WO2010031588A2

WO2010031588A2 - Maldi matrices

Info

Publication number: WO2010031588A2
Application number: PCT/EP2009/006838
Authority: WO
Inventors: Ales Svatos; Rohit Shroff; Lubomír RULÍSEK
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.; Institute Of Organic Chemistry And Biochemistry, Ascr,
Priority date: 2008-09-22
Filing date: 2009-09-22
Publication date: 2010-03-25
Also published as: WO2010031588A3; EP2166560A1

Abstract

The present invention relates to novel MALDI matrices for the analysis of low- molecular-weight compounds in both positive and negative ion mode. In addition, the present invention relates to the rational selection of the MALDI matrices for predetermined analytes, a process which has remained empirical since the invention of the technique. In another aspect, the present invention provides a new, fast and high-throughput method for analyzing low-molecular-weight compounds by MALDI analysis as well as systems therefore.

Description

MALDI matrices

The present invention relates to novel MALDI matrices for the analysis of low- molecular-weight compounds in both positive and negative ion mode. In addition, the present invention relates to the rational selection of the MALDI matrices for predetermined analytes, a process which has remained empirical since the invention of the technique. In another aspect, the present invention provides a new, fast and high-throughput method for analyzing low-molecular-weight compounds by MALDI analysis as well as systems therefore. Prior art

The 1980s saw the development of a new ionization technique, Matrix- Assisted Laser Desorption/lonization (MALDI), which when coupled to mass spectrometry revolutionized the field of analytical biochemistry. Today, MALDI-MS finds far reaching applications in various fields including, but not exclusive, proteomics, nucleic acid analysis, analysis of lipids, glycans and polymers. More recently, biomarker detection using imaging MALDI MS has been developed. Despite the plethora of literature available on MALDI MS, only a very restricted number of matrix compounds are used; these were discovered very early during development of the MALDI technique. Typical matrix compounds include α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxbenzoic acid (DHB) which are suitable for peptide, protein, lipid and oligosaccharide analysis and sinapinic acid for protein analysis.

These matrices gave excellent results with high-molecular-weight (> 1000 Dalton) analytes, however, said matrixes are not suitable for studying low-molecular- weight analytes of mass below 1000 Daltons. The reason is that conventional matrices produce interfering signals in the low mass region interfering with the signals of the analytes.

Several efforts have been made to obviate the problem of low mass region interference. For example, the use of a high-molecular-weight matrix, e.g. meso- tetrakis (pentaflourophenyl) porphyrin having a molecular mass of 974 Dalton has been suggested, said matrix could enable fatty acid analysis because there were no matrix-related ions in low-mass region. However, the said technique only worked with saturated compounds. In the presence of unsaturated compounds, all the peaks seem to shift by 14 Daltons for reasons which are largely unknown. Recently, 9- aminoacridine was demonstrated to be an efficient matrix for analyzing low-molecular

CONRRMATION COPY weight acids by various groups. Said compound forms only mono and double deprotonated matrix ions, although sometimes certain alkali metal adduct ions were observed. However, the perfect matrix should be totally devoid of any matrix-related ions, thus, making spectral interpretation straightforward. On the other hand, several matrix-free approaches involving laser desorption ionization from a specific surface have also been introduced. For example, the applicability of desorption/ionization on porous silicon-mass spectrometry (DIOS-MS) for analyzing the deprotonated ions of fatty acids have been demonstrated (Wei J. et a/., Nature 1999, 399, 234-246). However, the sensitivity on DIOS appears to be rather poor (i.e. in the high picomole range) and extensive formation of alkali ion adducts have been observed. In addition, clathrate-nanostructure-based surfaces have been described to study a wide range of analytes; other structures suggested are graphite surfaces and carbon nanotubes. However, small molecules analysis having a mass of below 2000 Daltons, in particular of below 500 Daltons via MALDI remains an analytical challenge.

For the preparation of the MALDI sample, the analyte to be investigated is typically co-crystallized with the matrix whereby the matrix is used in a 100 to 100,000 times molar excess to the analyte. The co-crystallization of the sample takes place on the sample support, thus, incorporating the analyte into the matrix. Typically, successful co-crystallization requires a matrix to analyte ratio of about 5000 fold for peptide analysis.

Other techniques applied for analysis of low-molecular-weight analytes, for example, of biological significant markers for diagnosing infectious diseases, exploring organismal response to environmental stresses and taxonomical classification of species, based on particular lipids, are low throughput gas- chromatography with electron ionization/mass-spectrometry or chemical ionization. Typically, a pre-preparatory step is included for enrichment of said lipids from biological mixtures including thin-layer chromatography or high-performance liquid chromatography followed usually by derivatization. In addition, free fatty acids have been analyzed using fast atom bombardment and Electro-Spray Ionization (ESI), however, said methods are work-intensive and time consuming.

Although the high-throughput nature of MALDI-MS makes it an ideal tool for large scale metabolomic studies, its application in the filed has been rather limited. This is because all conventional matrices produce a forest of interfering low-mass ions (<500 Da) obscuring the detection of metabolites in the range. Despite several novel approaches, challenging MALDI-MS based metabolomic tasks remains an unmet challenge.

The limitations call for novel matrices devoid of interfering ions, (so called ionless matrices), yet still assisting an efficient ionization/desorption of the analytes.

An ideal solution would be to have a rational selection protocol for such matrices whereby depending on the properties of the analytes of interest, appropriate matrices could be designed. Such a development would not only cross a long lasting hurdle of empirical selection of MALDI matrices but would also provide a powerful, fast and easy use tool to the biological community to selectively probe into the metabolomes of living organisms.

Thus, there is an ongoing need for MALDI matrices allowing fast and high- throughput analysis of small molecules having masses of < 2000 Daltons, like <1000 Daltons or <700 Daltons, in particular, of a mass < 500 Daltons. Hence, an object of the present invention is to provide matrices useful for analyzing low-molecular-weight compounds by matrix assisted laser desorption/ionization (MALDI) analysis in negative as well as positive ion mode. Another object of the present invention is directed to methods for analyzing low- molecular-weight compounds, in particular, of allowing quantitative analysis of said low-molecular-weight compounds. o

In addition, another object of the present invention relates to a method for rational selection of the appropriate MALDI matrices for the analysis of predetermined analytes.

These and other objects are achieved by the present invention.

Brief description of the present invention

Firstly, the present invention relates to the use of matrix compounds of general formula I

wherein Y is a nitrogen or a phosphorus atom,

Ri and R₂ are independent of each other selected from hydrogen, an aliphatic, alicyclic or aromatic group whereby at least one of R1 or R2 is not a hydrogen R₃ is an aromatic group; and n is an integer > 1 , as a matrix for matrix assisted laser desorption/ionization (MALDI) analysis for the detection of low-molecular-weight acidic compounds of ≤ 2000 Daltons in negative ion mode.

In a further aspect, the present invention relates to the use of a matrix compound which is a compound of the general formula IV

R₅ -( Z - H)_n (IV)

Z is selected from SO₃, BO₂, or PO₃, R₅ is an aromatic group which may be substituted, and n is an integer ≥ 1 as a matrix for MALDI analysis in positive ion mode for detection of low-molecular-weight basic compounds of ≤ 2000 Daltons.

Another aspect of our invention relates to the optimization of matrix to analyte ratio for analysis of low-molecular-weight compounds allowing ionless, namely with no matrix-related interfering ions detection of the analyte. Typically, a huge molar excess of the matrix is used for peptide and protein analysis. However, it has been noted herein that equimolar concentrations of matrix to analyte is optimal for maximal analyte signal and complete matrix suppression by comparing different matrix to analyte ratios from 0.1 :1 to 100:1.

Finally, the present invention relates to a system for the analysis of low- molecular- weight compounds with MALDI comprising the matrix according to the present invention and, optionally, a sample support.

Moreover, a method is provided allowing quantification of an analyte using MALDI analysis.

The present invention is based on the effect of forming a salt between the analyte and the matrix compound and said salt is allowed to crystallize before analysis by MALDI. Further, the present invention is based on a rational protocol for matrix selection based on Bronsted-Lowry acid-base theory and its application to metabolomics, biological screening/profiling/imaging and clinical diagnostics. Brief description of the figures

Figure 1 : Deprotonation of a fatty acid by DMAN and schematic representation of the stable hydrogen-chelated DMAN cation.

Figure 2: [M-H]^" signals for a) palmitic acid; b) stearic acid; c) arachidic acid; e) oleic acid; f) linoleic acid; g) linolenic acid.

Figure 3: Observed signal for 1 pmol (on plate) of stearic acid. S/N = 3:1. Figure 4: MALDI TOF MS negative ion spectra for 100 pmol of a) toluene sulfonic acid; b) trifluoroacetic acid; c) cysteine; d) ascorbic acid; e) margaric acid; f) arachidic acid; g) linolenic acid; h) gibberellic acid; i) 15,16-epoxylinolenoylglutamic acid; j) alprostadil. * denotes the peak at m/z 335.2 corresponding to loss of one water molecule from the deprotonated alprostadil [M-H-H₂O]^". + denotes the peak corresponding to m/z 317.2 [M-H-2H₂O]\

Figure 5: Fig. 5 a) Three-dimensional illustration of the signals obtained for different amounts of stearic acid in the negative ion mode with DMAN as matrix. 5b) TOF-detector response curves for increasing concentration of stearic acid. The dotted lines above and below the line of the best linear fit are 99% confidence bands for the data to be linear, p < 0.0001. As all the data points lie within this confidence interval, they show excellent linearity. Inset shows the signal for stearic acid at 15 pmol (S/N = 5:1). Statistics were performed using Origin v 7.0 software. Figure 6: A calibration plot for trifluoro (full dots) and trichloro (asterix) acids.

The measured signal intensity is plotted over used concentrations (n = 5). Full lines are the lines of the best linear fit. The dotted lines above and below the linear fit lines are 99% confidence bands for the data to be linear, p < 0.0001.

Figure 7: MALDI TOF MS negative ion spectra for a) GIu-VaI-OH at m/z 245.0; b) Phe-Phe-Phe-OH at m/z 458.1 ; c) Glu-Val-Phe-OH at m/z 392.1. * marked peaks are sodium adducts of the corresponding deprotonated peaks; d) TOF detector response curves for Phe-Phe-Phe-OH from 500 pmol to 400 fmol (over 3 concentration orders). The dotted lines above and below the lines of the best linear fit are 99% confidence bands for the data to be linear, p < 0.0001. Figure 8: 8a) An average of 20 scans of MALDI TOF MS data acquired from the mixture of the C-18 Zip-tip cleaned regurgitate of Manduca sexta and DMAN. Each peak is annotated with the chemical structure of the corresponding deprotonated analyte. CID spectra of 8b) m/z 279.2; 8c) m/z 384.1 ; 8d) m/z 406.2; 8e) m/z 408.2. Figure 9: Proposed mechanism for the gas phase fragmentation of deprotonated FACs (see Fig. 8b-e).

Figure 10: Averaged (30 scans with 20 laser shots per scan) MALDI TOF/MS negative ions spectra for 2.5 nmol stearic acid using: a) 1 ,8- bis(dimethylamino)naphthalene b) N,N-dimethylaniline c) 1 ,8-diaminonaphthalene d) aniline as MALDI matrixes. Inset in each section shows the mass spectra in the region 0-200 Th. Peaks marked 'A' correspond to stearate anion at m/z 283.2. Peaks in 'c' marked as 'M' correspond to matrix peaks, first peak at 313.1 corresponding to [2M-3H]^', and second peak at 336.0 corresponding to [2M-3H+Na]\ e) Comparison of the stearate monoisotopic signal for 2.5 nmol of stearic acid when mixed with 2.5 nmol of the four matrices. Red peak corresponds to the signal obtained with DMAN as matrix, green peak with N,N-dimethylaniline as matrix, pink peak with 1 ,8- diaminonaphthalene as matrix and blue peak with aniline as the matrix.

Figure 11 : Fig. 11 a) and b) A plot of relative intensity of trifluoroacetate (solid line) and stearate (dashed line) anions versus MALDI matrix (DMAN in a and DMA in b) concentrations plotted for clarity as Iog2 [matrix]. The amount of analytes was kept constant at 500 pmol and the matrix concentrations were increased to have the following matrix to analyte molar ratios: 0.02:1 , 0.05:1 , 0.1 :1 , 0.2:1 , 0.5:1 , 1 :1 , 2:1 , 5:1 , 10:1 and 100:1 for which MALDI TOF/MS measurements were made; a) 100% = 4000 counts; b) 100% = 35000 counts. Error bars represent s.e.m (n=5, 20 scans with 20 laser shots per scan). Fig 11 c) A plot of pKa versus limits-of-detection for four different acids namely, TFA, TCA, PA, SA. The solid line represents the LOD studies with DMAN as matrix (TFA 300 fmol; TCA 750fmol; PA 7.8 pmol; SA 15.6 pmol). The dashed line represents the LOD studies with Λ/,Λ/-dimethylaniline as matrix (TFA 1pmol; TCA 2.5 pmol; PA 10 pmol; SA 20 pmol). Inset shows the same curve for just two acids, TFA and TCA to highlight the difference in the LOD obtained for the two acids.

Fig 12: MALDI TOF/MS positive ion spectra for 250 pmol of a) triethylamine; b) diisopropylamine; c) N-ethyldiisopropylamine; d) 1 ,8-diazabicyclo[5.4.0]undec-7-ene. e) Limits-of-detection curves for DBU (solid line, 7.8 pmol) and triethylamine (dashed line, 31.25 pmol).

Figure 13: Comparision of mass spectra of 1 ,8- bis(dimethylamino)naphathalene (DMAN) with conventional matrices; 4-hydroxy- alpha-cyanocinnamic acid (α-CHCA) and 2,5-dihydroxybenzoic acid (DHB) when mixed with steaic acid (SA) in 1 :1 molar ratios. Positive mode analysis: a)spectra for alphay-CDCA + SA, b) for DHB + SA, c9 for DMAN + SA. Spectra in panels a) and b) are moinated by copius matrix ions with no signal for [M+H]⁺ of SA at m/z 285. clearly, none of the three matrices show the protonated analyte. Panel c) shows DMAN [M+H]⁺ at m/z 215 resulting from DMAN protonated with SA. Negative mode analysis: d) spectra for alpha-CHCA + SA, e) for DHB + SA, f) for DMAN + SA. Spectra in panels d) and e) show only copius matrix clusters with no signal for the deprotonated stearate ion at m/z 283; single ion at m/z 283 corresponding to stearate anion is observed, with no additional matrix peaks (f). Figure 14. Sections of negative mode mass spectra obtained according to the present invention from diverse biological materials. Matrix DMAN was dissolved in chloroform/methanol (2/1) mixture was applied either on extracts or directly on tissues, (a) Spectral profile of A. thaliana Col-4, 4 week-old leaf scratched with a scapel and with DMAN applied. The identified compounds are shown in table 3. (b) Overlaid mass spectra measured from extracts of D. . melanogaster males and females show distinct sex-related peaks, (c) Mass spectra oftained from D. melanogaster¹ s body (upper panel) and A. pisum wing (lower panel), (d) A drop of human blood deposited on the target covered with the matrix solution provided a rich spectrum of blood-associated fatty acids. Detailed description of the present invention

The present invention relates to novel and rational matrix development for Matrix Assisted Laser Desorption/lonization (MALDI) analysis of low-molecular- weight compounds. In particular, the present invention relates on one hand to a matrix for MALDI analysis in a negative ion mode and, on the other hand, to a matrix for MALDI analysis in positive ion mode. Both allowing detection of various analytes, in particular low-molecular-weight compounds of ≤ 2000 Daltons based on the formation of a salt between the analyte and the matrix compound. Further, the present invention relates to rationalization of the MALDI matrix selection process depending on the polarity of analysis and the polarity of the compounds to be studied and the ability ot form a salt between the analyte and the matrix compound. This represents a significant advance since matrix selection, which is the heart of the

MALDI process, has remained an empirical approach since the birth of the technique.

In case of MALDI analysis in negative ion mode, the matrix compounds according to the present invention are of general formula I

wherein

Y is a nitrogen atom or a phosphorus atom, Ri and R₂ are independently of each other selected from hydrogen, aliphatic, alicyclic or aromatic group whereby at least one of R₁ or R₂ is not a hydrogen; R₃ is an aromatic group, and n is an integer >1 allowing the detection of analytes of ≤ 2000 Daltons. Alternatively, the matrices for MALDI analysis in positive ion mode according to the present invention are of general formula IV

R₅ -( Z - H)_n (IV)

Z is selected from SO₃, BO₂ or PO₃, R₅ is an aromatic group which may be substituted, and n is an integer >1 allowing the detection of analytes of < 2000 Daltons.

The term "aliphatic group" as used herein refers to carbon atoms joint together in a straight-chain or branched chain which may be substituted, including alkanes, alkenes and alkynes. Substituents are typical substituents of carbon atom groups, like hydroxy groups, carboxylate groups, nitrogen containing groups, sulphur containing groups, oxygen containing groups and halogens.

The term "alicyclic group" as used herein refers to carbon atoms forming a non-aromatic ring system which may be substituted. Substituents of the alicyclic group include hydroxy groups, nitrogen containing groups, oxygen containing groups, sulphur containing groups, halogens, aliphatic groups etc.

The term "aromatic group" as used herein refers to groups having aromaticity, namely having a conjugated ring of unsaturated bonds or ion pairs of electrons and satisfying the Huckle's rule which states that the an aromatic system should have 4n + 2 electrons, where n is an integer > 0. Said aromatic group includes aryl and heteroaryl groups whereby said heteroaryl groups may contain heteroatoms of N, O, B, P, or S. Preferably, the residue Y is a nitrogen atom. That is, preferably, the compound is a tertiary amino group, like dialkylamino group. Furthermore, n is preferably an integer of 1 , 2, 3, or 4.

In a preferred embodiment, the matrices of general formula I for MALDI analysis in negative ion mode are matrix compounds of general formula Il or III

wherein

Ri and R₂ are as defined above. In a particular preferred embodiment, the residues Ri and R2 are independently selected from C-| to C12 aliphatic groups, like C-| to C12 alkane, in particular Ci to CQ alkane, like methyl, ethyl, propyl, butyl, pentyl or hexyl.

Particular preferred matrix compounds for MALDI analysis in negative ion mode are 1 ,8-bis(dimethylamino)naphthalene (DMAN), or N,N-dimethylaniline. The matrix for MALDI analysis in negative ion mode is preferably characterized in that the pKa of matrix protonation is above 3, likely above 5, preferably above 11 and simultaneously, the pKa of deprotonation of residues Ri, R2 or R₃ bound to Y of the matrix, namely, the central N or P atom, is above 35, likely above 40, preferably above 50. pKa is defined as the negative logarithm of the acid dissociation constant Ka.

Preferred, the matrix compounds useful for MALDI analysis in a negative mode do not have any acidic protons. That is, the aromatic residue R₃ does not contain any group liberating positively charged hydrogen atoms (protons) during MALDI analysis (acidic protons) and so forming anions. In case of the matrix for MALDI analysis in positive ion mode according to the present invention, matrix compounds of general formula IV are used.

R₅ -( Z - H)_n (IV) wherein Z is selected from SO₃, BO₂, or PO₃, R₅ is an aromatic group which may be substituted, and n is an integer > 1.

Preferably, the residue Z is SO₃ and n is an integer of 1 , 2, 3, or 4, in particular, 1.

For all matrix compounds according to the present invention, the aromatic group is preferably a mono or bicyclic aromatic group, like a phenyl group or naphthalene group. Of course, other aromatic groups may be used. Said aromatic groups may be aryl groups or heteroaryl groups, preferably, aryl groups are present. Preferably, the aromatic residue R₃ or R₅, respectively, is able to absorb UV light of the frequency of 300nm to 400nm.

In case of compounds of general formula IV, the substituent R₅ is preferably a mono or bicyclic aromatic group whereby said aromatic group is substituted with at least one substituent Re whereby R₆ is selected from a hydroxy group, an aliphatic group, an alicyclic group or an aryl group. In a preferred embodiment, the aromatic group R₅ is not substituted or has substituents which will enhance the acidity of the matrix compounds through inductive and mesomeric effects without themselves having an exchangeable acidic proton or a basic functional group capable of protonation. The matrix for MALDI analysis in positive ion mode is preferably characterized in that the pKa of matrix protonation is below -12, like below -20, preferably below -25 and simultaneously pKa of deprotonation of matrix Z-H group is below 5, like bellow 4, preferably below 3..

The matrix compounds useful for MALDI analysis in a positive mode do not have any basic functions. That is cannot attracts any positively charged species like proton, alkali metal cations. In other words the matrices for analyse in positive mode are not forming positively charged ions The matrix compounds according to the present invention are particularly useful for the analysis of low-molecular-weight compounds using MALDI. In this connection, the term "low-molecular-weight compounds" refers to compounds of ≤2000 Daltons. Preferably, the method allows analyzing compounds ≤1000 Daltons, like <700 Daltons, preferably, <500 Daltons. Typical low-molecular-weight analytes include fatty acids, amino acids, fatty acid- amino acids conjugates, plant and animal hormones, vitamins, short peptides, aliphatic, cyclic and aromatic acids including but not exclusive very volatile acids like trifluoroacetic acid and trichloroacetic acid,

On the other hand, analytes to be studied using MALDI analysis in positive ion mode include basic low-molecular-weight analytes like extremely volatile bases like triethylamine, short and long chain aliphatic, cyclic and aromatic bases.

Said analytes can be measured at physiologically relevant concentrations. That is, the present invention relates in a further aspect to a method for analysing low-molecular-weight compounds containing acidic function(s) as well as basic function(s) in a range as low as one picomole or even in the femtomole range. Thus, the present invention provides a new possibility for analyzing said low-molecular- weight compounds, like biologically significant markers for various purposes using MALDI analysis with high-sensitivity and specificity. As demonstrated in the examples and the figures, the matrix itself have no peaks in the spectrum and, additionally, no peaks arising from neutral losses of water or carbon dioxide are observed.

Hence, the matrix compounds according to the present invention are particularly useful for analyzing said low-molecular-weight compounds. One of the representative classes of said low-molecular- weight compounds include fatty acids. The present invention allows the analysis of all types of fatty acids, saturated as well as unsaturated fatty acids which was not possible before. Fatty acids are important biomolecules which have been studied extensively as biologically significant markers for diagnosing infectious diseases, exploring organismal response to environmental factors and for taxonomic species classification. Moreover, the present invention is not only limited to fatty acid analysis, which by itself is a significant advance, but also encompasses other chemically diverse analytes including short peptides, amino acids, vitamins, plant and animal hormones, aliphatic cyclic and aromatic acids and even extremely small volatile acids and bases like trifluoroacetic (TFA) and trichloroacetic (TCA) acids, triethylamine (TEA) which were never thought to be amenable under conventional vacuum MALDI conditions. Not to be bound to theory, it is assumed that the matrix according to the present invention allows the formation of salt/ion pairs between the analyte and the matrix. Fig 1 describes the ion formation using the matrix compounds of the nature according to the present invention in the negative ion mode, specifically using DMAN. Briefly, DMAN belongs to the class of compounds called "proton sponges". The name comes from the ability of the compounds to "mop up" any available protons. Hence the present theory of ion formation is that on mixing with acidic analytes even the weakly acidic proton on the -COOH group of the analytes is taken up by the DMAN, more specifically, it chelates between the two nitrogen atoms on DMAN forming a 2- electron 3 centre bond. This creates a stable salt/ion pair between the analyte and the matrix in solution and the charge state of the respective compounds is retained in the solid crystalline phase. Thus, the mode of action according to the present invention is different to the action of ionic fluids typically applied in MALDI technology. The salt formed by the analyte and the matrix crystallize on the support and, then, UV laser desorbs the salt due to UV-absorbing properties of the matrix compounds. The added energy dissociates the salt into ions in the gas phase, which are then detected in mass spectrometer.

Thus, it is possible to allow an "ionless" analysis of the analyte. In this connection, the term "ionless" means that no matrix-related interfering ions are present during detection of the analytes by MALDI.

Another embodiment of the present invention relates to a method for selecting appropriate matrix compounds to be used for MALDI analysis of predetermined analytes. Said analytes are predetermined in the fact that the acidity or basicity is known. The method for selecting an appropriate MALDI matrix for the analysis of either acidic or basic analytes comprises the following steps:

- determining the pK of the analytes to be analysed with MALDI,

- selecting the matrix compounds based on the pKa value of the matrix, the pK of the deprotonation and protonation, respectively, of substituents of the matrix compounds, characterized in that for acidic analytes the matrix compound selected has no acidic protons and a pK for deprotonation >20 while for basic analytes, the matrix compounds have a pK for protonation <0 and no basic function.

All parameters mentioned above are available by quantum mechanic calculations, especially using DFT (density functional theory) methods. The values for new matrix candidates could be calculated "in silico" and new lead compounds can be then tested experimentally. For example, for DMAN the values for pKa is 13.6 (protonation) and for deprotonation 64.6. (both in ethanol) That is, preferably, the MALDI matrix compounds are selected on the parameters that none of substituents of the central Y atom, namely, P or N, of general formula I contain acidic hydrogen atoms having a pKa of deprotonation higher than 40 and, in addition, the pKa of the matrix is same or higher then pKa of an analyte.

In case of basic analytes, the MALDI matrix is selected on the basis that the Z or R₅ groups of general formula IV do not contain a basic atom or group with pKa of protonation below -10 and in addition, the pKa of the matrix is same or lower then pKa of an analyte. For example for 2-naphthylsulfonic acid the pKa is 2.3 and pKa for matrix protonation is -20.5.

The pKa of the MALDI matrix and of the substituents R-| , R2 and R3 as well as R₅ and Re are typically calculated using quantum mechanical calculation in gas and condensed phase for example as illustrated for the four bases in table 1.

In addition, the present invention relates to a method for analyzing analytes, in particular low-molecular-weight compounds, containing acidic function(s) by MALDI analysis comprising the step of

- mixing the sample to be analyzed with the MALDI-matrix according to the present invention a ratio of 0.01 :1 to 100:1 matrix/sample;

- depositing said mixture on a MALDI-support; - ionising the matrix sample mixture using UV-laser in the range of 200 to

400 nm wavelength,

- detecting ions formed in the ionising step before whereby no matrix-related interfering ions are produced for detection. In case of compounds having basic function(s), the method for analyzing compounds, in particular, low-molecular weight compounds, by MALDI comprise the step of

400 nm wavelength,

- detecting ions formed in the ionising step whereby no matrix-related interfering ions are produced for detection. Particularly preferred, the wavelength of the laser is 337 or 355 nm, respectively.

A further aspect relates to a system for the analysis of analytes, in particular, of low-molecular-weight compounds with MALDI comprising the matrix compounds according to the present invention. Preferably, said system further comprises a sample support wherein the mixture of the matrix compounds with the analytes are deposited. Optionally, said system contains further components required for preparing the matrix compound/analyte mixture including solvents, instruction for use etc.

In addition, the present invention allows for quantification of an analyte in a sample by MALDI analysis using the matrix compounds according to the present invention. The method for quantification comprises the steps of

- providing a plurality of matrix-analyte molar mixtures with known amounts of bases in case of studying acids as analytes and with known amounts of acids in case of studying bases, respectively, - determining the relative intensity of the analyte with the MALDI technique,

- Construct x,y plot of matrix/analyte mixtures as x axis and intensity obtained for the analyte as y axis as illustrated in fig 11a and 11 b.

- determining the amount of the analyte based on the point of equimolarity of analyte and matrix compounds. The above method for the quantification of analytes, relative or absolute quantification enables quantification without using an extra standard whether external or internal standard. Moreover, no backgrounds ions of the matrix interfere with the quantification.

A wide variety of analytes including extremely volatile compounds can be analyzed using the matrix compounds according to the present invention.

The diverse biological applicability and versatility of the present invention will be further illustrated by means of examples. However, the invention is not restricted on the examples provided but the skilled person is well aware of the fact that modifications thereof are possible without leaving the scope of the present invention. Examples Materials

Palmitic, stearic, arachidic, oleic, linoleic, linolenic acids and DMAN were purchased from Sigma-Aldrich (St. Louis, MO, USA). The peptides were purchased from Bachem (Bubendorf, Switzerland). PEG 600 Sulfate was purchased from TCI (Antwerp, Belgium). Alprostadil was purchased from Tocris Bioscience (Elisville, MO, USA). HPLC-grade solvents, methanol, ethanol, acetone and chloroform were purchased from Roth (Karlsruhe, Germany). Synthetic FACs were kindly provided by the Department of Bioorganic Chemistry and the regurgitate of Manduca sexta by the Department of Molecular Ecology, both at the Max Planck Institute for Chemical Ecology, Jena, Germany. Sample preparation

Stock solutions of all the analytes were made in respective HPLC-grade solvents at 1nmol/μl. Fatty acids and other analytes were prepared in ethanol. The peptides were first dissolved in a small amount of acetic acid and then made up with ethanol to the desired concentration. DMAN was made up at the same concentration as the analytes in ethanol. One μl of the analyte was premixed with 1 μl of the matrix in an Eppendorf tube and 1 μl of the resulting mixture was spotted on a 96-well MALDI plate (Waters/Micromass, Manchester, UK) and allowed to dry under a gentle stream of argon.

Serial dilutions were made from the stock solution for limits-of-detection studies. DMAN was dissolved in ethanol (approx. ~ 2.1mg/ml). MALDI mass spectrometry

A MALDI micro MX mass spectrometer (Waters/Micromass, Manchester, UK) fitted with a nitrogen laser (337 nm, 4 ns laser pulse duration, max 330 μJ per laser pulse, max 20 Hz repetition rate) was used in reflectron mode and negative polarity for data acquisition. The instrument operated with voltages of 5 kV on the sample plate, 12 kV on the extraction grid, pulse and detector voltages of 1.95 kV and 2.35 kV, respectively. The laser frequency was set to 5 Hz and energy was optimized for different analytes (fatty acids at 80 μJ per pulse, peptides at 90 μJ per pulse). The extraction delay time was optimized to 150 ns. PEG 600 sulfate was used to calibrate the mass spectrometer for a mass range of 100-1200 Th in the negative ion mode. For positive mode calibration, a mixture of PEG 200 and 600 was used. The chemical identity of the FACs observed in the M. sexta regurgitate was confirmed by tandem mass spectrometry on an ion trap (LTQ) instrument (Thermo Fisher, San Jose, CA, USA) with an AP-MALDI source equipped with a solid-state Neodymium- Doped Yttrium-Aluminium-Garnet (Nd-YAG) laser (MassTech, Columbia, MD, USA) and running Target 6 (MassTech) and Excalibur v.2.0 (Thermo) software for data acquisition. Example 1

DMAN as matrix compound and fatty acids as analytes

Clear signals for acid anions were observed for all the tested fatty acids, namely, palmitic;16:0, stearic;18:0, arachidic;20:0, oleic;18:1 , linoleic;18:2 and linolenic;18:3 acids, as shown in Figure 2. Here it is worth noting that it was also possible to observe clear signals for fatty acids with multiple unsaturations (linoleic and linolenic, Figure 2e and f) which was not possible before with MALDI. Also, unlike in DIOS analysis, no extensive alkali adducts formation was observed; the spectra were thus clearer and more suitable for studying complex biological samples. Further, a considerable improvement in analyte sensitivity compared to any previous reports has been observed. Standard limits-of-detection tests showed that clear signals could be observed for as low as 1 pmol of stearic acid with a signal-to-noise of 3:1 (Figure 3). Another notable feature is the total suppression of any matrix- related ions in the entire low mass region (< 1000 Daltons). This can be attributed to the fact that after being mixed with a carboxylic acid, the proton sponge, here DMAN, gets protonated and forms an extremely stable hydrogen-chelated cation easily observable in the positive mode MS spectrum. The ability to completely suppress matrix related ions makes DMAN an excellent matrix for studying complex biological fluids. Example 2

DMAN as matrix compound and diverse low-molecular-weight compounds as analytes

Furthermore, the detection of volatile analytes was investigated. As shown in figure 4, single amino acids (fig. 4c, cysteine), vitamins (fig 4d, ascorbic acid), plant hormones (fig 4h, gibberellic acid) and animal hormones (fig 4j, alprostadil) and even short and extremely volatile acids (fig 4b, trifluoroacetic acid) can be analysed. Again negligible fragmentation was observed except for polyhydroxylated compounds. Elimination of one or two water molecules was noticed only for alprostadil (prostaglandin E₁), (Fig 4j), possible due to allylic and the homoallylic nature of two hydroxyl groups.

Limits-of-detection studies (S/N = 5:1) with stearic acid showed linearity for nearly 1.5 concentration orders over the entire picomole range (Fig. 5a and b). Here it should be noted that no alkali adduct formation was observed in the entire detection range of stearic acid (Fig. 5a) which is a notorious problem of DIOS technology. Volatile acids such as TFA and TCA, which cannot generally be studied using vacuum MALDI, could be detected with femtomole sensitivity (TFA - 300 fmol and TCA - 750 fmol; S/N = 5:1). Limits-of-detection studies with both TFA and TCA showed excellent linearity over 2 concentration orders from 500 pmol to 3 pmol for both the acids (Fig.6). The ability to analyze such a wide range of metabolites makes it possible to detect these low-molecular-weight analytes from biological systems (extracted fluids or even direct tissue analyses). Moreover, that even extremely volatile acids such as TFA and TCA were detected is extremely interesting not only from a mechanistic point of view, but also since such volatile analytes have not previously been reported to be detectable under the high vacuum environment of the MALDI systems. Example 3 Analysis of short peptides

Following the method described above several short di- and tri-peptides were analyzed using DMAN as the matrix. Since the peptides were not soluble in ethanol alone, they were first dissolved in a small amount of acetic acid and then made up to the desired concentrations in ethanol. Once again, clear singly charged anionic signals were obtained. Fig. 7 shows the deprotonated ions for GIu-VaI-OH at m/z 245.0 (Fig. 7a), Phe-Phe-Phe-OH at m/z 458.1 (Fig. 7b) and Glu-Val-Phe-OH at m/z 392.1 (Fig. 7c). The peptides showed sodium adduct formation ([M-H+Na]^") for higher amounts (> 250 pmol). Limits-of-detection studies with Phe-Phe-Phe-OH indicated that the peptide could be identified up to 400 fmol (S/N = 5:1). Moreover, the dynamic range of linearity was over two orders of magnitude, from 500 pmol to 400 fmol (Fig. 7d). Example 4

Determination of lipids in a regurgitate of Manduca sexta by MALDI analysis Preparation of regurgitate of M. sexta for MALDI analysis

The crude regurgitate of M. sexta was desalted with the following C-18 Zip-Tip procedure prior to MALDI analysis. 1) The Zip-Tip (Millipore, MA, USA) was pre-wetted by aspirating the wetting solution (50% ethanol in MiIIi-Q grade water) into the tip. The solution was dispensed to the waste.

2) The Zip-Tip was equilibrated for binding with washing solution (1% acetic acid in IiIIi-Q grade water). 3) The analytes were bound to the Zip-Tip by aspirating and dispensing the sample 10 times.

4) The Zip-Tip was washed twice for desalting with washing solution (1 % acetic acid in MiIIi-Q grade water). 5) The analytes were eluted from the Zip-Tip by dispensing 2 μl of elution solution (100% ethanol, 1% acetic acid) into a clean vial. The elution procedure was repeated three times to collect three fractions.

Sample preparation and MALDI measurement were performed as described in example 1. Namely, the eluent from the Zip-Tip was directly mixed with DMAN (ethanolic solution) and spotted on the MALDI target. The dried spots were then analyzed as mentioned in the methods section. Fig. 8a shows the averaged mass spectrum from 20 scans obtained from the spot. Almost all the peaks observed in the spectrum were found to be either fatty acids or fatty acid-glutamic acid conjugates (Fig. 8a). The identity of the peaks was confirmed by carrying out Collision-lnduced- Dissociation (CID) experiments and by comparing the MS² spectra observed to those obtained from standard compounds (Fig. 8b, c, d and e and Table 1). Eleven different analytes were positively identified through the CID experiments: 5:0-Glu at m/z

230.1 , 6:0-Glu at m/z 244.1 , 16:0 at m/z 255.1 , 18:2 at m/z 279.2, epoxy-18:3 at m/z

293.2, 18:3 at m/z 277.2, 21 :0 at m/z 325.2, 16:3-Glu at m/z 378.1 , 16:0-Glu at m/z 384.1 , 18:3-Glu at m/z 406.2 and 18:2-Glu at m/z 408.2 (all singly charged anions).

The ions at m/z 230.1 and 244.1 were too weak in the AP-MALDI spectra to obtain decent CID spectra. An interesting fragmentation pattern was observed with the FACs according to which they cleaved to certain ions with masses corresponding to the free acids. A fragmentation pattern for the same explaining how the free acid could be obtained from the fragmentation of FACs is proposed (Fig. 9). Table 1

Example 5

To study the effects of matrix structure and basicity, additional bases structurally similar to DMAM, were selected and stearic acid (SA), as a representative medium strong acid (pK 10.15), was analyzed using above mentioned conditions. Strong dependency of stearate ion abundance on pK values of used bases was observed (Fig. 10). Like DMAN, no matrix ions were observed for Λ/,Λ/-dimethylaniline (Fig. 10b, DMA) and surprisingly, for aniline (Fig. 1Od). The negative MALDI spectrum using 1,8-diaminonaphthalene shows, beside expected m/z 283 of stearate, copious matrix cluster ions obscuring both high and low-mass regions (Fig. 10c). It seems that gas-phase basicity of the matrix is important and the suitable MALDI matrix for negative mode must have low tendency for deprotonation under MALDI source conditions. The gas-phase pK of all four bases used here were calculated and the obtained values correlate well with the observed matrix ion formations (Table 2). Table 2

Example 6

A novel quantification strategy without the use of internal standards

Stock solutions of two acids, namely, TFA and Stearic acids were made at 500 pmol/μl. Two matrices, DMAN and DMA were made at different concentrations. Individual matrices were mixed at different amounts with individual analytes at fixed amounts so as to have a plurality of molar ratios ranging from 0.02:1 , 0.05:1 , 0.1 :1 , 0.2:1 , 0.5:1 , 1 :1 , 2:1 , 5:1 , and 10:1 to 100:1 (Matrix/Analyte). MALDI-TOF measurements were made in negative ion mode for each mixture for each set of matrix/analyte mixtures, namely, for DMAN/TFA, DMAN/SA, DMA/TFA and DMA/SA at the above mentioned variable molar ratios. A plot of the relative intensity of analyte ions (here, TFA, solid line and SA₁ dashed line) were made against MALDI matrix concentrations (DMAN in 11a and DMA in 11b) plotted for clarity as Iog2 [matrix]. It is clear from the figure 11a and b that maximum analyte signal was observed at the point of equimolarity. The signal steadily increased with increasing matrix concentrations, reaching a maximum at equimolar amounts and then sharply dipping beyond that. This strategy could be used for quantifying a diverse group of analytes by simply providing a plurality of matrix/analyte molar ratios and determining the point of equimolarity. Example 7

Stock solution of 2-naphthylsulfonic acid (Sigma-Aldrich) was made at 1mM in ethanol. The four simple bases used as analytes, triethylamine, diisopropylamine, N- ethyldiisopropylamine and 1 ,8-diazabicyclo[5.4.0]undec-7-ene were also made at 1mM in ethanol. The spectrum depicted in the fig. 12 is for 250 pmol of analyte mixed with 500 pmol of the matrix (on plate). For calibration curves with DBU and TEΞA (Fig. 12e) stock solutions of the two were made at 1mM. Serial dilutions were made in ethanol. Each acquisition was an average of 20 laser shots. Each point in the spectrum is an average of three such measurements. Example 8

Comparison of DMAN with conventional matrices Using stearic acid as a test analyte, negative mode analysis and positive analysis was performed. Matrices were mixed in a 1 :1 molar ratio with stearic acid. DHB and alpha-CHCA were used for comparison. Example 9 Diverse biological material was analysed with matrices according to the present invention. The results are shown in figures 8 and 14, respectively. Example 10

Analysis of free and bound fatty acids in biofluids Important metabolites are found in nature both in free form or bound to other molecules. For example fatty acids are esterified with diverse alcohols like glycerol and the formed glycerol fatty acids esters are furthed functionalized to diverse and important lipids like phospholipids, glycolipids etc. Our invention open a possibility to determine both the free and bound (esterified) fatty acids in a sample. Using of one of the above mentioned matrices working in negative ion mode and by stable isotopic labeling. During the labeling experiment, where the bound acids are liberated by alkaline hydrolysis using ¹⁸O labeled hydroxide in ¹⁸O water one of the oxygen in resulting free acid will bear isotopic signature of ¹⁸O. Thus the mass of the free acid and the acids of the same molecular composition liberated by the above mentioned hydrolysis will differ by two nominal mass units and so being distinguishable mass means of mass spectrometry analysis. This labeling allows for simultaneous determination of free/bound fatty acids in biofluids. Additionally, known amounts of the ¹³C Of ²H (deuterium) labeled internal standard(s) could be added for absolute quantification. The same method could be used for other acids (steroid hormones, phytohormones, plant metabolites etc) which are bound to other molecules by ester or amide bond. Alternatively we can perform two independent experiments, the first one without hydrolytic ¹⁸O labeling just with addition of internal standards to determine the free acids and other compounds ionizable in the presence of one of the matrices working in negative ion mode. Subsequently the same sample including internal standards is hydrolyzed with ¹⁸O labeled water in presence of base, neutralized and analyzed for total acid(s) content. Human plasma sample (10 μl) was spiked with ¹³C labeled fatty acid internal standard (myristic, palmitic, stearic acid) and an three aliquots (1 μl ) were mixed with DMAN matrix dissolved in ethanol (1 μl, 100 μM). One μl of the individually prepared sample/matrix mixtures using was separately deposited on metallic MALDI target and analyzed as described in above examples. The peak intensities of measured metabolites (acids) were compared to the intensities of internal standards and quantification was performed using correction factors obtained from 5-point calibration curve using the same internal standards. The left plasma sample (7 μlwas treated with a solution of in 1 M Na¹⁸OH in H₂ ¹⁸O (prepared from 99% H₂ ¹⁸O and sodium metal) at 60⁰C for 1 h upon vigorous shaking. The reaction mixture was acidified (1 M -HCI) to pH 4 and extracted tree times with hexane. Hexane extracts were evaporated to dryness and re-dissolved with ethanol (7 μl).Three one μl aliquots were analyzed as described in the above paragraph. The proportions of signals of unlabeled acids (which were free in the sample) and those bearing ¹⁸O label (being bound and not ionized in the first paragraph measurement) were determined using high resolution capabilities of mass analyzers available (time- of-flight, Orbitrap, or ion-synchrotron-resonance). Using the above obtained calibrations using internal standards the total fatty acid content was determined.

Brønsted-Lowry concept was successfully applied for the selection on "ionless" matrices with the potential to study a wide variety of metabolites. Conventional matrices such as 4-hydoxy-α-cyanocinnamic acid (α-CHCA) and 2,5- dihydroxybenzoic acid (DHB) produce copious interfering ions in the m/z < 500 Th range, making detection of metabolites in that range rather difficult, if not impossible (Fig. 13a, b, d, e). We reasoned that mixing an acidic or basic analyte with a strong base or acid employed as a matrix, respectively and crystallization of the salt formed on a target, followed by desorption of the formed salt by a laser pulse having frequency similar to the absorption maximum of base/acid matrix, should produce ions that are oppositely charged to those of the absorbing partner Fig. 13c, f. To explore this concept, we investigated ion pairs of four aromatic bases with aliphatic acids monitoring the formation of anions using TOF mass spectrometry. The first experiments were performed with a "proton sponge", which is a strong base (pK_a of its conjugate acids 12.5, 1 ,8-bis(dimethylamino)naphthalene (DMAN), never previously used as a matrix. Analytes (fatty acids, fatty acid-amino acid conjugates and other anionic species) were mixed in a 1 :1 molar ratio with DMAN in ethanol and allowed to dry on a MALDI metallic target. Under both vacuum and atmospheric pressure, gas-phase anions were formed upon U.V.-laser irradiation (337 or 355 mm) and detected at physiologically relevant concentrations. Negligible fragmentation was observed except in polyhydroxylated compounds such as water losses in prostaglandin El In contrast to common MALDI matrices, no matrix ions were detected. The absence of matrix ions in mass spectra affords a crucial advantage over existing matrices. To ensure that we were observing a matrix-assisted process and not later desorption/ionization (LDI), mixtures of an aliphatic base (triethylamine) and an inorganic base (KOH) with strong aliphatic acids (e.g. trifluoroacetic acid) were made and tested for the presence of anion signals. No analyte signal was observed even at ablative laser fluences, thus confirming the importance of the U.V. absorbing partner in the ionization process.

Potential of "ionless" matrices for targeted metabolomics was demonstrated on plant, insect and blood samples. In-situ profiling of a damaged Arabidopsis thaliana leaf spotted with DMAN solution provided rich negative ion- mode mass spectra (108 monoisotopic peaks; Fig. 14) with the mass accuraey suitable for identifying at least 46 metabolites using the KNApsAcK database (Table 3), which exceeds the number of metabolites identified from a single biological tissue by the existing state-of-art LAESI-MS method. Most of the intermediates of important plant metabolic pathways (Krebs cycle, fatty acid biosynthesis and glucosinolate biosynthesis) were detected (Fig. 14a inset and Table 3). In addition to primary metabolites, secondary metabolites such as kaempherol glycosides were identified (Table 3). The analysis of individual or pooled extracts of Drosophila melanogaster males and virgin females showed distinct sex-related profiles (Fig. 14b) composed of short, medium and long-chain fatty acids, many more than were detected using GC/MS. Direct profiling of male D. melanogaster's body (Fig. 14c; upper panel) and pea aphid's (Acyrthosiphon pisum) wing (Fig. 14c; lower panel), showed clear ion signals, presumably lipids, thus documenting the potential of our method for tissue profiling/imaging. Clear spectra were also obtained from sub μL volumes of human blood (Fig. 14d), thus highlighting the potential of the new matrices for clinical diagnostics by direct analysis of biofluids. Overall, our approach has a wide range of applications - high-throughput metabolomic screening, tissue profiling/imaging, direct biofluids analysis, which can be further developed for biomedical studies. Table 3: List of 46 metabolites identified in negative ion MALDI-TOF/MS profiling of Arabdopsis thaliana CoI-O 4-week-old leaf damaged with a scalpel and spotted with DMAN (10 mg/mL in 2:1 CHCI₃:CH₃OH)) matrix. Data obtained on externally calibrated instrument were internally recalibrated; the mass accuracies for identified metabolites are within ± 20 mDa.

a identified as [M-H]^" ions, ^b using KNApsAcK database

(http: //prime. psc.nken. jp/?action=metabolites_index ), TCA = tricarboxylic acid cycle = Krebs cycle

The acid-base model was further validated by a combination of MS, NMR, X-ray experiments and supported by density functional quantum chemical calculations. To study the effects of matrix structure and basicity, additional bases structurally similar to DMAN were selected, and stearic acid (SA) as a representative acid (pK_a = 10.15), was analyzed. A strong dependence of the stearate ion abundance on the pK_b values of the studied bases was observed. As in DMAN, no matrix ions were observed for N,N-dimethylaniline (DMA) and aniline. The negative- ion TOF mass spectrum obtained using 1 ,8-diaminonaphthalene shows, in addition to the expected m/z 283 of stearate, copious matrix cluster ions obscuring both high- and low-mass regions. It seems that the gas-phase basicity of the matrix is important and that the suitable matrix in the negative-ion mode must not tend towards deprotonation. The gas-phase pK_b of all four bases used here were calculated and the obtained values correlated well with the observed matrix ion formations. Given the results of these preliminary experiments, we suggest a mechanistic model (Eq. 1) to explain our observations.

pAT_a/ pKb ion pair complex

(Eq I) AH + M «→ A^" ÷ MH⁺ → A ... MH⁺ / AH ... M -+ U.V. ablation -♦ A- liquid phase crystal phase gas phase

The equilibrium between the ion pair and the associated base-acid complex in the liquid phase is characterized by pK_a and pK_b values of base or acid in the solution in terms of the Brønsted-Lowry concept. This equilibrium is reflected in the crystal phase and the amounts of observed ions are manifested by the ion pair/complex equilibrium. We are not considering gas-phase ionization/re-ionization. According to the proposed model, the ion yield should be dependent on both the matrix/analyte ratio and the pK_a (Eq. 1). Based on the suggested mechanismus it is proposed to name it as Matrix-Assisted Ionization/Laser Desorption, abbreviated as MAILD mass spectrometry.

In further experiments this model was tested using: [1] different matrix/analyte molar ratios and [2] diverse ion pairs form bases and acids with different pK_a values. The effect of increasing amounts of DMAN and DMA co-crystallized with 1 molar equivalent of trifluoroacetic acid (TFA. 11a) or SA (Fig. 11b) on trifluoroacetate and stearate intensity, respectively, is summarized in Fig. 11. As predicted by the model, the ion intensity increased from a 0-1 molar equivalent of bases, reaching a maximum at equimolar proportions. Upon further increase of the amount of base, signals for both anions decreased, probably due to the gas-phase ion re- neutralization. DFT/B3LYP calculations show high stability of the AH... M complex with respect to its dissociation into A^" and MH⁺ species in the gas phase (106 kcal.mol^'1 for a simple acetic acid and DMAN model), preventing gas-phase reionization. A similar trend was observed for the much weaker base, DMA (Fig. 11a, b), suggesting that these phenomena might be rather general. This observation differs from the widely accepted idea that excess matrix/analyte ratio has beneficial effects. Here it must be noted that for the analysis of large biomolecules excess matrix may act as an energy receptacle or a buffer zone preventing analyte degradation. Furthermore, the observed maximum in Fig. 11a, b at equimolar proportions of base and acid could be used to quantify acids/bases using a series of mathx-analyte mixtures with known amounts of the matrix. This would be the first use of a matrix-assisted method for absolute analyte quantification. The effect of pK_a/pK_b on the ion yield was investigated using two bases as matrixes and four acids with a pK_a range of 0.5-10.2. The limit of detection (LOD) at signal-to- noise (S/N) ratio 5 was determined for studied pairs and plotted on a pK_a scale (Fig. 11c). An unambiguous dependence of LOD values on decreasing pK_a was observed. This agrees with the Brønsted-Lowry concept, which predicts increased ion pair stability with decreasing pK_a - pK_b difference. This trend can be supported by DFT calculations. Only two halogenated acids are strong enough to form A^" ...MH⁺ pair in ethanol solutions of the studied acids and DMAN. The Gibbs free energy difference for the ionization process as described in equation 1 favors a strong add.

For two base-acid systems (DMAN + SA and DMAN + TFA), the conjugated acid- base was examined more carefully using crystallization, X-ray diffraction and melting point experiments. The crystallization of equimolar DMAN mixtures with weak acids (acetic and stearic) did not provide salts with sharp melting points. True salt of DMAN with TFA exhibited a strong fluorescence and a sharp melting point (188⁰C), which was much higher than that of DMAN (65⁰C). The NMR measurements on isolated crystals of DMAN + TFA salt confirmed the equimolar proportions of CF₃COO^' and DMAN+H+. Critical data obtained using X-ray crystallography (deposited in the Cambridge Structural Database under FO3376) further confirmed the salt nature of the crystal. Further confirmation was acquired by DFT calculations. Excellent surface coverage was observed for the matrix crystals alone and when mixed with analytes, limiting the "sweet spot" phenomena of other matrices like DHB. Inspired by the successful use of strong bases for MAILD analysis of acidic compounds, we tested the suitability of several sulfonic acids as MAILD matrices for the positive ion mode. 2-Naphthalenesulfonic acid was successfully applied for MAILD analysis of simple amines (Fig. 12). Clear spectra with no interfering matrix signals were obtained. Again, stronger bases gave higher ion-count manifested in the low LOD observed for diaza(1 ,3)bicyclo[5.4.0]undecane (Fig. 12e). the protonation of 2-naphthalenesulfonic acid in the gas phase is negligible an supports the calculated pK_a value.

A novel ionization mechanism termed MAILD is introduced. The novel matrices developed herein are "ionless", in other words they produce no matrix- related interfering io9ns solving the problem of conventional matrices and allowing the detection of small molecules (0-1000Da). Furthermore, the enormous applicability of our novel matrices in targeted metabolomic studies is illustrated. The analysis of biological tissues/extracts/biofluids shown here clearly demonstrates numerous possible metabolomic applications; a range of biomedical applications, namely high- throughput clinical diagnostics and drug distribution/imaging studies are likely to be developed. Our model, which reflects situations in equi- and subequimolar amounts of a matrix, is a crucial addition to photoionization/protonation and "lucky survivor" MALDI models. Several important physico-chemical characteristics for the rational design of matrices are illustrated: negative-ion mode matrices should have an absorption maximum matching the laser frequency, high basicity (pK_a > 10), and no acidic protons, as ours does. Furthermore, high acidity with minimal protonation in the gas phase seems to be an important characteristic of matrices designed for positive-ion mode analysis. Such simple matrix attributes can be calculated using DFT methods, which opens the door for rational in-silico MAILD matrix design.

Claims

1. The use of matrix compounds of general formula I

wherein

Y is a nitrogen atom or a phosphorus atom,

Ri and R₂ are independently from each other selected from hydrogen, an aliphatic, alicyclic or aromatic group whereby at least one of Ri or R₂ is not hydrogen;

R₃ is an aromatic group whereby said aromatic group does not contain any hydrogen atoms being ionized during Matrix Assisted Laser Desorption/lonization (MALDI) analysis; and n is an integer >1 as a matrix for MALDI analysis in a negative ion mode for detection of low- molecular-weight compounds of < 2000 Daltons.

The use according to claim 1 wherein Y is nitrogen and n is 1 ,

2,

3, or 4.

The use according to claim 1 or 2, wherein the matrix compound of general formula I is a matrix compound according to general formula Il or III

wherein Ri and R₂ are defined as above.

4. The use according to any one of the preceding claims wherein Ri and R₂ are independently selected from the group of C1 to C12 aliphatic groups.

5. The use according to any one of the preceding claims characterized in that the pKa of matrix protonation is above 3, likely above 5, preferably above 11 and simultaneously the pKa of deprotonation of residues R-i, R₂ or R₃ bound to Y of the matrix compound is above 35, likely above 40, preferably above 50, the pka is defined as the negative logarithm of the acid dissociation constant Ka.

6. The use of a matrix compound of general formula IV

R₅ -( Z - H)_n (IV)

wherein

Z is selected from SO₃, BO₂, or PO₃; R₅ is an aromatic group which may be substituted whereby said aromatic group does not contain any hydrogen atoms being ionized during Matrix Assisted Laser Desorption/lonization (MALDI) analysis; and n is an integer >1 as a matrix for MALDI analysis in a positive ion mode for detection of low- molecular weight compounds of ≤ 2000 Daltons.

7. The use according to claim 6 wherein Z is SO₃.

8. The use according to claim 6 or 7 wherein R₅ is a mono- or bicyclic aromatic group and R₅ is substituted with at least one substituent R₆ whereby R₆ is selected from hydroxy, aliphatic, alicyclic or aryl group, and n is 1 , 2, 3, or 4.

9. The use according to any one of preceding claims 6 to 8, characterized in that the pKa of matrix protonation is below -12, like below -20, preferably below -25 and simultaneously the pKa of deprotonation of matrix Z-H group is below 5, like below 4, preferably below 3.

10. The use of the matrixes according to any one of claims 1 to 9 in Matrix Assisted Laser Desorption/lonization mass spectrometry of low molecular weight compounds of ≤ 1000 Daltons.

11. The use according to any one of the preceding claims wherein the matrix compounds do no produce matrix-related interfering ions during detecetion.

12. A method for analyzing low molecular weight compounds of ≤ 2000 Daltons, preferably ≤ 1000 Daltons containing acidic function(s) or basic function(s), respectively, by MALDI analysis comprising the step of

- mixing the sample to be analyzed with the MALDI-matrix according to any one of claims 1 to 5 or 6 to 9, respectively, in a ratio of 0.01 :1 to 100:1 matrix/sample;

400 nm wavelength,

- detecting ions formed in the ionising step before whereby no natrix-related interfering ions are produced for detection.

13. The method according to claim 12 wherein the UV-wavelength are 337 or 355 nm.

14. A method for selecting an appropriate MALDI matrix for the analysis of either acidic or basic analytes, respectively, comprising the steps of: - determining the pK of the analytes to be analysed with MALDI

- selecting the matrix compounds based on the pKa value of the matrix, the pK of the deprotonation and protonation, respectively, of substituents of the matrix compounds, characterized in that for acidic analytes the matrix compound selected has no acidic protons and a pK for deprotonation >20 while for basic analytes, the matrix compounds have a pK for protonation <0 and no basic function whereby the matrix is selected that no matrix-related interfering ions are present during detection.

15. The method according to claim 14 wherein the matrix compounds are matrix compounds as defined in any one of claims 1 to 11.

16. A method for quantification of analytes in MALDI based analysis comprising the steps of

- providing a plurality of matrix-analyte mixture with known amounts of bases in case of determining acids and with amounts of acids in case of determining bases, respectively,

- determining the relative intensity of the analyte with the MALDI based technique,

- determining the amount of the analyte based on the point of equimolahty of analyte and matrix compounds.

17. System for the analysis of low molecular weight compounds with MALDI comprising matrix compounds according to any one of claims 1 to 9.

18. System according to claim 17 further comprising a sample support and/or instructions for use.