EP1549958A2

EP1549958A2 - Maldi-matrix

Info

Publication number: EP1549958A2
Application number: EP03793665A
Authority: EP
Inventors: Bernd Stahl; Günther Boehm; Marko Mank
Original assignee: Nutricia NV
Current assignee: Nutricia NV
Priority date: 2002-08-19
Filing date: 2003-07-30
Publication date: 2005-07-06
Also published as: WO2004023147A2; JP2005536759A; DE10238069A1; CA2496018A1; CN1675554A; WO2004023147A3; US20050158863A1; AU2003258549A1

Abstract

The invention relates to matrixes for ultraviolet matrix-assisted laser desorption-ionisation mass spectrometry made of the salt of an amine which reacts as a proton acceptor and an organic substance which reacts as a proton donator. Either the amine or the organic substance absorbs UV light. Said matrixes are characterised in that they represent an ionic liquid at room temperature and in that the amine is selected from the group which is made up of 3-aminoquinoline, pyridine, a primary amine whose N-Atom is bound to a phenyl radical or a straight or branched, saturated C1-C11-alkyl radical which can be substituted by an OH-group, a secondary and tertiary amine whose N-Atoms are bound to two or three radicals which can be the same or different and represent a straight or branched, saturated C1-C8-alkyl radical which can be substituted by an OH-group and a phenyl radical, imidazole and the C and/or N-alkylated imidazole derivatives and also characterised in that the organic substance is selected from the group which is made up of 2,5-dihydroxybenzoic acid and the isomers thereof, 2-hydroxy-5-methoxy-benzoic acid and the isomers thereof, picolinic-acid, 3-hydroxypicolinic acid, nicotinic acid, 5-chloro-2-mercapto-benzo-thiazole, 6-aza-2-thiothymine, 2',4',6'-trihydroxyacetophenone-monohydrate, 2',6'-di-hydroxyaceto-phenone, 9H-pyridol[3,4-b]indole, dithranole, trans-3-indolacrylic acid, osazones, ferulic acid, 2,5-dihydroxyacetophenone, 1-nitrocarbazole, 7-amino-4-methylcumarine, 2-(p-hdroxyphenylazo)-benzoic acid, 8-aminopyrene-2,3,4-trissulfonic acid, 2-[2E-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malonitril (DCTB), 4-methoxy-3-hydroxycinnamic acid and 3,4-dihydroxycinnamic acid. Said liquid matrixes enable reproducible, error-free analysis values to be obtained. The combination of pure mass spectrometric analysis with additional knowledge of enzymatic reactions/modifications with the possible monitoring is also described.

Description

MALDI matrix

DESCRIPTION

The invention relates to matrices for ultraviolet matrix-assisted laser desorption / ionization mass spectrometry comprising a salt of an amine reacting as a proton acceptor and an organic substance reacting as a proton donor, either the amine or the organic substance absorbing UV light, and the use of such matrices ,

Ultraviolet matrix-assisted laser desorption / ionization mass spectrometry is generally abbreviated as UV-MALDI-MS.

The analysis principle of UV-MALDI-MS is based on matrix-assisted laser desorption and ionization of molecules, including biomolecules from a cocrystallization from an analyte and a UV light-absorbing matrix substance. Such UV light-absorbing substances are also simply called MALDI matrix.

Typical MALDI matrices are, for example, 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA) and trans-3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid).

The cocrystallization of analyte and matrix is achieved, for example, by usually mixing and drying aqueous solutions of the aforementioned UV light-absorbing matrix substances and the analyte on a metal sample carrier. The ions generated from the solid phase of the cocrystallization are then detected using mass spectrometric analyzers, which are based, for example, on the TOF, quadrupole, ion trap or FTICR principle or on a combination of these techniques.

The UV-MALDI-MS is mainly used for mass spectrometric analysis of molecules or biomolecules, e.g. carbohydrates, proteins, peptides, Nucleotides and lipids including their corresponding conjugates such as glycoproteins, lipoproteins etc. It is also possible to directly characterize whole cells and microorganisms using MALDI-MS analysis. In this regard, reference is made, for example, to Alomirah HF, Alli I, Konishi Y..Applications of mass spectrometry to food proteins and peptides, J. Chromatogr. A. 2000 Sep 29; 893 (1): 1-21. Review; Kussmann M, Roepstorff P. Sample preparation techniques for peptides and proteins analyzed by MALDI-MS, Methods Mol. Biol. 2000; 146: 405-24; Bonk T., Humney A., MALDI-TOF-MS analysis of Protein and DNA, Neuroscientist, 2001, 27 (5), 465-72.

The MALDI-MS has many advantages. This includes the high sensitivity of the analysis (femto - to attomol range for isolates), a high tolerance to impurities and various buffer substances, the simple sample preparation ("Dried Droplet method") and the integration of the MALDI-MS principle in high-throughput analyzes.

Furthermore, it is possible to use enzymes on the MALDI targets or the MALDI sample carriers in order to carry out modifications to the molecules to be examined and to demonstrate them by mass spectrometry. For this purpose, a solution of enzyme, analyte / substrate and a neutral conventional MALDI matrix such as ATT is applied directly to the MALDI target. The enzymatic reaction taking place is then stopped by drying and cocrystallization of the batch. The analysis of the reaction products using MALDI can then be carried out as usual after the actual enzymatic reaction. Alternatively, an acidic matrix such as DHB can also be added to the reaction mixture at the end of an enzymatic reaction.

A major disadvantage of MALDI-MS analysis with a fixed matrix or a fixed analyte preparation is, however, the often high variance of the signal intensities of the analyte when desorbing different ones Make the same preparation or the same preparation. This variance is based on the one hand on the fact that the analyte molecules are distributed differently over the surface of the dried preparation. On the other hand, the reason for this can be seen in the different incorporation of different substance classes into the crystal lattice of the dried cocrystallization of analyte / sample and matrix. Various preparation methods have now been proposed in order to achieve more homogeneous crystallizations. These include, for example, thin-film preparation (Vorm O., Roepstorf, P., Mann M., Anal, Chem., 64, 1992, 1879-1884), the preparation on thin layers of matrix crystals, which serve as crystallization nuclei, the use of nitrocellulose additives as well as fucose and the dissolution of conventional MALDI matrices such as DHB or CHCA in glycerol (Sze ET, Chan TW, Wang G. Formulation of matrix Solutions for use in matrix-assisted laser desorption / ionization of biomolecules. J. Am. Soc. Mass. Spectrom. 1998 Feb; 9 (2): 166-74). However, these optimized methods only ever solve problems from sub-areas of MALDI MS analysis.

Ionic liquids from an amine salt of an organic acid have also been proposed as matrices for MALDI-MS, see D. W. Armstrong et al, Anal. Chem. 2001, 73, 3679-3686 ,. These are amine salts of various cinnamic acid derivatives or from aminoquinoline and CHCA (Kolli VSK, Orlando R, Rapid Commun. Mass Spectrom. 1996, 10: 923-926).

The object of the present invention is to provide improved matrices for UV-MALDI-MS with which more error-free and reproducible analysis values can be obtained, in particular the combination of the pure mass spectrometric analysis with the additional knowledge from enzymatic reactions / modifications with the possibility of monitoring should be given. This object is achieved by the matrices according to the teaching of claims 1 to 4 and by the use of these matrices in the form of an ionic liquid according to the teaching of the use claims.

The invention relates to new matrices, namely those which are ionic liquids and are therefore liquid at room temperature. These matrices are composed of a salt of an amine reacting as a proton acceptor and an organic substance reacting as a proton donor, either the amine or the organic substance absorbing UV light. The amine is 3-aminoquinoline, pyridine, a primary amine, to the N atom of which a phenyl radical or a straight or branched, saturated Ci-Cn-alkyl radical, which can be substituted by an OH group, is bonded is a tertiary or secondary amine, to the N atom of which two or three radicals are bonded, which may be the same or different and which are a straight or branched, saturated C 1 -C ₈ -alkyl radical which is formed by an OH Group may be substituted, and a phenyl radical is imidazole and the C- and / or N-alkylated imidazole derivatives.

The C 1 -C ₈ -alkyl radicals mentioned can thus have 1, 2, 3, 4, 5, 6, 7 or 8 C atoms, the C 1 -C 3 -alkyl radicals in the primary amines 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10 or 1 1 carbon atoms.

The amines used according to the invention thus include the following:

• a primary amine, on the N atom of which is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-pentyl, iso-pentyl, n-hexyl -, iso-hexyl, n-heptyl, iso-heptyl, n-octyl, iso-octyl, n-nonyl, iso-nonyl, n-decyl, iso-decyl, n-undedcyl or iso-undecyl radical, which may be substituted by an OH group, or a phenyl radical is bonded.

A secondary or tertiary amine, to the N atom of which two or three radicals are bonded, which may be the same or different, and which are selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-pentyl, iso-pentyl, n-hexyl, iso -Hexyl, n-heptyl, iso-heptyl, n-octyl and iso-octyl residues, which can be substituted by an OH group, and a phenyl residue.

• 3-amino-quinoline, pyridine, imidazole and the C- and / or N-alkylated imidazole derivatives, where the alkyl radicals can be the same or different and in particular have 1, 2, 3, 4, 5 or 6 carbon atoms.

The iso radicals listed above include all possible isomers.

** I The organic substance is 2,5-dihydroxybenzoic acid and the isomers (especially 2,6-dihydroxybenzoic acid) thereof, 2-hydroxy-5-methoxy-benzoic acid and the isomers thereof, picolinic acid, 3-hydroxypicolinic acid, nicotinic acid , 5-chloro-2-mercaptobenzothiazole, 6-aza-2-thiothymine,

15 trifluoromethanesol fonate, 2 ^I , 4 ', 6'-trihydroxyacetophenone monohydrate, 2', 6'-dihydroxyacetophenone, 9H-pyridol [3,4-b] indole, dithranol, trans-3-indolacrylic acid, Osazone, ferulic acid, 2, 5-dihydroxyacetophenone, 1 -nitrocarbazole, 7-amino-4-methylcoumarin, 2- (p-hydroxyphenylazo) benzoic acid, 8-aminopyrene-2,3,4-trissulfonic acid, 2- [2E-3- (4-tert-

20 butylphenyl) -2-methylprop-2-enylidenes] malonitrile (DCTB), 4-methoxy-3-hydroxycinnamic acid and 3,4-dihydroxycinnamic acid. In addition to the isomers listed for the individual organic substances, isomers and, in particular, positional isomers of the other organic substances can also be used, provided that they are able to use a

25 amine to form an ionic liquid, which will be discussed in more detail below. Thus, those materials are claimed that are present as an ionic liquid at room temperature. This ionic liquid is generated due to an acid-base reaction from an amine with the function of a proton acceptor and a

30 mentioned organic substance with the function of a proton donor. The amine is preferably aniline, ethanolamine, ethylamine. n-butylamine, N, N-diethylamine, N, N-diethylaniline, N, N-diethylmethylamine, N, N-dimethylamine, triethylamine, tri-n-propylamine, tri-n-butylamine, 3-aminoquinoline and pyridine.

The new matrices preferably include

2,5-dihydroxybenzoic acid-butylamine and 2-hydroxy-5-methoxy-benzoic acid ^{j-butylamine.}

The new matrices are obtainable by reacting a proton accepting amine described above with an organic reacting proton donor in a molar ratio of 0.5: 1 to 1: 0.5 and preferably in a molar ratio by for example, these two reactants are brought into contact with one another. If only one of the reactants is liquid, then the solid reactant can be added to the liquid reactant and vice versa. In the case of two liquid reactants, these can be combined. However, the two reactants are preferably brought into contact with one another in a solvent which is removed after the reaction, preferably by stripping off the solvent, in particular in vacuo. If liquid reaction products are obtained in this reaction or after removal of the solvent at room temperature, then they are ionic liquids or matrices according to the invention.

It is thus readily ascertainable by a simple experiment whether an ionic liquid can be prepared from an amine described here as a proton acceptor and an organic substance described here as a proton donor at room temperature. If this is the case, it is a matrix according to the invention.

The invention further relates to the use of the new matrices and known matrices in the form of ionic liquids from one Salt from an amine reacting as a proton acceptor and from cinnamic acid. or a cinnamic acid derivative, the amine being one on whose N atom one, two or three methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl radical (E), which may be substituted by an OH group and / or a phenyl radical is / are pyridine or 3-aminoquinoline, as a medium for carrying out reactions with (bio) polymers and for Monitoring of these reactions and for analysis of the resulting reaction products by means of the ultraviolet matrix-assisted laser desorption / ionization mass spectrometry.

These known liquid matrices are described at the location already mentioned, namely by D. W. Armstrong et al in Anal. Chem. 2001, 73, 3679-3686.

The new matrices and the known matrices are referred to as matrices according to the invention in the context of the present documents.

Since the matrix according to the invention is liquid, there is a homogeneous distribution of the analyte in this matrix. Therefore, the problems with the fixed matrices described above do not occur. For example, there is no segregation of different analyte components at different points in the preparation. Therefore, a UV-MALDI-MS with a liquid matrix can also be used for the purposes of a quantitative analysis that was previously only possible in a few exceptional cases, for example through the use of isotope-marked internal standards.

Another advantage of using ionic liquids as a matrix is that the matrix / analyte mixture does not have to be dry. This leads to a time saving in the preparation of the preparation.

However, it is also possible to use the matrices according to the invention or the ionic liquids according to the invention together with a solvent such as ethanol, isopropanol and long-chain alcohols and acetonitrile, dimethylformamide, dimethyl sulfoxide and tetrahydrofuran can be used in order to reduce the viscosity of the ionic matrix and make it easier to handle, for example by pipetting. Furthermore, the solubilization of the analytes in the matrix can be improved by using the solvents mentioned.

The matrices according to the invention can be used without problems in already existing UV-MALDI mass spectrometers which are equipped, for example, with N ₂ lasers.

With a liquid matrix, it is also possible to analyze a wide range of molecules. These include technical polymers and biopolymers such as carbohydrates, e.g. B. oligosaccharides, proteins, peptides, lipids, nucleic acids, secondary metabolites, pharmaceuticals and their conjugates, for example glyco- and lipoconjugates and also secondary plant metabolites (e.g. flavonoids including procyanidins etc.).

With the matrices according to the invention, it is also possible to examine reaction sequences and reaction curves, including kinetics, without having to stop the reaction at an arbitrary point in time. This preferably applies to reactions which are catalyzed by enzymes, in particular glycosidases, proteases, nucleases, lipases or lyases. In particular, enzymatic cleavages of peptides / proteins by means of proteases (eg trypsin, chymotrypsin, pepsin, aminopeptidase, carboxypeptidase) as well as cleavages of carbohydrates and glycoconjugates by means of glycosidases (eg: fucosidases, sialidases, galactosidases, hexosaminidases, and pectinases) of lipids (triglycerides, phospholipids, glycolipids) and nucleic acids (DNA, RNA) are examined. Furthermore, synthesis reactions in the MALDI matrix can also be monitored using transferases such as transglycosidases. Analytes with a high concentration can be directly prepared and measured undiluted. In addition, simultaneous detection of different analytes with little variance depending on the desorption site is possible. This also applies to analytes of different substance classes with possibly different physico-chemical properties.

The use of a liquid matrix also enables the analysis of labile analytes. It is assumed, without being bound to this explanation, that the desorption from a liquid matrix proceeds more gently, since the analyte does not pass from the crystal lattice but from a liquid into the gas phase.

Due to the fact that the pH of the matrices according to the invention is closer to the physiological values of non-covalent complexes and acid-labile molecules, such analytes can also be measured.

In addition, the liquid matrices also enable the coupling of analytical and preparative chromatographic methods such as HPLC, GPC, HPAEC with the MALDI-MS. In the Atmospheric Pressure MALDI mode, for example, the matrix and the column eluate can be mixed in or before the source. With offline MALDI analysis, suitable sample application robots can apply column eluates in parallel with a liquid matrix to targets, in order to guarantee an improved analysis continuously under "mapping" of a high chromatographic separation.

In addition to LC, ultraviolet matrix-assisted laser desorption / ionization mass spectrometry can also be carried out using electrophoresis techniques (such as FFE, PAGE or CE techniques), if necessary using sample application robots, or using (micro) preparation

/ Separation techniques, in particular μTAS, GYROS ^® and Lab-on-Chip ^{® can be} combined / coupled. The invention also relates to a method according to the teaching of the method claims.

The invention is described in more detail below on the basis of preferred exemplary embodiments. In principle, the matrices according to the invention can be produced by adding an amine reacting as a proton acceptor in an equimolar amount to an organic substance reacting as a proton donor, with either the amine or the organic substance absorbing UV light. The reaction is carried out at room temperature. An ionic liquid or a liquid salt is obtained which is generally highly vacuum stable and can be used as a UV-MALDI matrix.

example 1

Production of 2,5-dihydroxybenzoic acid-butylamine (DHB-Bu):

308.4 mg of 2,5-dihydroxybenzoic acid (DHB) was dissolved in 10 ml of ethanol. Then 198.4 ul of butylamine (Bu) was added.

The solvent was drawn off at about 40 ° C. and about 43 mbar until there was no further reduction in volume (about 30 minutes). The final volume of the DHB-Bu obtained was approximately 200 μl.

Example 2

Preparation of 5-methoxysalicylic acid butylamine (MSA-Bu):

336.4 mg of 5-methoxysalicylic acid (MSA; also referred to as 2-hydroxy-5-methoxybenzoic acid) was dissolved in 10 ml of ethanol. 198.4 μl of butylamine (Bu) were added. Working up was carried out in accordance with Example 1. Approx. 200 μl of MSA-butylamine (MSA-Bu) were obtained.

By mixing butylamine DHB with butylamine MA (10: 1) (v / v) butylamine DHBS can be produced. Example 3

Preparation of α-cyano-4-hydroxycinnamic acid butylamine (CHCA-Bu):

378.4 mg of α-cyano-4-hydroxycinnamic acid (CHCA) was dissolved in 10 ml of methanol. Then 198.4 ul of butylamine (Bu) was added. Working up was carried out in accordance with Example 1. 200 μl • α-cyano-4-hydroxycinnamic acid butylamine (CHCA-Bu) were obtained.

Example 4

Production of sinapic acid triethylamine:

378.4 mg of sinapic acid (trans-3,5-dimethoxy-4-hydroxy-cinnamic acid) was dissolved in 10 ml of ethanol. Then 278.4 ul triethylamine were added. Working up was carried out in accordance with Example 1. 200 μl of sinapic acid triethylamine were obtained.

Example 5

Preparation of 6-aza-2-thiothymine-butylamine (ATT-Bu):

286.4 mg of 6-aza-2-thiothymine (ATT) was dissolved in 10 ml of ethanol. Then 198.4 ul of butylamine (Bu) was added.

The solvent was drawn off at about 40 ° C. and about 43 mbar until there was no volume reduction (about 30 minutes). The final volume of the ATT-Bu obtained was approximately 200 μl.

Example 6

Preparation of 2 ', 4', 6 ^, -Trihydroxyacetophenone Monohydrate-Butylamine (THAP-Butylamine):

372.4 mg of 2 ', 4', 6'-trihydroxyacetophenone monohydra (THAP) was dissolved in 10 ml of ethanol. Then 198.4 ul of butylamine (Bu) was added. The solvent was drawn off at about 40 ° C. and about 43 mbar until there was no volume reduction (about 30 minutes). The final volume of the THAP-Bu obtained was approximately 200 μl.

To reduce the viscosity of the ionic matrices obtained, they can each be diluted with a solvent, for example pure ethanol in a ratio of 1: 1 V / V, and are then readily pipettable

Example 7

A matrix analyte preparation can be prepared according to the following general procedure:

1 . 1 μl of liquid ionic matrix A (undiluted or 1: 1 (v / v) in EtOH or solvent B) are mixed with 1 μl of analyte C in a suitable solvent D on a MALDl sample plate (stainless steel target).

2. Alternatively, the preparation can be carried out as under 1., but after prior desalting of the analyte by incubation in a 1: 1 (v / v)

Dilution with a. Crown ether or with ion exchangers.

3. The MALDI sample plate with the premixed sample is transferred directly to the MALDI high vacuum. Then the MALDI MS analysis is carried out.

All ionic liquids described here and in particular the following can be used as matrix A: butylamine-CHCA, butylamine-DHB, butylamine-MA, butylamine-DHBS and triethylamine-sinapic acid.

The following can be used as solvent B: acetone, acetonitrile, methanol (MetOH), ethanol (EtOH), butanol, isopropanol, chloroform and H ₂ O.

The analyte C can be of the following substance classes:

1.Technical polymers (e.g. PEG, polyacrylamides, PE),

2. carbohydrates (e.g. mono-, di-, tri-, oligo- and polysaccharides, homopolymeric and heteropolymeric composition),

3. proteins and peptides,

4. lipids,

5. conjugates of the above analytes, 6. mono-, di, -tri-, oligo- and polynucleotides,

7. secondary plant metabolites (phenolic substances, flavonoids etc.),

8. Atoms that are not volatile in a high vacuum (e.g. alkali, alkaline earth metals (such as Na, K, Ca, Mg), metals (such as Fe, Zn, Sn, Cu, Cr, etc.).

As a solution! D can serve the following:

Aqueous or acidified solution of 10-80% MetOH, EtOH, H ₂ O, acetone, acetonitrile, isopropanol, butanol, chloroform, DMSO, DMF, glycerin and THF.

The solution can e.g. acidified with 0.1-5% TFA, acetic acid or formic acid.

Example 8

Preparation of 2,6-dihydroxybenzoic acid butylamine (2,6-DHB-Bu):

308.4 mg of 2,6-dihydroxybenzoic acid (DHB) was dissolved in 10 ml of ethanol. Then 198.4 ul of butylamine (Bu) was added.

The solvent was drawn off at about 40 ° C. and about 43 mbar until there was no further reduction in volume (about 30 minutes). The final volume of the 2,6-DHB-Bu obtained was about 200 μl.

Example 9

Preparation of 2,5-Dihydroxybenzoic Acid-1-HexyI-3-Methylimidazole (DHB-HMIM): 308.4 mg of 2,5-dihydroxybenzoic acid (DHB) was dissolved in 10 ml of ethanol. Then 371.4 mg of 1-hexyl-3-methylimidazole (Bu) was added.

The solvent was drawn off at about 40 ° C. and about 43 mbar until there was no further reduction in volume (about 30 minutes). The final volume of the DHB-HMIM obtained was approximately 200 μl.

Example 10

Production of 3-aminoquinoline triflate (AC triflate):

288.4 mg of 3-aminoquinoline (AC) was dissolved in 10 ml of ethanol. Then 298.2 mg of trifluoromethanesulfonate was added.

The solvent was drawn off at about 40 ° C. and about 43 mbar until there was no further reduction in volume (about 30 minutes). The final volume of the 3-aminoquinoline triflate obtained was approximately 200 μl.

In this matrix, the 3-aminoquinoline and thus the amine or the proton acceptor is the compound which is able to absorb UV light, while in the other examples the amine is not able to do this, but rather as a proton donor functioning organic substance that absorbs UV light.

Example 1 1

In the matrices described above, reactions of (bio) polymers and in particular enzymatic reactions can be carried out. The matrix also serves as a medium or as a “reaction container”. The course of the reaction can then be followed directly and continuously by mass spectroscopy.

The preparation can be carried out as follows (general procedure):

0.5μl liquid ionic matrix A (undiluted or 1: 1 (v / v) in EtOH or another suitable solvent) with 0.5 μl one Enzyme B_ (approx. 1 μg / μl) in a suitable solvent / buffer C and homogeneously mixed with 0.5 μl of a substrate D (approx. 1 μg / μl) in a suitable solvent / buffer on a MALDl sample plate. This is followed by a direct transfer of the MALDI sample plate with the premixed enzymatic batch in the ionic liquid MALDI matrix into the MALDI high vacuum.

Those listed in Example 7 can be used as matrix A.

The following enzyme classes can be used as enzyme B: hydrolases, isomerases, lyases, transferases, oxidoreductases and ligases.

The following can be used as solvent / buffer C: H ₂ 0, carbonate buffer, ammonium acetate buffer, Tris buffer or another suitable and MS compatible buffer system or solvent.

The enzyme / substrate ratio in the finished matrix-enzyme substrate solution is 1: 1 to 1: 300 (w / w) or more.

Application Examples:

• Desialization of a trisaccharide from human milk: 0.5 μl sialidase from Clostridium Perfringens 1 μg / μl (approx. 0.1 U / μl) in 25 mM

Mix NH ₄ acetate buffer at pH 5.0) with 0.5 μl sialyl lactose 1 μg / μl in H ₂ O and with 0.5 μl DHB-Bu (1: 1 in EtOH) on a MALDl sample plate and transfer directly to the MALDI-MS high vacuum.

• Defucosylation of a pentasaccharide from human milk: 0.5μl - fucosidase from bovine kidney 1 μg / μl (approx. 2mU / μl) in 25mM NH -

Mix acetate buffer at pH 5.0) with 0.5μl LNFP 1 μg / μl in H ₂ O and with 0.5μl DHB-Bu (1: 1 in EtOH) on a MALDl sample plate and transfer directly to the MALDI-MS high vacuum. Deglycosylation of a glycoprotein: 0.5μl PNGase F from Flavobactenum Meningosepticum, recombinant 1 μg / μl (approx. 0.5U / μl) in 20mM Tris buffer pH 7.5 with 0.5μl RNAse B 1 μg / μl in H ₂ O and Mix with 1 μl CHCA-Bu or DHB-Bu (1: 1 in EtOH) on a MALDl sample plate and transfer directly to the MALDI-MS high vacuum.

• enzyme. Hydrolysis of human casein: 0.5μl trypsin with TPCK from bovine pancreas 0.01-1 μg / μl (approx. 0.074 -7.4 U / μl) with 0.5μl ß-casein 1 μg / μl in imidazole buffer 12.5mM Mix pH 7.6 and with 1 μl CHCA-Bu (1: 1 in EtOH) on a MALDl sample plate and transfer directly to the MALDI-MS high vacuum.

• Carboxypeptidase digestion for sequence analysis of a peptide: 0.5μl carboxypeptidase from Saccharomyces Cerevisiae (CPY) 0.2μg / μl (approx. 20mU / μl) with 0.5μl peptide 0, 1-1 μg / μl in 60mM diammonium hydrogen citrate buffer pH 5 and with 1 Mix μl CHCA-Bu (1: 1 in EtOH) on MALDl sample plate and directly into MALDI-MS

Transfer high vacuum.

• Aminopeptidase digestion for sequence analysis of a peptide: 0.5μl aminopeptidase from Aeromonas Protolytica 0.2μg / μl (approx. 20mU / μl) with 0.5μl peptide 0.1-1.1 μg / μl in tricine buffer pH 8 + ZnCI ₂ and with 1 μl Mix CHCA-Bu (1: 1 in EtOH) on MALDI sample plate and transfer directly to the MALDI-MS high vacuum.

• Dephosphorylation of a phosphoglycopeptide: 0.5μl acid phosphatase from potatoes 0.01-1 μg / μl (approx. 0.02 -2 U / μl) with 0.5μl ß-casein 1 μg / μl in 25mM NH ₄ acetate buffer Mix pH 5 and with 1 μl CHCA-Bu (1: 1 in EtOH) on MALDl sample plate and add directly to

Transfer MALDI-MS high vacuum.

• Dephosphorylation of a phospholipid: 0.5μl phospholipase A ₂ from honeybee venom 0.01 μg / μl (approx. 10mU / μl) with phosphatidylcholine-diheptadecanoyl 1 μg / μl in MetOH / CHCI ₃ (2: 1) and with 1 μl DHB- Bu Mix (1: 1 in EtOH) on the MALDI sample plate and transfer directly to the MALDI-MS high vacuum.

Deglycosylation of a glycolipid / ganglioside: 0.5μl ceramide glycanase from Marobdella Decora 1 μg / μl (approx. 10 mU / μl) in 20mM Tris buffer pH 7.0 with bovine ganglioside GM1 1 μg / μl in H ₂ O and with 1 μl CHCA -Bu or DHB-Bu (1: 1 in EtOH) mix on MALDI sample plate and transfer directly to the MALDI-MS high vacuum.

• Sequencing of an oligonucleotide: 0.5μl phosphodiesterase I from Crotalus Adamanteus Gift approx. 1 mU / μl in 20mM Tris buffer pH

Mix 8.0 with 0.5μl oligonucleotide in H ₂ O 1 μg / μl and with 1 μl CHCA-Bu (1: 1 in EtOH) on a MALDl sample plate and transfer directly to the MALDI-MS high vacuum.

The MALDI-MS spectra are recorded e.g. after 0, 5, 10, 20, 30, 60 and 120 minutes. If necessary, the MS analysis can also be extended to a few days after the mixture has been prepared

In the manner described above, simple screening of substrates or reaction products is also possible, for example. For example, several hundred different substrates can be incubated with an enzyme on just one sample plate. Only the smallest amounts of enzyme and substrate (0.5 μl at 1 μg / μl, for example) are required. For example, mass spectrometric analysis can be used to track kinetics in a vacuum. This application hides _. using existing automatic measurement programs, a very high potential with regard to automation and cost savings.

This applies, for example, when using sialidase to desialize sialyllactose in DHB-Bu and when using PNGaseF to deglycosylate glycopeptides / proteins in DHB-Bu. Furthermore, the matrices described above can be used in combined LC-MALDI-MS or in electrophoresis-MALDI-MS methods (PAGE, CE, FFE). A combination with (micro) preparation / separation techniques, in particular μTAS, GYROS ^® and Lab-on-Chip ^® , is also possible

Claims

1. Matrices for the ultraviolet matrix-assisted laser desorption / ionization mass spectrometry of a salt of an amine reacting as a proton acceptor and an organic substance reacting as a proton donor, wherein either the amine or the organic substance absorbs UV light, characterized in that the Matrices at room temperature represent an ionic liquid, that the amine is selected from the group consisting of 3-aminoquinoline, pyridine, a primary amine, on the N atom of which a phenyl radical or a straight or branched, saturated Ci-Cn-alkyl radical, which can be substituted by an OH group, is bound, a secondary and tertiary amine, to the N atom of which two or three radicals are bound, which may be the same or different, and which are a straight or branched, saturated C 1 -C ₈ alkyl radical, which may be substituted by an OH group, and a phenyl radical, imidazole and the C- un d / or N-alkylated

Imidazole derivatives and that the organic substance is selected from the group consisting of 2,5-dihydroxybenzoic acid and the isomers thereof, 2-hydroxy-5-methoxy-benzoic acid and the isomers thereof, picolinic acid, 3-hydroxypicolinic acid, nicotinic acid, 5-chloro -

2-mercaptobenzo-thiazole, 6-aza-2-thiothymine, 2 ', 4', 6 ^, -Trihydroxyaceto-phenon monohydrate, 2 ', 6'-Di-hydroxyaceto-phenone, 9H-pyridol [3,4-b ] - indole, dithranol, trans-3-indolacrylic acid, osazone, ferulic acid, 2,5-dihyd _. roxyacetophenone, 1-nitrocarbazole, 7-amino-4-methylcoumarin, 2- (p-hydroxyphenylazo) benzoic acid, 8-aminopyrene-2,3,4-trissulfonic acid, 2- [2E-3- (4-tert- butylphenyl) -2-methylprop-2-enylidene] malononitrile (DCTB), 4-methoxy-3-hydroxycinnamic acid, trifluoromethanesulfonate and 3,4-dihydroxycinnamic acid.

2. Matrices according to claim 1, characterized in that. the amine is aniline, ethanolamine, ethylamine. n-butylamine,

N, N-diethylamine, N, N-diethylaniline, N, N-diethylmethylamine, N, N-dimethylamine, triethylamine, tri-n-propylamine, tri-n-butylamine, 3-aminoquinoline and pyridine.

3. Matrices according to claim 1 or 2, characterized in that the matrices are 2,5-dihydroxybenzoic acid butylamine or 2-hydroxy-5-methoxybenzoic acid butylamine.

4. Matrices according to one of the preceding claims, characterized in that they are mixed with a solvent.

5. Use of the matrices representing ionic liquids according to one of the preceding claims in the ultraviolet matrix-assisted laser desorption / ionization mass spectrometry and in particular for quantitative or qualitative ultraviolet matrix-assisted laser desorption / ionization mass spectrometry.

6. Use of the matrices representing ionic liquids according to one of the preceding claims 1 to 4 and of matrices representing ionic liquids which are liquid at room temperature and which comprise a salt of an amine reacting as a proton acceptor and cinnamic acid or a cinnamic acid derivative, the amine being a such, on the N atom of which one, two or three methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl radical (s) are formed by an OH group may be substituted, and / or a phenyl radical is / are 3-aminoquinoline and pyridine, as a medium for carrying out reactions with (Bio) polymers and for monitoring these reactions as well as for analyzing the resulting reaction products using ultraviolet matrix-assisted laser desorption / ionization mass spectrometry.

7. Use according to claim 6, characterized in that the reactions are catalyzed by enzymes, in particular by glycosidases, proteases, nucleases, lipases or lyases, and the (bio) polymers are peptides, proteins, carbohydrates, lipids, nucleic acids , Secondary metabolites and / or pharmaceuticals and their conjugates.

8. Use according to one of claims 5 to 7, characterized in that the ultraviolet matrix-assisted laser desorption / ionization mass spectrometry with analytical or preparative liquid chromatography, in particular HPLC, GPC and HPAEC, electrophoresis techniques, in particular CE, CEC, PAGE and FFE, or (micro) preparation / separation techniques, in particular μTAS, GYROS ^® and Lab-on-Chip ^® , is combined.

9. A process for carrying out reactions with (bio) polymers and for monitoring these reactions and for analyzing the resulting reaction products, these reactions being catalyzed in particular by enzymes, preferably glycosidases, proteases, nucleases, lipases or lyases, characterized in that these reactions in a matrix representing an ionic liquid according to one of claims 1 to 4 or in a matrix representing an ionic liquid consisting of a salt of an amine reacting as a proton acceptor and of cinnamic acid or a cinnamic acid derivative, the amine being a such, at whose

N atom one, two or three methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl radical (s) which are substituted by an OH group be / can, and / or a phenyl radical is / are bound, is 3-aminoquinoline or pyridine, and is monitored by means of the ultraviolet matrix-assisted laser desorption / ionization mass spectrometry.

10. The method according to claim 9, characterized in that the ultraviolet matrix-assisted laser desorption / ionization mass spectrometry with analytical or preparative liquid chromatography, in particular HPLC, GPC and HPAEC, electrophoresis techniques, in particular CE, CEC, PAGE and FFE, or

(Micro) preparation / separation techniques, in particular μTAS, GYROS ^® and Lab-on-Chip ^® , is combined.