WO2001094910A2 - Method for the qualitative and quantitative analysis of complex mixtures of chemical compounds, using maldi-tof mass spectrometry - Google Patents

Abstract

The invention relates to a method for the qualitative and quantitative analysis of complex mixtures of chemical compounds, using MALDI-TOF mass spectrometry, preferably in the presence of an internal standard on a special supporting material. The inventive method can be used in an advantageous manner to carry out biochemical flow analyses.

Description

Process for the qualitative and quantitative analysis of complex mixtures of chemical compounds with MALDI-TOF mass spectrometry

description

The present invention relates to a method for the qualitative and quantitative analysis of complex mixtures of chemical compounds with the aid of MALDI-TOF mass spectrometry, preferably in the presence of an internal standard on a special carrier material.

A precise knowledge of the biochemical flows of the various chemical substances or compounds within the organisms is advantageous for the optimization of fermentative processes. These so-called bioflux studies require the analysis of a complex mixture of chemical compounds inside and outside the cells. For these bioflux studies, that is to say the determination of substance flows inside and outside the organism, a wide variety of separation methods such as thin-layer chromatography (= DC), high-pressure liquid chromatography (= HPLC), gas chromatography (= GC), enzyme assays are usually used; MR and other analytical methods are used. These methods are time-consuming and allow only a low sample throughput.

Complex mixtures of chemical compounds or substances also need to be analyzed in combinatorial chemistry. These mixtures arise, for example, in the so-called classic split and combine method (= synthesis of mixtures in a reaction vessel). The analysis methods mentioned above are also used for analysis in this case.

The above-mentioned analysis methods are also used when screening for new enzymatic reactions. This screening largely depends on chance. In such a screening, a large number of organisms have to be screened for the desired enzymatic activity until the desired enzyme activity is found. As a rule, complex mixtures must also be analyzed here.

For the bioflux studies, the combinatorial syntheses as well as for the screening for enzyme activities, quick, simple, highly sensitive and highly specific analysis procedures are required. The quick and easy identification of the chemical or enzymatic reaction or Compounds arising from the fermentative process and / or, if appropriate, the decrease in the starting material used and / or in a fermentative process the decrease in the carbon source used such as glucose or sucrose or the nitrogen source used such as protein hydrolyzates and the formation of new chemical compounds from these are important. As mentioned above, separation processes such as thin-layer chromatography (= DC), high-pressure liquid chromatography (= HPLC) or gas chromatography (= GC) are generally used to analyze the products. Methods such as MR, which can be used after working up, for example by salt precipitation and / or subsequent chromatography, can also be used for the analysis. These methods are time-consuming and allow only a limited sample throughput, so that such analysis methods are not used for the so-called high-throughput screening (= HTS), in which the desired reaction is first screened for. The advantage of these methods is that they provide information both about the products of the reaction and, if appropriate, about the decrease in the starting material.

In order to enable a higher sample throughput in the HTS, indirect, easily measurable methods such as color reactions in the visible range, turbidity measurements, fluorescence, conductivity measurements etc. are often used. In principle, these are very sensitive, but they are also prone to failure. The main disadvantage here is that many false positive samples are analyzed using this procedure and, since these are indirect detection methods, no information about the product and / or starting material is available. In addition, these methods generally provide little information about the flow distributions. In order to be able to exclude these false positives in the further procedure, further analysis methods such as DC, HPLC or GC are generally used after the first screening. Again, this is very time consuming.

In general, it can be said that the improvement in the sensitivity and the informative value of the detection methods with regard to the reaction products leads to a slowdown in the speed of a test.

For so-called bioflux studies, in which the metabolic substance flows are determined, such methods, in particular NMR, are used despite their high expenditure of time due to their meaningfulness about the most diverse chemical compounds that arise through the metabolism of the organisms. However, to simplify these studies and thus determine the metabolic material flows would be a fast, highly sensitive and highly specific analytical method desirable.

MALDI-TOF-MS (= matrix-assisted laser desorption ionization with time-of-flight mass spectrometry) is a quick and easy 5 method that is widely used for the analysis of large, non-volatile biomolecules such as peptides, proteins, oligonucleotides and oligosaccharides or other polymers are used. High molecular weight materials such as coal tar, humic acid, fulvic acid or kerogens were also analyzed with MALDI 10 (Zenobie and Knochenmus, Mass Spec. Rev., 1998, 17, 337-366).

A problem with the MALDI-MS is the quantification of the measurement results, this is because the signal intensity in

15 depends to a large extent on the homogeneity of the sample applied and the radiation density of the laser (Ens et al., Rapid Commun. Mass Spectrom, 5, 1991: 117-123), the intensity increasing exponentially with increasing laser energy (Ens et al., Rapid Commun. Mass Spectrom, 5, 1991: 117-123). Will the

If the signal intensity is too high, the signal at the detector may be saturated, which also precludes quantification. In addition to these physical-technical problems, there are other reasons that make quantitative analysis of MALDI measurements difficult. So can

For example, fragments of the ions or molecular adducts sought occur. The biggest problem with quantitative MALDI-MS, however, is the inhomogeneity of the samples. The quality of a MALDI spectrum depends to a large extent on the morphology of the sample examined (Garden & Sweedler, Anal. Chem. 72,

30 2000: 30-36). Significant differences regarding the occurrence of signals, the intensity, the resolution and the mass accuracy can be observed as soon as a MALDI sample is examined at different locations (Cohen & Chait, Anal. Chem., 68, 1996: 31-37 ; Strupat et al., Int. J. Mass Spectrom. Ion

35 Processes, 111, 1991: 89-102; Amado et al. , Rapid Commun. Mass Spectrom., 11, 1997: 1347-1352). These inhomogeneities are due to an uneven distribution of matrix and analyte on the sample target, which is caused by the uneven crystallization behavior of these two components. To this inhomogeneity

To eliminate or minimize 40 actions - which is equivalent to the formation of a microcrystalline, homogeneous sample topology - a number of proposals have already been developed. These include, for example, the use of comrices (Gusev et al., Anal. Chem., 67, 1995: 1034-1041), multi-layer

45 preparations, the use of solvent mixtures and electrospray preparations (Hensel et al., Rapid Commun. Mass Spectrom., - 11, 1997: 1785-1793) (a compilation of these Experiments can be found in the Garden & Sweedler, Anal reference. Chem. 72, 2000: 30-36). However, all of these approaches have limitations and are therefore not widely applicable. Therefore, quantification is still a problem.

For MALDI (= matrix-assisted laser desorption ionization), the samples are usually applied in a thin layer to a metal surface and then irradiated with a pulsed laser. By focusing the emitted ions, the resolution of the mass spectra in the lower mass range can be increased to about 5000 D.

Duncan et al. (Rapid Communications in Mass Spectrometry, Vol. 7, 1993: 1090-1094) describes the analysis of the low molecular polar compounds 3, 4-dihydroxyphenylalanine, acetylcholine and the peptide Ac-Ser-Ile-Arg-His-Tyr-NH ₂ with the help of MALDI in the presence of the corresponding compounds labeled via ¹³ C or ² H or via a similar peptide as an internal standard.

By Goheen et al. (J. Mass Spec., Vol. 32, 1997: 820-828) describes the use of MALDI-TOF-MS for the analysis of the following low-molecular compounds:

Citric acid, propionic acid, butyric acid, oxalic acid and stearic acid, ethylenediaminetetraacetic acid (= EDTA), N- (2-hydroxyethyl) - ethylenediaminetriacetic acid (= HEDTA), ethylenediamine-N, N'-diesiacetic acid (= EDDA) and nitrilotriacetic acid (= NTA) and sulfate -, nitrate, nitrite and phosphate salts. 2, 5-Dihydroxybenzoic acid is used as the matrix in all experiments.

A disadvantage of both methods is that they are only suitable for the measurement of pure substances. This problem is discussed in the discussion by Duncan et al. addressed, there is proposed to overcome this difficulty, the cleaning of the test samples.

Overall, however, the MALDI-TOF-MS is an interesting simple and rapid method which provides specific information about the analyzed compounds, so that the use of the MALDI-TOF-MS is desirable for measuring chemical or enzymatic reactions or fermentative processes with low molecular weight substances would. In particular, their use in high-throughput screening and in bioflux analysis would be desirable.

The task was therefore to develop a method for the qualitative and quantitative analysis of complex mixtures of chemical compounds using MALDI-TOF mass spectrometry. This object was achieved by a method for the qualitative and quantitative analysis of complex mixtures of chemical compounds, characterized in that the compounds are analyzed using MALDI-TOF mass spectrometry. These complex mixtures can arise in a chemical or enzymatic reaction or in a fermentative process.

Complex mixtures of chemical compounds are mixtures which contain at least two, preferably at least three, particularly preferably at least four, very particularly preferably at least five chemical compounds which are to be identified in the analysis using MALDI-TOF mass spectrometry and, if appropriate, quantified. Also particularly preferred complex mixtures of chemical compounds are mixtures that arise before, during or after a fermentative process inside and outside the organisms. The complex mixture of chemical compounds can contain polymeric compounds (= substances) such as proteins such as enzymes, receptors etc., polysaccharides such as glycoproteins, glycolipids etc., polynucleotides such as DNA or RNA and / or non-polymeric compounds. These chemical compounds can be analyzed using the method according to the invention alone or in combination with the non-polymeric compounds. Only the non-polymeric compounds are preferably analyzed, particularly preferably the non-polymeric, organic compounds.

Non-polymeric, organic compounds in the process according to the invention are compounds which are above all not peptides, proteins, oligo- or polynucleotides or oligo- or polysaccharides or artificial or natural polymers. These non-polymeric organic compounds (= low molecular weight compounds) have a molar mass of less than 4000 D (= dalton), advantageously less than 3000 D, preferably less than 1000 D, particularly preferably less than 800 D, very particularly preferably less than 600 D. As such low molecular weight organic compounds are, for example, sugars such as monosaccharides such as glucose or fructose, disaccharides such as sucrose or maltose, tri or tetra saccharides, amino acids such as natural amino acids such as lysine, threonine, alanine, phenylalanine, isoleucine, arginine, aspartic acid, glutamic acid to name a few or artificial amino acids, di- or tripeptides, lower carboxylic acids such as monocarboxylic acids such as acetic acid, propionic acid or dicarboxylic acids such as oxaloacetic acid or tricarboxylic acids such as citric acid, terpenes, steroids, carotenoids, vitamins, antibiotics such as nucleoside antibiotics, polyketides, ß- Lactams such as penicillins or chepalosporins or fats acids mentioned as examples. Chemical reactions in the sense of the invention are understood to mean all chemical reactions of organic and inorganic chemistry, preferably organic chemistry.

Such chemical reactions in which complex mixtures occur occur, for example, in combinatorial chemistry. These mixtures are created, for example, in combinatorial chemistry using the so-called classic split and co-bine method (= synthesis of mixtures in a reaction vessel). Such reaction solutions or mixtures can be analyzed using the method according to the invention.

Enzymatic reactions are to be understood as enzyme-catalyzed reactions with whole cells, which can be of plant, animal, bacterial or fungal origin, and yeast cells are also suitable. The enzymatic conversion can take place with resting, growing, permeabilized or immobilized cells or microorganisms. Enzymes are also suitable for the enzyme-catalyzed implementation. These enzymes can still be contained in the permeabilized cells or microorganisms or they can also be present in so-called crude extracts. Purified or purified enzymes are used for a faster and generally also by-product-free conversion, which can be used in the conversion as free or immobilized enzymes. The reaction is preferably carried out with free, purified, purified or immobilized enzymes.

Enzymes of enzyme classes 1 to 6 (International Union of Biochemistry and Molecular Biology = IUB) are advantageously used in the process according to the invention, preference is given to enzyme classes 1 to 4, particularly preferably enzyme class 3 such as classes 3.1 (reaction with ester bonds), 3.2 (glycosidases ), 3.3 (reaction with ether bonds), 3.7 (reaction with carbon-carbon bonds), 3.11 (reaction with carbon-phosphorus bonds), enzymes such as lipases, esterases or phosphatases such as phytases are very particularly preferred. Further advantageous enzymes can be found in enzyme class 6.

Fermentative processes in the sense of the invention are processes in which microorganisms such as plant or animal cells, bacteria, fungi, yeasts, algae or ciliates prefer bacteria, fungi or yeasts in the presence of a carbon source, a nitrogen source, a phosphate source, salts such as alkali and alkaline earth metal salts and possibly vitamins and trace elements in a manner known to the person skilled in the art at temperatures, depending on the organism, from 10 ° C. to 1-10 ° C., with pH control or without pH control, with or be attracted without fumigation. In these fermentative processes, a number of chemical compounds are produced by the metabolism of the organisms (both the anabolic and catabolic metabolism) that are present inside or outside the cell. To optimize such fermentative

Processes, an exact knowledge of the biochemical flows of the different chemical substances or compounds inside and outside the organisms is an advantage. That means a precise knowledge of the metabolism of the organisms is required. This knowledge is gained through so-called bioflux analyzes or studies. These bioflux studies require the analysis of a complex mixture of chemical compounds inside and outside the cells. This is possible with the method according to the invention in a quick, simple and specific way. With the help of MALDI-TOF mass spectrometry, chemical compounds such as amino acids such as lysine or alanine or sugar and sugar degradation products such as glucose or sucrose or lipids can be easily and highly specifically detected qualitatively and quantitatively. Through these analyzes, the connections and thus the internal so-called pools of the various connections present in the cell can be precisely determined. Flow distributions and reactions can advantageously be determined or determined via measured intensity distributions of the different isotopomers or groups of isotopomers. In this way, possible bottlenecks in metabolism, that is to say enzymes which, for example, have too little activity in the metabolism, can be recognized. These bottlenecks can then be optimized using genetic engineering methods such as overexpression of the associated genes.

Mixtures of chemical compounds in the sense of the method according to the invention are also present, for example, in food samples, pharmaceutical samples or environmental samples, in which certain ingredients, ie certain chemical compounds, are to be detected, for example the contamination of food with pesticides or the contamination of soils with environmental toxins. Mixtures of chemical compounds from plants such as plant extracts or from animals or humans, for example blood samples or organ samples, can also be analyzed in the method according to the invention. For example, blood glucose can be measured. The method according to the invention is therefore suitable for trace analysis, for the quantification of substances and for diagnostics. The method according to the invention is particularly suitable for the bioflux analysis of fermentation samples, environmental samples and food samples.

The measurement error in the determination of the chemical compounds in the method according to the invention is below 10%, preferably below 7.5%, particularly preferably less than 5%.

In addition to the analysis of chemical compounds, the method according to the invention can also advantageously be used to monitor enzyme reactions, ie enzyme kinetics can be set up. Furthermore, the K _m value, the V _max value, the selectivity of the enzyme, the yield of the reaction can be determined and the effect of inhibitors on the enzyme reaction can be monitored in vivo and in vitro. Possible reaction parameters such as temperature or pH for the enzyme-catalyzed reaction can also be examined.

A cleaning of the reaction or fermentation solution is not necessary for the method according to the invention before analysis with MALDI-TOF mass spectrometry. The reaction can be measured directly. This applies in particular to complex sample mixtures on fermentation broths as part of bioflux analyzes. Also, no pure substances have to be used for the reaction, although this is of course possible.

Chemical compounds which are poorly or not at all detectable in MALDI-TOF-MS can advantageously be derivatized before the analysis (see examples) and thus finally analyzed. The derivatization can take place before or after the chemical or enzymatic reaction or the fermentative

Processes take place. A derivatization is particularly advantageous in cases in which hydrophilic groups are introduced into hydrophobic or volatile compounds, such as esters, amides, lactones, aldehydes, ketones, alcohols, etc., which advantageously also have an ionizable functionality. Examples of such derivatizations are reactions of aldehydes or ketones to oximes, hydrazone or their derivatives or alcohols to esters, for example with symmetrical or mixed anhydrides. This extends the detection spectrum of the method significantly. The derivatization after the chemical or enzymatic reaction or the fermentative process has taken place enables the direct measurement of the original substrate of the reaction or the process. By using the MALDI-TOF-MS, substances that do not contain a chromophore can also be analyzed. This is a considerable advantage over other methods, since for example conventional detection methods, for example by means of visual recognition, as a rule artificial substrates which, for example, contain a chromophore, must be used. When optimized for this reaction, these may not lead to one

Improvement of the desired natural enzymatic implementation, since the optimization of this artificial reaction does not reflects relationships.

An internal standard is advantageously added in the method according to the invention for analyzing the chemical or enzymatic reaction or the fermentative process. This internal

Standard advantageously enables the quantification of the low molecular weight compounds in the reaction solution. This standard can be added to the chemical or enzymatic reaction or the fermentative process before the start, during or after the completion of the chemical or enzymatic reaction or the fermentative process. The internal standard is advantageously added to the fermentative process before the start, for example in the form of labeled carbon or nitrogen sources. These carbon or nitrogen sources serve as a substrate, i.e. the internal standard in this case serves as a substrate in the metabolism of the organisms. In this case, the internal standard is a so-called "tracer substance", with the aid of which a marking pattern of the resulting compounds in the organism can be created by anabolic and / or catabolic reactions in the metabolism. With the aid of this marking pattern, the specific metabolism of an organism, for example a microorganism such as a bacterium or a fungus or a plant or an animal or human, can be elucidated. In this case, the marking of the internal standard is transferred to the metabolites of the metabolism. In this way, the metabolic flows in the organism can be studied and identified. In this way, intermediate products in the metabolism can be detected. This also applies to intermediate products of chemical or enzymatic reactions. In the foreground of these bioflux analyzes is the quantification of the isotopomer distribution of the different labeled intermediate products of the metabolism. The intermediate products can also be quantified in addition to the qualitative detection. To this end, other differently marked internal standards can be added after the end of the reaction or fermentation. The intermediate products are ultimately also to be understood as products of the starting material used at the start of the reaction. Accordingly, the method according to the invention can also be used to monitor or analyze enzyme reactions which catalyze the successive reaction. These can be catalyzed by one or more enzymes. By-products can also be analyzed.

Labeled substances are advantageously used as the internal standard, which are advantageously implemented in the metabolism in an organism or which are implemented in a chemical or enzymatic reaction. But there are. also chemical compounds similar to the starting materials and / or products in principle internal standard suitable. Similar chemical compounds of this type are, for example, so-called compounds of a homologous series, the members of which differ only by, for example, an additional methylene group. As an internal standard, at least one isotope is preferably selected from the group ² H, ¹³ C, ¹⁵ N, ¹⁷ 0, ¹⁸ 0, ³³ S, ³ S, ³⁶ S, ³⁵ C1, ³⁷ C1, ²⁹ Si, ³⁰ Si, ⁷⁴ Se or their mixtures of labeled starting material, product, where starting material and / or product can be an organic compound which can be used as a carbon and / or nitrogen source or in any other way in the organism, or uses a further labeled chemical compound. The chemical compound is advantageously selected by at least one isotope from the 2 _H 1 ³ C, ISN, ¹⁷ 0, 18 _0f 33 _{S /} 34g, 36 _S 35 _C1 _, 37 _{C1 |} 29 _Sij 30 _Si

⁷⁴ Se or their mixtures marked. For cost reasons and for reasons of accessibility, ² H, ¹⁵ N or ¹³ C is preferably used as the isotope. Examples of such labeled substances are L-α ¹⁵ N-lysine, Lll ³ C-alanine and I- ¹³ C-glucose. These internal standards do not need to be complete for analysis, that is, they must be fully marked. A partial marking is completely sufficient. A distance of the marking from greater than or equal to 3 daltons to less than or equal to 10 daltons is advantageously sufficient. However, a measurement is also possible in principle under 3 daltons or over 10 daltons, although overlapping with the isotopes of the analyte may occur at small distances or isotope effects may occur at larger distances. This complicates the measurements but does not make them impossible. In the case of a marked internal standard, it is also advantageous to choose a substance which has the highest possible homology, that is to say structural similarity, to the chemical compound to be measured. The higher the structural similarity, the better the measurement results and the more accurately the connection can be quantified.

For the method according to the invention and in particular for the quantification of the chemical compounds present in the reaction, such as starting materials, products, intermediate products or by-products, it is advantageous to use the internal standard in a favorable ratio to the compound to be measured, such as starting material, product, intermediate product or by-product , Ratios of analyte (= connection to be determined) to internal standard greater than 1:15 do not improve the measurement results, but are possible in principle. A ratio of analyte to internal standard is advantageously set in a range from 0.1 to 15, preferably in a range from 0.5 to 10, particularly preferably in a range from 1 to 5, very particularly preferably a ratio of 1: 1 , If flow distributions and reversibility of reactions are determined in the bioflux analysis, the internal standard is advantageously added in such amounts that the resulting intensity distributions of the analytes are advantageously in a range from 0.1 to 10, preferably in a range from 0.2 to 5, particularly preferably in a range from 0.5 to 2, very particularly preferably in a ratio of 1: 1.

The method according to the invention enables the measurement of non-polymeric compounds in an advantageous concentration from 1 μM to 500 mM, preferably from 10 μM to 100 mM.

The analysis samples are advantageously concentrated in the smallest possible space or on the smallest possible diameter in order to achieve a further improvement in the measurement point resolution or measurement accuracy.

The reaction samples in the method according to the invention can be prepared manually or advantageously automatically using conventional laboratory robots. The analysis with MALDI-TOF-MS can also be carried out manually or advantageously automatically. By automating the method according to the invention, MALDI mass spectrometry can advantageously be used for the rapid screening of enzyme-catalyzed reactions in so-called high-throughput screening. The MALDI-TOF-MS is characterized by a high sensitivity with the lowest sample consumption. For example, rapid screening for metabolic activities in microtiter plates can be performed. With _. The method can be used as rapid new enzyme activities and new mutants of known enzymes after a mutagenesis, for example via a classic mutagenesis with chemical agents such as NTG, radiation such as UV radiation or X-rays or after a so-called site-directed mutagenesis, PCR mutagenesis or the so-called gene shuffling can be found. This is reflected in a change in the bioflux analysis compared to the corresponding starting organism.

Carrier materials with a roughness value or a roughness number of R _z greater than 1, preferably greater than 2, particularly preferably greater than 3, very particularly preferably greater than 4 are advantageously used for the method according to the invention. R _{z means} the average roughness depth (μm) as the arithmetic mean of the individual roughness depths of five adjacent individual measuring sections. The roughness depth is determined according to DIN 4768. These carrier materials are polished, coated or vapor-coated carrier materials or polished and coated or polished and vapor-coated carrier materials. The straps are made of a material selected from the group of glass, ceramics, quartz, metal, stone, plastic, rubber, silicon, germanium or porcelain. The material preferably consists of metal or glass.

5 To determine further analysis data, the analysis in the method according to the invention can additionally be carried out with the aid of the metastable decay after the ionization or the shock-induced decay. As a result, further mass data can be obtained which facilitate or enable the identification of the starting materials, products, by-products or intermediate products present and advantageously also expand the determination of the isotopomer distribution.

In the method according to the invention, the dynamics 15 of the marking pattern and the concentration of the chemical are advantageous

Compounds specifically measured in the enzymatic reaction or in the fermentative processes. As a result, enzyme kinetics or cell-internal or cell-external substance pools or other kinetic parameters can be analyzed. In this way, K _m and V _max 20 of an enzyme can be determined.

The invention is illustrated by the following examples:

Examples

25

The following examples show the quantification of metabolites of metabolism (= chemical compounds) during the fermentation of the lysine-producing microorganism Corynebacterium glutamicum ATCC 21253. Lysine production with micro-

30 organisms is one of the major biotechnological fermentation processes (Jetten and Sinskey, Crit. Rev. Biotechnol., 15, 1995: 73-103).

The following chemicals were used for the experiments: 35 95 to 99% L-α ¹⁵ N-lysine, 99% Ll- ⁱ C-alanine and 99% 1- ¹³ C-glucose from Euroisotop (Gif-sur-Yvette, France), Sugar molasses from Südzucker AG (Mannheim / Ochsenfurt, Germany) with a dry weight of 84% and a polarity of 51.4 and a pH of 7.4. All other chemicals come from Sigma-40 Aldrich (Deisenhofen, Germany).

The strain Corynebacterium glutamicum ATCC 21253 was on PMB medium (Vallino and Stephanopolus, Biotechnol. Bioeng., 41, 1993: 633-646) either with 18 g / 1 glucose as carbon source 45 (= mineral medium) or with 20 g / 1 Molasses (= complex medium) attracted. Washed with 0.9% NaCl, preformed on LBG5 medium (Vallino and Stephanopolus, Biotechnol. Bioeng., 41, 1993: 633-646) Raised cells were resuspended in 0.9% NaCl and taken as an inoculum. The experiments with molasses were carried out in 100 ml shake flasks. The cultivation in mineral medium was carried out in 100 ml bioreactors (Meredos, Nörten-Hardenberg, Germany). The cultivation was carried out at pH 7.0 and 30 ° C.

The biomass was determined gravimetrically at an optical density of 660 nm (Ultrospec 2000, Amersham Pharmacia Biotech Europe, Freiburg, Germany) and as a dry biomass. The correlation _factor between optical density and _{dry biomass} is 0.29 * OD ₆ 6 _0nm = _{dry biomass} in the unit g / 1. The amino acids were duplicated by HPLC using a derivatization kit (ACCQ-TAG, Waters, Milford, USA), an ACCQ-TAG column (Waters, Milford, USA) with a gradient of ACCQ-EluentA (Waters, Milford , USA) and acetonitrile / water (60:40) at a flow of 0.8 ml / min, 37 ° C and UV detection at 250 nm. The measurement errors in the lysine and alanine determination were 1, 6 or 2.5%. Glucose was determined enzymatically in duplicate (Sigma-Aldrich, Deisenhofen, Germany). The measurement error in the determinations was 1.7%.

The MALDI-TOF-MS measurements were carried out using a Bruker Reflex III (Bruker-Franzen, Bremen, Germany) with a 337 nm nitrogen laser and a right-angled 384-well scout target for sample collection. The device has an 8 bit digitizer. Various compounds were used as the matrix. 2.5-Dihydroxybenzoic acid (= DHB) was dissolved in methanol (200 mg / ml) and 5-aminoquinoline (= 5AQ) in deionized water (75 mg / ml). The following matrixes were created in

Saturation in a mixture of acetonitrile: water: trifluoroacetic acid (70:30:: 0.1) dissolved: sinapic acid (= SA), trihydroxyacetophenone (= TACP) and 4-hydroxy-azobenzene-2-carboxylic acid (= HABA ). In the case of the SA, HABA, TACP and 5AQ matrixes, 0.3 μl of matrix were pipetted into the middle of a measuring point and air-dried. Then 0.3 μl of the samples to be measured were pipetted onto each measuring point. The best results were achieved with DHB as a matrix according to the following scheme: A thin film of the matrix solution was applied to a metal support by slowly moving the pipette over neighboring measuring points. Due to the surface tension, the solution was automatically transferred from the pipette tip to the metal carrier, where it formed a thin film that dried within seconds. The samples (0.3 μl) were then pipetted into the middle of the measuring points. The metal carrier plate treated in this way was dried in air for about 10 minutes and then introduced directly into the vacuum chamber of the MALDI measuring device. Each measuring point was Shots analyzed from 5 different positions. Five measuring points were produced and analyzed for each sample to be measured. In some cases the shots gave overdriven or too weak signals (<10 counts per shot) due to the heterogeneous crystallization. Due to the fact that the integration of the surface units of such shots would lead to errors, they were excluded from the analysis. The direct use of the fermentation broth supernatant usually resulted in thick and dense crystals with poor signal efficiency even for matrix ions. This effect is probably due to the high salt content of the fermentation broths, which is known to reduce the sensitivity of the MALDI analyzes (Karas et al., Biomed. Environ. Mass Spectrom. 18, 1989: 841-843). For example, a 1: 5 dilution of the samples with, for example, deionized water before the MALDI analysis improves the results drastically.

Example 1: Detection of the metabolites with MALDI-TOF-MS

Lysine, alanine, glucose and sucrose were determined with positive reflector mode. Figure la shows the spectrum of a 2.5 mM mixture of these 4 compounds with a DHB matrix in deionized water. As a control, FIG. 1b shows pure deionized water. The ions formed could be clearly assigned to the individual analytes by individual analysis of the analytes and comparison with the spectra of more stable labeled isotopes of the analytes

(Results not shown). Alanine and lysine mainly form [M + H] ⁺ ions at m / z 90 and m / z 147. Adducts of the two compounds with sodium and potassium ions were detected with the corresponding mass at a lower intensity. Glucose (m / z 203, 219) and sucrose (m / z 365, 381) gave clear signals as [M + Na] ⁺ and [M + K] ⁺ adducts. No protonated ions of these sugars were observed. Lysine in particular gave strong signals. This is probably due to the two amino groups of the molecule, which can be easily charged by the ionization in positive mode. The control with pure DHB showed no isobaric overlays between analyte and matrix. The main ions detected with DHB were: [DHB-H ₂ 0] + at m / z 136, [DHB-H ₂ 0 + H] ⁺ at m / z 137, [DHB] ⁺ at m / z 154, [DHB + H] ⁺ at m / z 155, [DHB-H ₂ 0 + Na] ⁺ at m / z 159, [DHB-H + Na] ⁺ at m / z 176, [DHB + Na] ⁺ at m / z 177, [DHB-H + 2Na] ⁺ at m / z 199 and possibly the conversion product of the dimeric ions at m / z 273, 304 and 375. The ratio of the protonated ions to the ions with sodium and potassium depends depending on the sample and analysis conditions. The amount of ions with potassium and sodium increased significantly when the buffer with these ions was present in the samples. Usually the number of different ions increased with increasing laser energy, which is used for ionization. was applied. All four compounds could be determined simultaneously in one sample. The intensity of the individual compound varies, possibly due to its heterogeneous distribution in the matrix. The signal intensity of glucose and alanine can be increased by higher laser energies. Additional m + 1 signals were observed for all analytes. This reflects the presence of natural isotopes of carbon, nitrogen, hydrogen and oxygen in the samples. The experimental m + 1 / m signal ratios of 0.075 ± 0.005 (lysine), 0.038 ± 0.005 (alanine), 0.072 ± 0.004 (glucose) and 0, 140-ά: 0, 011 (sucrose) correlate very well with the theoretical values of 0.076 for lysine, 0.038 for alanine, 0.071 for glucose and 0.142 for sucrose. Among the matrixes tested, DHB gave the best results, especially for sugar. Sinapic acid also gave good results. No signals of the analytes could be obtained in the negative mode.

Example 2: Quantification with MALDI-TOF-MS and internal standard

The MALDI-TOF mass spectrum of an approximately 1: 1 mixture of 9 mM naturally labeled and 10 mM 95% α ¹⁵ N-lysine are shown in FIG. 1c. The signals at m / z 147, 148 and 149 represent unlabeled, single-labeled and double-labeled isotope fractions. The signals at m / z 147 and m / z 148 are clearly separated so that the associated area units are clearly separated. The same was observed for alanine and glucose (data not shown). The area units of the signals of m represent the amount of unlabeled lysine and that of m + 1 the amount of single-labeled lysine. The m + 1 fraction also contains natural isotopes, which are contained in the results and must be corrected by the theoretical values. The quantities are quantified using the following equations:

^s m + l, analyte "Canaiyte ^{+ s} m + l, standard" Cgtandard

I = (1) ^s m, analyze "Ganalyte ^{+ s} , standard ^* Cgtandard

( ^s m + l, standard ^s m, standard "1) Canaiyte ⁼ Cgtandard (2>

( ^s m, analyze "1 ~ ^s + l, analyze)

In the equations, the ratio of intentions is I = I _{m +} ι / It-. the concentration of analyte C _{ana e} iy and internal standard Cg _ta n _dard the sensitivity s. The senistivity reflects the different mass fractions of a compound. The example is Sensitivity of lysine reproduced. Naturally labeled lysine consists of 92.9% unlabeled and 7.1% single-labeled isotopomers, higher-labeled isotopomers are negligible. The internal standard used 95% α ¹⁵ N-lysine has three main mass fractions: unlabeled isotopomer (m), α ¹⁵ N-labeled isotopomer (m + 1) and naturally labeled isotopomer of the single-labeled α ¹⁵ N-lysine (m +2). These were quantified with 2.5%, 90.6% and 6.9%, respectively. This led to the following sensitivities for lysine: S _m , _a nalyte = 0.929, S _{m +} ι, _ana iy _te = 0.071, S _m , standard = 0.025 and S _{m +} ι, _S tan _d ard = 0.906.

The potential of the MALDI-TOF-MS for quantification was tested with aqueous mixtures of naturally labeled compounds with equal amounts of internal standard in a concentration range of the analyte from 10 μM to 100 mM. Comparison of the theoretical and experimental concentrations of lysine, alanine and glucose (Figures 2A-F, quantification in aqueous solution) shows a clear linear relationship with a regression coefficient of 1000 for each analyte. This shows the potential of MALDI-MS in combination with marked internal standards for the analysis of the concentration of small chemical compounds. The mean standard deviation across all tests carried out is 4.3% for lysine, 3.2% for glucose and 3.7% for alanine. The best results for quantification were achieved with a concentration of analyte to internal standard of approximately 1: 1. However, good quantifications were also possible with a concentration ratio of analyte to internal standard between 0.2 to 5.

Example 3: Application of MALDI-TOF-MS to C. glutamicum ATCC 21253 fermentation broths

MALDI-TOF-MS measurements were used to quantify metabolites in C. glutamicum ATCC 21253 fermentation broths. The lysine producing strain was grown in PMB mineral medium with an initial concentration of 20 g / 1 molasses and 18 g / 1 glucose (Figures 3a and 3b). In Figure 3a (molasses) and 3b (glucose) the mass spectra of 1: 5 diluted culture supernatants with DHB as a matrix are shown directly after the inoculation. Clear signals of the substrates could be determined. Both sodium and potassium adducts of the two compounds (sucrose and glucose) were found at m / z 203 and 219 (glucose) and m / z 365 and 381 (sucrose). No signals were found for the m / z values for lysine and alanine, which are only formed during cultivation. An example of a MALDI spectrum at the end of cultivation after 40 hours is shown in FIG. 3c for the growth on mineral medium with a Concentration of initially 18 g / 1 glucose is shown. The glucose signal has disappeared. The two main products of the alanine and lysine strains can be clearly detected. Particularly high ^'signals resulting from lysine, as its essential ions, the ions [M + H] ⁺ at m / z 147 [M + Na] ⁺ at m / z' shows 169 and [M + K] + at m / z 185 , Alanine was determined to be [M + H] + at m / z 90.

Figures 4a and 4b show the profile after 30 hours of cultivation of C. glutamicum ATCC 21253 in PMB medium with glucose (Figure 4a) and molasses (Figure 4b). Glucose, lysine and alanine were quantified as described above with MALDI-TOF-MS and internal standard and measurement of cell growth as optical density at PD6 _{60 nm} . The standard deviation of the measurements (5 * 50 shots) is shown in the figures. FIGS. 5A to 5D show the comparison of the MALDI-TOF-MS quantification of lysine, alanine and glucose with the results of conventional analytical techniques such as HPLC (lysine, alanine) and enzymatic determination (glucose). This comparison shows an excellent agreement between the MALDI-TOF-MS and the conventional methods over the entire course of the fermentation. The regression coefficient was close to 1 in all cases. Especially at higher concentrations above 1 mM, the standard deviations in the MALDI-TOF-MS measurement were less than 5%.

The strain excretes lysine in both media and initially grows exponentially in both media. With the consumption of the essential amino acids methionine, threonine and isoleucine in the medium (data not shown), the growth stops and the lysine production begins. The kinetics and stoichiometry of the two cultures were significantly different. 9.5 mM lysine and 1.1 mM alanine were formed on defined medium, only 3.5 mM lysine and 1.0 mM alanine were formed on molasses medium. The opposite was the case for the production of the biomass. With 4.3 g / 1 biomass, 22% less biomass was formed on mineral medium than on molasses medium (5.5 g / 1). C. glutamicum can grow on molasses medium without the amino acids threonine, methionine and leucine up to a biomass of 1.2 g / 1.

The good agreement between the results with conventional methods and MALDI-TOF-MS shows the possibility of using this method for the bioflux analysis of organisms. The MALDI-TOF-MS method has a number of advantages over conventional methods such as the faster analysis time, the simultaneous analysis of very different compounds such as amino acids and sugars Possibility of analysis with very small sample quantities or volumes.

Example 4:

¹³ C-tracer experiments with measurement of the labeling patterns of metabolic products by MALDI-TOF-MS and stoichiometric balancing for bioflux analysis of the metabolism of Corynebacteri m glutamicum ATCC 21253 in batch cultivation.

The following example shows the determination of bioflux parameters of the metabolism of the lysine-producing microorganism Corynebacterium glutamicum ATCC 21253.

Definition of individual terms used in the example for ¹³ C marking of substances

Different position isotopomers result from different numbers and positions of ¹³ carbon atoms in a substance. A substance with n carbon atoms therefore has 2 ⁿ different position isotopomers. Positional isotopomers of the same mass, which therefore have an identical number of ¹³ carbon atoms, but the exact position of which in the molecule can be different, form groups of mass isotopomers. The proportions of individual groups of mass isotopomers can be measured using mass spectrometry. The result is a characteristic mass spectrum with individual peaks that reflect different mass isotopomer groups. The relative frequency of two mass isotopomer groups of masses mi and m to one another is referred to as the intensity ratio I _m ι _{/ m2.} Intensity ratios can be determined by comparing peak heights or peak areas of the corresponding mass isotopomer groups.

The cultivation and analysis was carried out as described in the previous examples.

Complement to the cultivation: ¹³ C-labeled glucose was used as the substrate, two parallel batches being carried out. In an approach 99% as the substrate was ^L-13 C-glucose

(Euriso-top, Gif-sur-Yvette, France). In the other approach, a mixture of naturally labeled glucose (Sigma, St. Louis, USA) and 99% U- ¹³ C-glucose (Euriso-top, Gif-sur-Yvette, France) in a molar ratio of 37.5 was used as the substrate: 62.5 used. Samples were drawn after about 17 hours of cultivation time towards the end of the lysine production phase then analyzed with MALDI-TOF-MS for the marking patterns of the products formed lysine, acetate and trehalose.

The following changes were made to the MALDI analysis generally described in the examples: The signals from 100 x 5 shots were added up per sample. With the device software XMass (Bruker Daltonics, Bremen), the relative peak heights of the individual mass iso-topomers were determined for each metabolite.

Results :

Cultivation course of the batch fermentation of C. glutamicum ATCC 21253

After about 7 hours of cultivation, the consumption of the essential amino acids threonine, leucine and methionine led to the formation of lysine and various by-products such as alanine, valine, acetate, lactate, pyruvate and trehalose. Formation and consumption rates for the period of lysine formation were determined from the measured concentrations of substrate, products and biomass after 7 and 17 hours of cultivation. From this, yield coefficients related to the substrate (in mmol / mmol glucose) were calculated. The need for individual intermediate metabolites for biomass synthesis was determined using the experimentally determined biomass yield (in g of dry biomass / mmol glucose) and data on the biomass composition of C. srlutamicum (Marx, A., de Graaf, AA, Wiechert, W., Eggeling, L and Sahm, H., 1996, Bio-technology & Bioengineering, 49: 111-129). Tables 1 and 2 show the mean values from the tracer experiments carried out in parallel. The main product lysine is formed with a yield of 0.225 mol / mol. The strain also produces a number of by-products with lower proportions.

Determination of the labeling pattern of metabolites with MALDI-TOF-MS

Optimal signals were obtained for the measurement of the labeling patterns of trehalose and lysine with 2, 5-dihydroxybenzoic acid as a matrix. 6 shows a MALDI-TOF-MS spectrum of lysine from the tracer experiment with a mixture of U- ¹³ C-glucose and naturally labeled glucose (37.5: 62.5), which as [M + H] ⁺ adduct with 2, 5-DHB was recorded as a matrix in positive reflector mode. The various mass iso-isomers formed can clearly be seen, for example those of the masses 147 (m), 148 (m + 1), 149 (m + 2) or 150 (m + 3). Fig. 7 shows a MALDI-TOF-MS spectrum of acetate from a Tracer experiment with 99% l ¹³ C-glucose from the negative .Reflektormode with 2,5-DHB as the matrix. For both measurements Fementation broth from 17 h batch culture of C. glutamicum ATCC21253 was taken. In addition to the main peak of the unlabeled acetate at the mass charge number m / z 59 as an [MH] ^~ adduct, the other mass isotopomers m + 1 (m / z 60) and m + 2 (m / z 61) formed can be recognized. The intensity ratios were determined by comparing the respective peak heights with the Bruker software XMass. The following intensity ratios were used for metal METABOLITES with l ¹³ C-glucose determined as a tracer substrate: I _{m +} i _{/ m,} lysine = 1.113 and I _{m +} ι _{/ m +,} trehalose = 0.422 (see Tab. 3). The intensity ratios were for the mixture of U ¹² / U ¹³ C-glucose

In the + l / m _. Lysine ⁼ 0.89, I _m + 2 / m + l, lysine = 1-28, I _m + l /, acetate = 0.120 and l _{m +} ι _/ m, ₊₂ Ace _ta t = 0.211 measured. The errors of the respective measurements were mostly in the range of 5 - 10%. These intensity ratios were used for the bioflux calculation.

BIOFLUX calculation

Using a model of the central metabolism of

C. g-lutamicum in the software Matlab / Simulink (Wittmann and Heinzle, 1999, Biotechnology & Bioengineering 62: 739-750; Wittmann and Heinzle, Abstract, 9 ^th European Congress of Biotechnology, Brussels, Belgium, July 1999) was the intracellular flow distribution calculated from the experimentally determined labeling patterns of the metabolites and the stoichiometric data of the cultivations (see Tab. 1 and Tab. 2). As an exception, the experimental yield of lysine was not taken into account from the stoichiometric data. Rather, the lysine yield determined from the bioflux analysis was compared with the experimental value in order to check the consistency of the data.

The measured intensity ratios can be used to determine various flow parameters. This resulted in simulations of the metabolism of C. glutamicum, which had previously been carried out with a model developed in Matlab / Simulink. An overview of the results is shown in Tab. 3. Using l- ¹³ C-glucose as a tracer substrate, the branching ratio between glycolysis and pentose phosphate pathway (Φppp) can be determined from the intensity ratio I _{m +} i _m of lysine and the reversibility of glucose from the intensity ratio I _{m + 2 / m + l} ^on trehalose. Determine 6-phosphate isomerase (ζQi). With a mixture of naturally labeled and U- ¹³ C-glucose as a tracer substrate, the branching ratio between carboxylation of pyruvate / phosphoenol pyruvate and the introduction of pyruvate into the citrate cycle can be determined from the intensity ratio I _{m +} ι _{/ m} ^of lysine, (Φpyc ). the branching ratio between succinylase and dehydrogenase path in the lysine biosynthesis from the intensity ratio I _{m + 2 /} m ₊ ι of lysine, and from the intensity ratio Determine the exchange flow between pyruvate / phosphoenolpyruvate and oxaloacetate (ζpc _/ PEPCκ) in the _{+ l / m} of acetate or alanine or other metabolites formed from pyruvate.

The respective parameter adjustments were carried out using a gradient solver from the Matlab software (fmincon). The starting values of the parameters were varied in various adaptations to enable total Mini a to be found. In all cases, the algorithm converged to the same end values for the parameters to be determined. The criterion of the goodness of fit in all cases was the sum of the squares of the deviation between model results and experimental results (least square).

The stoichiometric data in Tab. 1 and Tab. 2 were taken into account in all adjustments. The following flow parameters were not determined and literature data describing tracer experiments of C. glutamicum in continuous culture with NMR were taken: reversibility of transketolase I (ζ _tk i = 2.50), reversibility of transketolase II (ζ _tk2 = 0.50 ), Reversibility of the transaldolase (ζ _ta = 1.00).

The flows were calculated from the experimental data as follows: For the tracer experiment with l- ¹³ C-glucose, the branching ratio between glycolysis and pentose phosphate pathway (Φ _PPP ) and from the intensity ratio I _{m + l /} m of lysine were first determined Intensity ratio I _{m + 2 / m +} ι ~ ^ on trehalose determined the reversibility of glucose-6-phosphate isomerase (ζpσi). The two flow parameters were then passed to the next parameter determination, the branching ratio between carboxylation of pyruvate / phosphoenolpyruvate for the experiment with the mixture of naturally labeled and U- ¹³ C-glucose as the tracer substrate from the intensity ratio I _{m +} ι _{/ m} of lysine and the introduction of pyruvate into the citrate cycle, (Φpyc). and calculated from the intensity ratio I _{+ 2 / m +} ι of lysine the branching ratio between succinylase and dehydrogenase pathway in lysine biosynthesis. The results were then adopted in a third loop and used to determine the exchange flow between pyruvate / phosphoenolpyruvate and oxaloacetate ((pc _/ p _EPC κ) from I _{m + l} of acetate and I _{m +} ι _{/ m + 2} of acetate. The three parameter adjustments were carried out in succession until no significant change in the results could be observed. This was achieved after 3 runs. 8 shows the resulting flow map for the phase of lysine formation. The following interesting conclusions for batch culture between 7 and 17 h cultivation time of C. glutamicum ATCC21253 can be drawn from the data:

(1) The relatively high flow in the pentose phosphate pathway (Φppp = 0.69) provides sufficient NADPH for lysine synthesis.

(2) Both alternative metabolic pathways in lysine biosynthesis are active.

(3) There is significant reflux of oxaloacetate to the pyruvate / phosphoenolpyruvate pools. The resulting "futile cycle" could be used to break down excess ATP.

(4) The citrate cycle runs with high activity, obviously a high amount of energy equivalents is generated, which due to the stopping growth during the

Lysine formation may be consumed via the "futile cycle" between oxaloacetate and pyruvate / phosphoenolpyruvate.

(5) The enzyme glucose-6-phosphate isomerase is highly reversible.

The lysine yield of 0.202 mol / ol determined via the bioflux analysis shows excellent agreement with the experimentally found value of 0.224 mol / mol. This underlines the consistency of the data.

Summary of example 4

With the present method, the central metabolism of the lysine producer Corynebacterium glutamicum can be almost completely quantified from a few targeted analyzes of intensity ratios of metabolites in tracer experiments. In particular, the key parameters of lysine formation such as the branching ratio between glycolysis and the pentose phosphate pathway (ΦP _P P), reversibility of glucose-6-phosphate isomerase (ζpσi), the branching ratio between carboxylation of pyruvate / phosphoenolpyruvate and the introduction of pyruvate into the citrate cycle (Φpyc). The developed method can be used to determine the branching ratio between succinylase and dehydrogenase path in lysine biosynthesis and exchange flows between pyruvate / phosphoenolpyruvate and oxaloacetate (C _PC / _P E _PCK ), which is essential for the characterization and strain improvement of lysine-producing bacteria. Tab. 1: Yield coefficients for biomass (in g / mmol Glc) and various products in (in mol / mol) in batch cultivations of C. glutamicum ATCC 21253 during lysine formation.

Tab. 2: Precursor requirement for biomass synthesis in batch cultivations of C. glutamicum ATCC 21253 during the formation of lysine.

Precursor for BTM consumption (mol / mol glucose)

Glucose-6-phosphate 0.012

Fructose 6-phosphate 0.004

Ribose 5-phosphate 0.053

Phosphoglycerate 0.077

Erythrose-4-phosphate 0.016

Phosphoenolpyruvate 0.032

Pyruvate 0.108

Glyceraldehyde-3-phosphate 0.077 a-ketoglutarate 0.102

Oxaloacetate 0.075

Acetyl-CoA 0.150 Tab. 3: Experimental design for bioflux analysis. Key parameters of the metabolism of C. glutamicum: suitable selection of tracer substrate and measured intensity ratio.

1,113

0,422

0.422

• m + 1 / m, lysine Φpγ _C 0.89

'm + 2 / m + 1, lysine ΦDH 1.28

'm+1/m, acetate PC/PEPCK 0,120

'm + 1 / m, acetate PC / PEPCK 0.120

Example 5:

Screening for metabolic activities in microtiter plates - ¹³ C-tracer experiments with measurement of the labeling pattern of metabolic products by MALDI-TOF-MS for bioflux analysis of various lysine-producing microorganisms.

The following example shows the screening for metabolic activities in microtiter plates with MALDI-TOF-MS using the example of determining the branching ratio between glycolysis and pentose phosphate pathway for 4 different lysine-producing microorganisms.

microorganisms

The lysine-producing microorganisms Brevibacterium flavum NRRL 11478, Corynebacterium glutamicum ATCC 21253, Corynebacterium glutamicum ATCC 21526 and Corynebacterium glutamicum ATCC 21544 were used as strains.

The cultivation was carried out as described in the previous examples, the following additions and deviations being made: The organisms were cultivated in microtiter plates, which were incubated in a reader (Fluoroscan Ascent FL, Labsystems, Finland) for 24 hours at 30 ° C. The inoculum and medium were mixed immediately before the experiment and filled into the microtiter plate with a pipette. Ual as carbon was 99% l- ¹³ C-glucose (Euriso-top, Gif-sur-Yvette, France) were used. The wells in the middle of the plate were used for cultivation. Unused wells of the plates on the outer edge were filled with water to increase the humidity in the reader. The plates were shaken continuously during the incubation (orbital shaker, shaking speed 1020 rpm, shaking diameter 1 mm). The cultivation volume was 200 μl at the beginning, and decreased by evaporation under test conditions about 2% per hour. For comparison, parallel experiments were carried out in 25 ml shake flasks with baffles and 5 ml PMB medium, which were incubated at 150 rpm and 30 ° C.

The incubations were terminated after 24 hours, 50 μl samples were taken from the wells and the shaking flasks and examined for the labeling pattern of the lysine formed with MALDI-TOF-MS.

analytics

The intensity ratio I _{m +} ι _{/ m} of lysine was measured with MALDI-TOF-MS from samples diluted 1: 5 with deionized water. The procedure was analogous to the example for the lysine marker measurement in the example of bioflux analysis.

In contrast to the analytics described above, the following changes have been made:

30 x 5 shots per sample were added up.

Determination of the labeling of lysine with MALDI-TOF-MS.

Optimal signals were obtained for the measurement of lysine with 2, 5-dihydroxybenzoic acid as a matrix. The measured values obtained in detail for the various strains examined are shown in Table 4.

BIOFLUX calculation

With the help of a model of the central metabolism of C. glutamicum in the software Matlab / Simulink (Wittmann and Heinzle, 1999, Wittmann and Heinzle, 1999), the branching relationship was nis between glycolysis and pentose phosphate pathway calculated from the experimentally determined labeling pattern of lysine. Stoichiometric information on biomass formation was not taken into account. As previously carried out simulations of the metabolism of C. glutamicum using a model developed in Matlab / Simulink, using l- ¹³ C-glucose as a tracer substrate, the branching ratio between glycolysis and pentose phosphate pathway can be determined from the intensity ratio I ₊ ι _/ m of lysine ( Φppp) determine. The good sensitivity of this approach results from the fact that the Ci atom of glucose is cleaved off as C0 ₂ in the pentosephosphate pathway, while it remains preserved in the glycolysis in the carbon skeleton of the metabolites. Rising flows in the pentosephosphate path thus lead to an increasing decrease in the labeling in the lysine.

The parameter adjustment was carried out with a gradient solver from the Matlab software (fmincon). The start values of the parameters were varied in various adaptations to enable total minima to be found. In all cases, the criterion of the goodness of fit was the square of the deviation between model results and experimental results (least square). In all cases, the algorithm converged to the same final values for the parameter to be determined. Flow parameters not determined in this experiment were taken from our own results or literature data (see patent example for Bioflux analysis).

Tab. 4 shows the values obtained for (Φppp). It can be seen that in all cases relatively high flows result in the pentose phosphate pathway, so that a limitation of the lysine formation by NADPH is unlikely. Interestingly, the various lysine producers examined showed good agreement with Φppp. There were also no significant differences in Φppp between the experiments in microtiter plates and in shake flasks. Further studies on the amount of lysine produced showed that strains NRRL 11478, ATCC 21526 and ATCC 21544 produced practically the same amount of lysine in shake flasks and microtiter plate cultivation (data not shown), whereas the strain ATCC 21253 in the microtiter plate after 24 hours only about 30% the amount of lysine produced in the shake flask.

Summary of example 5

With the present method, metabolic characterizations of microorganisms can be carried out on a microscale, which is based on a strain comparison of 4 different lysine products. ten was demonstrated. This is achieved by the MALDI-TOF-MS analysis used, which only requires the smallest sample amounts of <1 μl. With targeted analyzes of the intensity ratios of metabolites in tracer experiments, even flow distributions in the metabolism can be quantified. In addition, the MALDI-TOF-MS analysis can be used for determining the concentration, see example for determining the concentration from fermentation broths of C. glutamicum (Example 3), which can be used to determine product formation rates or yield coefficients for the cultivation of microorganisms in microtiter plates. This makes the method very suitable for screening strains.

Tab. 1: Screening of lysine-producing microorganisms in microtiter plates and shaking l- ¹³ C-glucose as substrate: intensity ratio I _{m +} ι _/ m of lysine and relative flow into the pentose phosphate pathway

Claims

claims 1. A method for the qualitative and quantitative analysis of complex mixtures of chemical compounds, characterized in that the compounds are analyzed using MALDI-TOF mass spectrometry. 2. The method according to claim 1, characterized in that the mixtures of chemical compounds arise in a chemical or enzymatic reaction or in a fermentative process. 3. The method according to claim 1 or 2, characterized in that the mixtures of the chemical compounds consist of polymeric or non-polymeric compounds or polymeric and non-polymeric compounds. 4. The method according to claims 1 to 3, characterized in that an internal standard, before the start of the chemical or enzymatic reaction or the fermentative process or during or after completion of the chemical or enzymatic reaction or the fermentative process is added and the mixture chemical compounds are analyzed in the presence of this internal standard. 5. The method according to claims 1 to 4, characterized in that the analysis of the mixtures takes place during or after the chemical or enzymatic reaction or the fermentative process with the MALDI-TOF mass spectrometry. 6. The method according to claims 1 to 5, characterized in that non-polymeric compounds with a molecular weight <4000 Dalton are analyzed in a mixture. 7. The method according to claims 1 to 6, characterized in that the non-polymeric compounds present in the mixture are present in a concentration of 1 μM to 500 mM. 8. The method according to claims 1 to 7, characterized in that the analysis is carried out on a carrier material with a roughness value R _Z > 1. Sign. 9. The method according to claims 1 to 8, characterized in that the analysis on a polished, coated or vapor-coated carrier material or on a polished and coated or polished and vapor-coated carrier material 5 is carried out. 10. The method according to claims 1 to 9, characterized in that the carrier made of a material selected from the group glass, ceramic, quartz, metal, stone, plastic, 10 rubber, silicon, germanium or porcelain. 11. The method according to claims 1 to 10, characterized in that the compounds are derivatized in a mixture before analysis. 15 12. The method according to claims 1 to 11, characterized in that the method is carried out manually or automatically. 13. The method according to claims 1 to 12, characterized in that the method is used in a high throughput screening. 14. The method according to claims 1 to 12, characterized in that the method is used for bioflux analysis. 15. The method according to claims 1 to 14, characterized in that an internal standard beforehand the fermentative pro 30 seconds is added and this standard is implemented as a substrate in the fermentative process, the isotopomer distribution of the resulting intermediates or products or intermediates and products being measured using MALDI-TOF mass spectrometry. 35 16. The method according to claims 1 to 15, characterized in that selected as an internal standard by at least one isotope from the group ² H, ¹³ C, ¹⁵ N, ¹⁷ 0, ¹⁸ 0, ³³ S, 34 _S 36 _S 35C1, 37th _C ι, 29 _S i _; 30 _S i, 74 _{Se or} their mixtures 40 labeled starting material, product or another labeled chemical compound is used. 17. The method according to claims 1 to 16, characterized in that the analysis additionally using the eta- 45 stable decay occurs after ionization or shock-induced decay. 8. The method according to claims 1 to 17, characterized in that the dynamics of the labeling pattern and concentration of the substances in the chemical or enzymatic reaction or in the fermentative process is measured.