALDI or ESI-MS) after incubation of the substrates with the immobilized proteins is also automated and the data obtained are managed and, if necessary, evaluated using corresponding software. Fractions with enzyme activity are checked for protein purity and identified for purity. For this purpose, the fractions obtained are analyzed for uniformity using techniques such as 2D electrophoresis or microbore HPLC and the corresponding fractions are identified by MALDI-MS and LC-ESI-MS / MS or by Edman degradation.
  • the automated system for carrying out the method according to the invention comprises the following components: a system (“scouting” system) for exploring the composition of the extract, which provides data for the combination of the cleaning steps, a multidimensional chromatography system for fractionating the protein extracts, a robot for the immobilization of the proteins, for the automatic incubation of substrates with fractions of the immobilized proteins and for the transfer of the reaction products to the mass spectrometer, a mass spectrometer and a data processing system.
  • a system for exploring the composition of the extract, which provides data for the combination of the cleaning steps
  • a multidimensional chromatography system for fractionating the protein extracts
  • a robot for the immobilization of the proteins
  • for the automatic incubation of substrates with fractions of the immobilized proteins and for the transfer of the reaction products to the mass spectrometer, a mass spectrometer and a data processing system.
  • Pig kidneys are selected as the source for the protein extract in the examples below. This choice has the advantage that the necessary organs can be obtained in sufficient quantities from slaughterhouses.
  • the kidney is used as an organ, because specific scientific questions can be connected to this organ (for example, searching for angiotensin II and urotensin-generating enzymes). This question is interesting for the project as a model for the evaluation of the system, since the detection of the enzyme activities via the mass spectrometric detection of the reaction products (angio- tensin I or angiotensin II; Urotensin) after incubation of renin substrate or pro-urotensin with immobilized protein fractions.
  • kidney as a source of the proteins is also advantageous because this tissue is known to synthesize renin.
  • the detection and identification of renin should serve as a control for the functionality of the invention.
  • Renin a specific protease formed in the kidney, catalyzes the hydrolysis of renin substrate, in which angiotensin I is formed as a reaction product.
  • An effective chromatography system with which it is possible to purify protein extracts into their individual components as quickly and gently as possible, is carried out by a) the systematic search ("scouting" experiments) for suitable parameters for the chromatographic separation of the protein extract and b) by combining different chromatography columns (Affinity, ion exchange, size exclusion, hydrophobic interaction, hydroxylapatite, immobilized metal ion affinity and reversed phase chromatography) based on orthogonal separation principles. Individual fractions are directly linked to the following by techniques such as column switching Chromatography column passed on without the protein fractions leaving the system, ie they have to be temporarily stored in fraction vessels.
  • the fractionation strategy includes the following steps: After creating a protein extract pool that is large enough to carry out a large number of fractionation experiments with identical samples, this pool is characterized in terms of its protein composition and enzymatic activities ("scouting" experiments).
  • SDS-PAGE electrophoresis, isoelectric focusing and 2D electrophoresis are the first to obtain a detailed picture of the proteins present in the extract, and this data is used to further plan fractionation, with the protein extract not only being separated into its components, but also
  • the eluates of the chromatography media are additionally examined with electrophoresis in order to document the effectiveness and selectivity of the respective chromatographic parameters, in order to check the influence of the chromatograph
  • Common steps for enzyme activities are to be used for various enzymes.
  • the enzyme Kinetics are investigated using methods of UV spectroscopy and fluorescence spectroscopy. The use of these techniques is necessary in order to be able to measure the activities not only after but also before the immobilization. By comparing the enzyme kinetics before and after immobilization, statements about the influence of immobilization on the enzyme activities are possible.
  • the problem with creating / building the protein library is to fractionate protein extracts with the aim of achieving the most effective possible separation of the proteins with high resolution - while maintaining as many enzymatic activities as possible. After successful separation, aliquots of the chromatographically obtained fractions have to be immobilized for the protein library.
  • the mass spectrometric investigations of the immobilized protein-substrate complexes for the detection of the enzymatic activity of the proteins are carried out with the methods of MALDI mass spectrometry, in particular with a reflector MALDI mass spectrometer. Both the work steps for immobilization and the mass spectrometric detection of enzymatic activities should be automated as much as possible, but of course can also be carried out “manually” (without automation) if necessary.
  • the protein libraries can be stored for a long time without loss of the enzymatic activities. It does not matter in which way the proteins are immobilized, for example as activated affinity chromatography gels or on magnetic gel particles. Such magnetic gel particles, so-called magnetobeads, are offered, for example, by chemagen (see www.chemagen.de). The coupling chemistry between magnetobeads and non-magnetobeads is essentially known. The advantage of using magnetobeads is that the particles are easier to automate using electromagnets.
  • the advantages of the automated mass spectrometry-supported system according to the invention for screening protein libraries for enzymatic activities are as follows:
  • the invention proves to be advantageous for functional proteome research in the sense of a comparison of two different life states (profiling of enzyme activities with substrate libraries to be developed, multifunctional probes), as well as for the search for new enzymes for the synthesis of biomolecules (with known biomolecules as substrates), for the metabolism of molecules (substrates: biomolecules, active substances) and for organic synthesis (substrates: synthesis building blocks);
  • biomolecules with known biomolecules as substrates
  • substrates for the metabolism of molecules
  • substrates biomolecules, active substances
  • organic synthesis substrates: synthesis building blocks
  • Metabolomics research beaten as new drug-relevant enzymes are recognized and metabolic pathways can be investigated with the invention.
  • the system can be used both for the targeted search for enzymes for the implementation of a given substance and for the exploration of natural synthetic routes.
  • the invention serves as a tool for the search for new enzymes for all biotechnological areas in which enzymes are used. This includes the pharmaceutical industry (search for new targets (enzymes) for drug development, search for new enzymes for the synthesis of drugs, decoding the metabolism of new drugs, development of new diagnostics), food technology
  • the invention serves as a tool for the search for enzyme activities of unknown enzymes for functional proteome research.
  • multiplex screening with multifunctional substrates and / or substrate mixtures / substrate libraries it is possible to create profiles of enzyme activities from two systems to be compared.
  • protein libraries of every prokaryotic or eukaryotic cell (or their cell cultures), every cell culture of human or animal cells, every human or animal organ or every conceivable plant can be created, which can be searched against substrate libraries for enzyme activities. Both known and unknown substances or mixtures can serve as substrates.
  • the invention has great potential for application and extends from the biochemistry of microorganisms to the biochemistry of plants to basic clinical research. Another area of application important for life science research is the possibility of being able to trace metabolic pathways, since not only unknown enzymes are detected and identified, but also new unknown metabolites can be recognized at the same time.
  • FIG. 2 dependence of the relative signal intensity of Lys-bradykinin (signal intensity of Lys-bradykinin:
  • FIG. 3 detection of urotensin-generating activity from a protein fraction mass spectrum of a reaction mixture of an incubation of a protein fraction bound to particles from kidney tissue with an urotensin substrate after an incubation time of 4 h.
  • External standard Saralasin.
  • Pig kidneys are used for the production of protein extracts. Immediately after being removed from the slaughterhouse, they are cooled in physiological saline (0.9% NaCI solution) until further processing. The kidney tissue is cut (at temperatures from 4 to 6 ° C) into pieces of approx. 1 cm 3 , filled into pre-cooled lyophilization tubes, frozen in liquid nitrogen and stored at -80 ° C overnight. The tissue pieces are completely dried in a lyophilization plant for about a week 1 week. The anhydrous pieces of tissue are then pulverized with a grain mill at the finest level. 10 g of the powder are dissolved in 200 ml of buffer (20 mM Hepes buffer in 50% distilled water, 50% glycerol. 10 "4 M dithiothreitol, pH 7.4). A homogenizer is used for this.
  • two different goals can be pursued, namely 1.) the targeted purification of a single enzyme or 2.) high-resolution fractionation while maintaining a large number of activities.
  • the chromatographic purification steps are combined in such a way that protein fractions only leave the chromatography system after the last purification step and are collected in collecting vessels (deep-well plates). Aliquots of the fractions are aliquoted for the reserve systems.
  • the coupling of the proteins to particles takes place, for example, according to Hermanson et al. (Academic Press, 1992), 53-56). In this regulation, bromocyanine-activated particles (from Amersham) are used for the binding of proteins to particles. The particles are dissolved in four times their own amount of 1 mM HCl (pH 2-3). The swollen gel is rinsed several times with 100 times its own volume with HCI. Then it is washed with water 5-10 times its own volume.
  • coupling buffer A 0.1 M NaHCO 3 buffer in 0.5 M NaCl, pH 8.3
  • coupling buffer A 5-10 mg Protein per ml of gel
  • Unbound protein is removed by rinsing with coupling buffer A.
  • unbound active groups of the gel are blocked with glycine.
  • the gel is incubated with 2 to 3 times the gel volume of coupling buffer B (0.2 M glycine buffer in coupling buffer A) at room temperature for two hours or at 4 ° C. overnight with constant swirling. It is alternated 3 times with Tris-HCI Buffer, pH 8.0 and Na acetate buffer, pH 4 rinsed.
  • the finished gel is stored at 2-8 ° C in 0.02% sodium azide solution.
  • the individual fraction In order to be able to make statements about the enzymatic activities of individual fractions, the individual fraction must be incubated with a substrate that is chemically changed by the activity to be detected. (In the example in FIG. 1a: proteolytic cleavage of kallikrein substrate by kallikrein to the reaction product lys-bradykinin). The activity can be verified by mass spectrometric detection of the resulting reaction product.
  • ESI electrospray
  • MALDI-MS the sample to be analyzed must be largely free of salts and other low molecular weight substances.
  • This condition can be met if a) the components of the fractions are previously bound to particles and freed of salts and b) the substrates are dissolved in high-purity water and the solution is incubated. The reaction products can then be analyzed directly by mass spectrometry without further sample preparation.
  • a kallikrein preparation was bound to particles and incubated with kallikrein substrate. After a defined time interval (30 min), an aliquot of the reaction solution (1.5 ⁇ l) is removed and pipetted onto the surface of a metal anchor plate (Bruker). Then 1.5 ⁇ l of the UV-absorbing desorption matrix hydroxy- ⁇ -cyano-cinnamic acid (saturated in ACN / H 2 O (50/50) with 0.1% trifluoroacetic acid) are added. After the sample has crystallized on the sample plate, it is brought into the mass spectrometer (Reflex III, Bruker) via a vacuum lock.
  • Reflex III Reflex III
  • the desorption of the sample ions takes place through the pulse of a nitrogen laser beam (VSL-337 ND, Laser Science, wavelength 337 nm, pulse duration 4 ns, energy 10 6 -10 7 W / cm 2 , diameter of the radiation field 50 - 100 ⁇ m).
  • the acceleration voltage U acc in the electrostatic field was 10 kV
  • the pressure in the system was 6-8 x 10 "7 mbar.
  • the mass spectra are recorded with a LeCroy 9400 transient recorder. 200 spectra are summed up during the detection. As a further example, the mass spectrometric detection of an activity generating urotens is shown.
  • a peptide (urotensin-generating enzyme substrate) with the sequence RIKKPYKKRGPPSECFWKY (2430.3 Da) is dissolved in HPLC-pure water in a concentration of 10 "5 mol / l.
  • GPPSECFWKY (here called "urotensin”).
  • a signal of mass 1214.33 Da can then be seen in the mass spectrum (see FIG. 3).
  • This signal corresponds to the simply protonated ion of a urotensin-like peptide
  • An aliquot of the proteins bound to particles was removed and incubated with the urotensin-generating enzyme-substrate solution After an defined time interval, an aliquot of the reaction solution (1.5 ⁇ l) was taken and pipetted onto the surface of a metal anchor plate (from Bruker) Then 1.5 ⁇ l of the UV-absorbing desorption matrix hydroxy- ⁇ -cyano-cinnamic acid (saturated in ACN / H 2 O (50/50) with 0.1% trifluoroacetic acid) are added Allization of the sample on the sample plate is brought into the mass spectrometer via a vacuum lock and the sample is analyzed as indicated above.
  • the relative enzyme activity is defined here as the amount of the reaction product that occurs per unit of time:
  • rel.lnt f i relative intensity of the analyte, based on the internal standard, based on the incubation period t ⁇
  • the signal of the MALDI-MS depends on the location of the laser on the crystallized sample. Due to the non-uniform alignment of the crystals to the laser, the radiation energy that the area hit by the laser absorbs varies. As a result, different intensities of the signal are le detected due to the different intensity of the absorbed radiation energy. As a result, there is often no direct correlation between signal intensity and analyte concentration in individual spectra.
  • the problem is avoided by the following measures:
  • the sample is measured by an automated process, in which the laser of the MALDI-MS shoots at the sample in accordance with prescribed trajectories in order to find areas that guarantee an optimal yield of signal intensity.
  • the laser intensity is automatically optimized. In this example, a maximum of 60 laser pulses were applied to the sample per area. If no signal was detected before the 60 pulses were reached, a new area on the sample is automatically searched for. 200 spectra were summed and averaged for a total spectrum.
  • an external standard of the matrix Saralasin (10 "5 M to 10 " 10 M) is added.
  • FIG. 2 shows the ratio of the relative, ie, the signal intensity of lys-bradykinin based on the internal standard to the concentration of the analyte.

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Abstract

L'invention concerne une technologie de plate-forme intégrée, selon laquelle on nettoie des extraits de biopolymère (de préférence des extraits de protéine) issus de sources biologiques (par ex. de cultures cellulaires de procaryotes et d'eucaryotes, de tissus et de cellules humains ou animaux, ou de cellules végétales), on conserve des échantillons de différents niveaux de nettoyage dans un système de réserve (sous forme de bibliothèque de protéines) et on extrait des parties aliquotes de ce système de réserve pour vérifier des activités catalytiques précises. Les échantillons qui présentent cette activité déterminée sont ensuite identifiés par des méthodes analytiques, notamment par spectrométrie de masse ou par des procédés de chimie des protéines. Les différentes étapes du procédé peuvent être réalisées de manière manuelle ou automatique.
PCT/DE2003/003109 2002-09-19 2003-09-15 Procede pour identifier des activites enzymatiques dans des extraits de proteine quelconques Ceased WO2004029286A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
DE10393828T DE10393828D2 (de) 2002-09-19 2003-09-15 Verfahren zur Erkennung von Enzymaktivitäten von beliebigen Proteinextrakten
AU2003273738A AU2003273738A1 (en) 2002-09-19 2003-09-15 Method for identifying the enzyme activities of any protein extract

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Application Number Priority Date Filing Date Title
DE10243530 2002-09-19
DE10243530.8 2002-09-19

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WO2004029286A2 true WO2004029286A2 (fr) 2004-04-08
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006049822A1 (de) * 2006-10-19 2008-04-24 Charité-Universitätsmedizin Berlin Verfahren zur Identifikation von Urotensin-2Konvertase-Inhibitoren
US8142996B2 (en) 2006-02-28 2012-03-27 Bruker Daltonik Gmbh Mass spectrometric determination of blood enzyme activity

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10027794A1 (de) * 2000-06-07 2001-12-13 Basf Ag Verfahren zur Analyse Enzym-katalysierter Umsetzungen mit MALDI-TOF-Massenspektrometrie

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8142996B2 (en) 2006-02-28 2012-03-27 Bruker Daltonik Gmbh Mass spectrometric determination of blood enzyme activity
DE102006009083B4 (de) * 2006-02-28 2018-05-30 Bruker Daltonik Gmbh Massenspektrometrische Bestimmung der Aktivität von Blutenzymen
DE102006049822A1 (de) * 2006-10-19 2008-04-24 Charité-Universitätsmedizin Berlin Verfahren zur Identifikation von Urotensin-2Konvertase-Inhibitoren
DE102006049822B4 (de) * 2006-10-19 2009-01-02 Charité-Universitätsmedizin Berlin Verfahren zur Identifikation von Urotensin-2-Konvertase-Inhibitoren

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WO2004029286A3 (fr) 2004-07-08
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