Maldi matrices

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

[0001] 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

[0002] The 1980s saw the development of a new ionization technique, Matrix-Assisted Laser Desorption/Ionization (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.

[0003] 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.

[0004] 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. mesotetrakis (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 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.

[0005] 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 (Wej J. et al., 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.

[0006] 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.

[0007] 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 prepreparatory 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.

[0008] 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.

[0009] Thus, 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.

[0010] 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.

[0011] These and other objects are achieved by the present invention.

Brief description of the present Invention

[0012] Firstly, the present invention relates to a matrix for matrix assisted laser desorption/ionization (MALDI) analysis of low-molecular-weight acidic compounds in negative ion mode wherein said matrix compounds are of general formula I

wherein

Y is a nitrogen or a phosphorus atom,

R₁ and R₂ are independent of each other selected from hydrogen, an aliphatic, alicyclic or aromatic group;

R₃ is an aromatic group; and

n is an integer ≥1.

[0013] In a further aspect, the present invention relates to a matrix for MALDI analysis in positive ion mode allowing detection of low-molecular-weight basic compounds, said matrix is a compound of the general formula IV

        R₅-(Z-H)_n     (IV)

Z is selected from COO, SO₃, BO₂, or PO₃, R₅ is an aromatic group which may be substituted, and n is an integer ≥1.

[0014] Another aspect of our invention relates to the optimization of matrix to analyte ratio for analysis of low-molecular-weight compounds. 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.

[0015] 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.

[0016] Moreover, a method is provided allowing quantification of an analyte using MALDI analysis.

Brief description of the figures

[0017] 

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) Glu-Val-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 log2 [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 N,N-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).

Detailed description of the present invention

[0018] The present invention relates to novel and rational matrix development for Matrix Assisted Laser Desorption/Ionization (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. 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. 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.

[0019] 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,

R₁ and R₂ are independently of each other selected from hydrogen, aliphatic, alicyclic or aromatic group;

R₃ is an aromatic group, and

n is an integer ≥1.

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 COO, SO₃, BO₂ or PO₃, R₅ is an aromatic group which may be substituted, and n is an integer ≥1.

[0020] 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.

[0021] The term "alicyclic group" as used herein refers to carbon atoms forming a nonaromatic 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.

[0022] 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.

[0023] 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.

[0024] In a preferred embodiment, the matrices of general formula I for MALDI analysis in negative ion mode are matrix compounds of general formula II or III

wherein

R₁ and R₂ are as defined above.

[0025] In a particular preferred embodiment, the residues R₁ and R₂ are independently selected from C₁ to C₁₂ aliphatic groups, like C₁ to C₁₂ alkane, in particular C₁ to C₆ alkane, like methyl, ethyl, propyl, butyl, pentyl or hexyl.

[0026] Particular preferred matrix compounds for MALDI analysis in negative ion mode are 1,8-bis(dimethylamino)naphthalene (DMAN), N,N-dimethylaniline, or aniline.

[0027] 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 pKa of deprotonation of residues R₁, 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.

[0028] 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 COO, SO₃. BO₂, or PO₃, R₅ is an aromatic group which may be substituted, and n is an integer ≥1.

[0029] Preferably, the residue Z is SO₃ and n is an integer of 1, 2, 3, or 4, in particular, 1.

[0030] 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.

[0031] 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 R₆ 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.

[0032] 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 above 35, like above 40, preferably above 50.

[0033] 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 below 1000 Daltons, like below 700 Daltons, preferably, below 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,

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 our 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The pKa of the MALDI matrix and of the substituents R₁, R₂ and R₃ as well as R₅ and R₆ are typically calculated using quantum mechanical calculation in gas and condensed phase for example as illustrated for the four bases in table 1.

[0044] 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.

[0045] 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

• 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.

[0046] Particularly preferred, the wavelength of the laser is 337 or 355 nm, respectively.

[0047] 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.

[0048] 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 11 a and 11 b.
• determining the amount of the analyte based on the point of equimolarity of analyte and matrix compounds.

[0049] 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.

[0050] A wide variety of analytes including extremely volatile compounds can be analyzed using the matrix compounds according to the present invention.

[0051] 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

[0052] 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

[0053] 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 anslyte 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.

[0054] 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

[0055] 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

[0056] 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 OMAN, 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

[0057] 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.

[0058] 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

[0059] 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 Glu-Val-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

[0060] 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 Milli-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 Milli-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 Milli-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.

[0061] 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-Induced-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

m/z	CID spectra	CID of standards	Identification
255.1	255.1 [M-H]-, 237 [M-H-H2O]-, 211 [M-H-CO2]-	255.1 [M-H]-, 237 [M-H-H2O]-, 211 [M-H-CO2]-	16:0
277.2	277.2 [M-H]-,259 [M-H-H2O]-, 233 [M-H-CO2]-	277.2 [M-H]-,259 [M-H-H2O]-, 233 [M-H-CO2]-	18:3
279.2	279.2 [M-H]-, 261 [M-H-H2O]-, 235 [M-H-CO2]-	279.2 [M-H]-, 261 [M-H-H2O]-, 235 [M-H-CO2]-	18:2
293.2	293.2 [M-H]-, 275 [M-H-H2O]-, 249 [M-H-CO2]-	na1	epoxy-18:3
325.2	325.2 [M-H]-, 307 [M-H-H2O]-, 281, [M-H-CO2]-	325.2 [M-H]-, 307 [M-H-H2O]-, 281, [M-H-CO2]-	21:0
406.2	406.2 [M-H]-, 388 [M-H-H2O]-, 362 [M-H-CO2]- 277, 145, 128	406.2 [M-H]-, 388 [M-H-H2O]-, 362 [M-H-CO2]-, 277, 145, 128	18:3-Glu
408.2	408.2 [M-H]-, 390 [M-H-H2O]-, 364 [M-H-CO2]-, 279, 145, 128	408.2 [M-H]-, 390 [M-H-H2O]-, 364 [M-H-CO2]-, 279, 145, 128	18:2-Glu
378.1	378.1 [M-H]-, 360 [M-H-H2O]-, 334 [M-H-CO2]-. 249, 145, 128	Na	16:3-Glu
384.1	384.1 [M-H]-, 366 [M-H-H2O]-, 340 [M-H-CO2]-. 255, 128, 145	Na	16:0-Glu

Example 5

[0062] 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 N,N-dimethylaniline (Fig. 10b, DMA) and surprisingly, for aniline (Fig. 10d). 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

	Gas Phase			Solvent	(EtOH)
	ΔG(prot)	ΔG(deprot)		ΔG(prot)	Pka	ΔG(deprot)	pKa(deprot)
Aniline	210.0	368.3		267.8	3.3	318.5	40.4
DAN	224.9	352.8		269.7	4.6	310.3	34.3
DMA	222.2	398.5		270.1	4.9	347.4	61.5
DMAN	245.3	393.0		282.5	13.6	351.5	64.6
DMAN_Ac		353.1				311.4	35.2

Example 6

A novel quantification strategy without the use of internal standards

[0063] 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 SA, dashed line) were made against MALDI matrix concentrations (DMAN in 11a and DMA in 11b) plotted for clarity as log2 [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

[0064] Stock solution of 2-naphthylsulfonic acid (Sigma-Aldrich) was made at 1 mM 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 TEA (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.

Claims

1. A matrix for Matrix Assisted Laser Desorption/Ionization (MALDI) analysis in a negative ion mode allowing detection of low-molecular-weight compounds wherein said matrix compounds are of general formula I

wherein

Y is a nitrogen atom or a phosphorus atom,

R₁ and R₂ are independently from each other selected from hydrogen, an aliphatic, alicyclic or aromatic group;

R₃ is an aromatic group; and

n is an integer ≥1.

2. The matrix according to claim 1 wherein Y is nitrogen and n is 1, 2, 3, or 4.

3. The matrix according to claim 1 or 2, wherein the matrix compound of general formula I is a matrix compound according to general formula II or III

wherein R₁ and R₂ are defined as above.

4. The matrix according to any one of the preceding claims wherein R₁ and R₂ are independently selected from the group of C1 to C12 aliphatic groups.

5. The matrix 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 pKa of deprotonation of residues R₁, 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.

6. A matrix for MALDI analysis in a positive ion mode allowing detection of low-molecular weight compounds said matrix are of general formula IV

        R₅-(Z-H)_n     (IV)

wherein

Z is selected from COO, SO₃, BO₂, or PO₃; R₅ is an aromatic group which may be substituted; and n is an integer ≥1.

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

8. The matrix 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 matrix 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 pKa of deprotonation of matrix Z-H group is above 35, like above 40, preferably above 50.

10. The use of the matrixes according to any one of claims 1 to 9 in Matrix Assisted Laser Desorption/Ionization mass spectrometry of low molecular weight compounds, in particular, compounds of ≤ 2000 Daltons, like ≤ 1000 Daltons.

11. A method for analyzing low molecular weight compounds 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 00.1:1 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.

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

13. 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.

14. The method according to claim 13 wherein the matrix compounds are matrix compounds as defined in any one of claims 1 to 10.

15. 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 equimolarity of analyte and matrix compounds.

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

17. System according to claim 16 further comprising a sample support and/or instructions for use.

Drawing