NL2022038B1 - Method for analysing an analyte sample and matrix material therefore - Google Patents

Method for analysing an analyte sample and matrix material therefore Download PDF

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NL2022038B1
NL2022038B1 NL2022038A NL2022038A NL2022038B1 NL 2022038 B1 NL2022038 B1 NL 2022038B1 NL 2022038 A NL2022038 A NL 2022038A NL 2022038 A NL2022038 A NL 2022038A NL 2022038 B1 NL2022038 B1 NL 2022038B1
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matrix material
analyte
alkoxy
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Veneman Tom
Christian Van Schaik Nicholas
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Biosparq B V
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

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Abstract

4-alkoxy-0L-cyano substituted cinnamic acid compounds and corresponding sulfuric acid and phosphonic acids are used as matrix material for use in MALDI Mass Spectrometry, wherein the analytes are micro-organisms such as bacteria, fungi, Virus and pollen. The matrix material is also suitable for a sample pre-treatment Wherein a dispersion or solution of the analyte and the matrix material is dried on the fly Without deposition on a plate. Such a pre-treatment enables a so-called single cell MALDI matrix procedure.

Description

METHOD FOR ANALYSING AN ANALYTE SAMPLE AND MATRIX MATERIAL THEREFORE
FIELD OF THE INVENTION
The present invention relates to a cyano substituted cinnamic acid compound, its use and method for analysing an analyte sample, particularly in MALDI mass spectrometry, and preferably for micro-organisms.
BACKGROUND OF THE INVENTION
MALDI mass spectrometry is a powerful analysis method for detection of analytes and more particularly analytes of biological origin, such as proteins, cells, micro-organisms such as bacteria and the like. MALDI is an abbreviation for matrix assisted laser desorption/ionisation, which already indicates that use is made of a matrix material. This is a compound that is deposited onto an analyte, which facilitates the subsequent ionisation.
Matrix materials to perform MALDI are known per se. Well-known examples include 4hydroxy- α-cyano-cinnamic acid (also known as CHCA and HCCA), 2,5-dihydroxybenzoic acid, sinapic acid, ferulic acid, 2-aza-5-thiothymine, 3-hydroxypicolinic acid. The matrix material has a major impact on the efficiency of the mass spectrometry. Moreover, different matrices are of first choice for different classes of analytes and analytical problems, as is stated in F. Hillenkamp, T. Jaskolla & M. Karas, “The MALDI Process and Method”, in F. Hillenkamp et al (ed), MALDI MS: A Practical Guide to Instrumentation, Methods and Applications (2nd ed, 2014), pages 1-40, therein pages 24-25.
Already minor substitutions to the HCCA matrix material turn out to change sensitivity for specific analytes. The HCCA-matrix material and the 4-chloride substitution are specified for the analysis of peptide-mass fingerprints generated by protein enzymatic digests, as well as for MS/MS fragmentation experiments. The 4-chloride variant is stated in the above mentioned handbook as particularly suitable with laser lights with 337 nm. The 2,4-difluoro-substituted HCCA-matrix is suggested for phospholipids. According to US8,980,642, the latter matrix w'ould be suitable tor MALDI MS of negative and positive ions. The 4-methyl substituted HCCA-matrix is suitable, according to said patent, for doubly charged ions. Jaskolla et al, J. Soc. Mass Spectrometry, 2009, 20, 1104-1114, furthermore mentions 4-hydroxy-3-methyl and 4-hydroxy-3-methoxy substituted HCCA-matrices, leading to crystals of sub-micrometer dimensions. These are not mentioned as providing something worth further investigations. Again another HCCA-variant, the propoxy-ester is disclosed in S. Wang et al, J. Am. Soc. Mass Spectrometry, 2016, 27, 709-718. However, according to the said handbook on page 5, the use of neutral HCCA-derivatives such as esters or amides would lead to weaker ion-dipole interactions between protonated analytes and the matrices, and strongly diminished analyte-signal intensities.
Still, it appears that there is quite some optimization possible. While the handbook suggested the 2,4-difluor-substituted HCCA as the matrix of choice for positive and negative ions, EP2542897 (with Jaskolla and Karas as inventors) presents data showing (see Fig. 3, 4 5) that some individual matrix materials perform quite weak, but that mixtures of halogenated cyano substituted cinnamic acid derivatives and HCCA are to be used as matrix material in MALDI mass spectrometry, particularly for operation in the negative ion mode. Specific examples of HCCA derivatives mentioned are for instance 4-bromo-a-cyanocinnamic acid, a-cyano-2,4dichlorocinnamic acid, a-cyano-2,4-difluorocinnamic acid and also tri-, tetra- and pentahalogenated a-cyano-cinnamic acid derivatives.
It is clear from Fig. 2 and Fig. 4 of this patent that the matrix materials and the mixtures thereof with CHCA do not perform equally well for analytes. Fig. 2 shows the intensity of the signal of the mass spectrometry for several analytes and chosen matrix material (mixtures). The analytes are peptides and have a molecular weight in the range of 800 to 2200 g/mol. The signal intensity is too low at 820 g/mol, becomes sufficient to very good for some of the matrix material mixtures at roughly 1100-1400 g/mol, but decreases again for peptides of higher molecular weight. At 2200 g/mol, the signal intensity is again as weak as at 800 g/mol, except for a mixture with a pentahalogenated derivative that performs acceptable. In Fig. 4, a similar variation of the signal intensity is shown with a dependence on the molecular weight of the analyte and the composition of the matrix material (mixture). The analytes shown in this Figure are well known analytes DesArg Bradykinin, Angiotensin 1, Glu-Fibrinopeptid B, Neurotension and two parts of the ACTH polypeptide (1-17 and 18-39). The molecular weights again increase from about 800 to about 2400.
Therefore, it does not become clear from EP2542897 which matrix material will provide a sufficient signal to noise ratio for analytes with a molecular weight above 2500 g/mol. Such higher molecular masses are however very relevant when using MALDI mass spectrometry for identification of diseases, micro-organisms such as bacteria and health-relevant biological compounds such as inulin. Particularly when analysing micro-organisms and the like, the mass spectrogram will contain sufficient peaks so as to distinguish common, irrelevant micro-organisms from relevant (for instance infectious) micro-organisms, provided that the signal to noise ratio is sufficiently strong. Moreover, the matrix material mixtures of this patent are specified for use in the negative ion mode, whereas one typically uses the positive ion mode for analysis of bacteria.
Furthermore, as mentioned in said handbook on page 4, the overall process of the desorption and ionization of the matrix material has not yet been fully described, almost 30 years after its invention. As mentioned on page 13, the mechanisms leading to the formation of charged matrix and analyte molecules in the MALDI process are even more poorly understood than the physics of the material ablation/desorption. One typically refers to co-crystallisation of matrix material and analyte. This limited understanding complicates the search for a matrix material suitable for use in combination with micro-organisms as analytes in MALDI mass spectrometry. A further desire is that the matrix material is also applicable for MALDI versions, wherein the matrix material is to be deposited on the analyte during a flight of a droplet containing both, rather than on a plate. An example of such a method is for instance described in the non-prepublished application PCT/EP2018/063203 in the name of the Applicant, which is herein incorporated by reference.
It is observed that the use of MALDI mass spectrometry for the analysis of bacteria is known per se from Y. Hotta et al, FEMS Microbiol Letters, 330 (2012), 23-29. The analysis was made with isolated bacterial cells. Sinapic acid was used as the matrix material. Sample and matrix mixture was herein spotted onto the MALDI target, and spectra were obtained by averaging 1000 individual laser shots. Nine ribosomal subunit proteins were commonly detectable subunits by MALDI MS analysis of the bacteria used, Sphingomonadaceae. Sinapic acid is however only suitable for use in MALDI MS analysis on a plate, not for the application w'herein droplets are dried on the fly.
SUMMARY OF THE INVENTION
Therefore, it is a first object of the invention to provide a matrix material suitable for positive ionisation and/or forming a co-crystal of the matrix material and the analyte, wherein the analyte is a micro-organism such as bacteria and/or health-relevant biological compounds such as insulin. In relation thereto, it is a further object of the invention to provide a matrix material suitable for analysis of ribosomal proteins and providing a sufficiently good signal to noise ratio.
It is a further object of the invention to provide a composition of a matrix material that is suitable for use in methods, wherein the matrix material is applied on the particulate, high-molecular analyte on the fly, rather than when disposed on a plate.
It is again a further object to provide the use of compounds as a matrix material in MALDI mass spectrometry.
It is also an object to provide a method of identifying a micro-organism by means of a MALDI mass spectrometry using a suitable matrix material.
The first object is achieved with an α-cyano substituted cinnamic acid compound according to formula (I) and/or an α-cyano substituted cinnamic sulfonic acid compound according to formula (II) and/or an α-cyano substituted cinnamic phosphonate compound according to formula (III), for use as a matrix material in MALDI mass spectrometry
Figure NL2022038B1_D0001
formula III wherein R1, Rla and/or Rlb are chosen from group of: hydrogen;
wherein R2, R3, R4, R5 and R6 are independently chosen from the group of: hydrogen, hydroxyl, C; - C15 alkoxy, chloride, bromide, fluoride, Cj-Ca alkyl;
wherein R2 is a C, - CV alkoxy.
It has been observed in experiments leading to the invention, that a matrix material with an alkoxyside group in the para-position (R2-substituent) and preferably with an acid group can be deposited effectively onto bacteria and other micro-organisms, including proteins with a comparatively high molecular weight. The deposition and any subsequent removal of solvent of the matrix material leads to crystallisation of the matrix material on the analyte. Herein, co-crystallisation occurs, wherein ribosomal proteins co-crystallize with the matrix material. This co-crystallisation process effectively involves that such proteins are extracted out of the cell and into the matrix material. As a result, a very good signal to noise ratio was observed, which by far outperformed the result for CHCA. This enables identification of individual peaks in the spectrum to at least 10,000 g/mol and preferably 20,000 g/mol or more. As a consequence, it becomes feasible to distinguish microorganisms that are structurally similar but biologically different.
The inventors believe, without desiring to be bound thereto, that the acid group facilitates interaction with proteins in the analyte, whereas the alkoxy-substituted ring system favours crystallisation on the analyte surface. The alkoxy-chain extends a rather hydrophobic portion of the molecule, which accelerates crystallisation from rather polar solvent mixtures, such as water/alcohol mixtures. The alkoxy-chain furthermore may limit the intramolecular hydrogen bonding between molecules of the matrix material, i.e. between hydroxy groups and carboxylgroups, and therewith to better alignment during crystallisation. Moreover, in order to explain the enhanced signal-to-noise ratio as compared to HCCA, also the electron system and its delocalisation might play a role. An alkoxy-substituent is a more strongly electron-withdrawing group than the corresponding hydroxyl-group. As a consequence, it is believed that the electron distribution in the ring (and adjacent delocated portions) differs, leading to other preferred mesomeric groups and thus different chemical and physical properties. Particularly, as the delocalization extends outside the benzene ring, it is not impossible that some mesomeric groups are based on further ring-shapes involving the cyano-group.
An advantage of the α-cyano substituted cinnamic acid compound according to the invention is that the compound can interact with positively charged protein functional groups on the basis of its negatively charged conjugated acid group. This is deemed to facilitate extraction of proteins. The compound is furthermore feasible of formation of hydrogen bridges. Such hydrogen bridges are important to form crystal structures and influence the crystallisation rate.
Another advantage of the α-cyano substituted cinnamic acid compound according to the invention is that the micro-organisms such as bacteria and/or health-relevant biological compound co-crystallises below 40 °C, preferably co-crystallises below 30 °C, more preferably co-crystallises at room temperature.
In another embodiment according to the invention, the Cj - C)5 alkoxy is an C]-C5-alkoxy, such as methoxy, ethoxy, propoxy, Zso-propoxy, butoxy, Zso-butoxy, terfbutoxy, CH3(CH2)4O, CH^CIDsO, CH3(CH2)7O, CH3(CH2)9O. Preferably, the alkoxy is chosen from the group of ethoxy, propoxy, Zso-propoxy, butoxy and isobutoxy. Most preferably wherein the alkoxy is propoxy. A short or moderate length of the alkoxy-side chain has an impact on hydrophobicity. This is especially relevant when crystallisation of the matrix material on the analyte occurs on the fly, i.e. by evaporation of solvent from a droplet, from a mixture of water and ethanol. Herein, the solubility of the matrix material in the alcohol should be sufficient, so that a composition with the matrix material is stable and droplets can be generated therefrom, for instance by means of inkjet printing. Furthermore, the solubility should reduce upon evaporation of the ethanol at a rate that allows development of the crystal rather than generating an amorphous matrix or a matrix with a high number of individual crystallites. It has been observed that the signal-to-noise ratio of a spectrum obtained without proper crystallisation is rather low.
In a primary embodiment according to the invention, the R3 - Rft substituents are hydrogen. The matrix material with such a substitution pattern can be easily prepared by means of a Knoevenagel condensation. Furthermore, good results as to the signal-to-noise ratio have been obtained. Finally, it has turned out effective for MALDI versions wherein the analyte and matrix material are dispensed in the form of a droplet towards a location for ionization, in which path the matrix material is to crystallize onto the analyte.
In a further embodiment according to the invention, at least one of the R3 - R6 substituents is chosen from hydroxyl, C) - C5 alkoxy, chloride, bromide, fluoride, Ci - C3 alkyl, and the remaining of R’ - R° is hydrogen. The provision of a further substituent to the benzene ring can be used to further improve the properties of the matrix material with reference to a desired target group of analytes. Preferably, the number of halogens as substituents is at most one, as multiple halogens tend to reduce UV-absorption of laser energy. By further preference, the alkyl if any is methyl, as any alkyl but certainly larger alkyls require more space which may hamper crystallisation. Any additional Ci-Cj-alkoxy is preferably CrC; alkoxy, or even methoxy or ethoxy for the same reason. In one specific implementation, the further substituent is a hydroxyl or C|-C2alkoxy group. Thus is believed to increase the effect of the alkoxy group already available. In another specific implementation, the further substituent is a halogen. Generally, it is preferable that there is mere a single further substituent, and the remaining of R3-Rö is hydrogen.
It is to be understood that the matrix material of this further embodiment is not merely suitable as an alternative to the primary embodiment. Rather, the matrix material may also be used for specific purposes, such as for a specific class of analytes. Such specific use could be implemented in that spectrum analysis for an analyte is repeated: first with the primary matrix material, and thereafter with a further matrix material that is useful for certain properties of the analyte, or rather to confirm the findings with the primary matrix material.
In one further embodiment, a combination of matrix materials is used, wherein the substituents R-R6 to the benzene ring mutually vary. In one beneficial implementation, the combinations include different 4-alkoxy, a-cyano cinnamic acids and/or corresponding sulfuric acids and phosphonic acids (as shown in Figures 1-3). More preferably, the 4-alkoxy substituent is chosen from Ci-C5-alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, and pentoxy. N-alkoxy substituents are considered to be especially preferable. In this manner, the resulting degree of hydrophobicity can be tuned, for instance to being comparable to a single matrix material with R2 being ethoxy, propoxy or π-butoxy or anything in between. It is deemed that the use of such combination does not have a negative impact on the development of the crystals, as the R2-substituent is believed to be arranged away from the analyte, i.e. on the outside. Rather, it may well be that such combination is effective for co-crystallisation, as the crystallisation may start more quickly (due to the presence of rather hydrophobic species) and continue longer (due to the presence of less hydrophobic species).
According to a further preferred implementation, the number of different alkoxysubstituted derivatives is two or three. By further preference, at least one of n-propoxy and isopropoxy is present. Most preferable combinations are deemed to be n-propoxy/n-butoxy, npropoxy/iso-propoxy, n-propoxy/iso-butoxy, n-propoxy/ethoxy, n-propoxy/iso-propoxy/ethoxy, methoxy/ethoxy/n-propoxy, methoxy/ethoxy/iso-propoxy, ethoxy/n-propoxy/n-butoxy, ethoxy/npropoxy/iso-butoxy.
In yet a further embodiment according to the invention, wherein the compound is a cyanosubstituted cinnamic acid according to formula (I). While the corresponding sulfuric acids and phosphonic acids are suitable, cyano-substituted cinnamic acids are best known as matrix materials and thus have the preference in the field.
The second object is achieved in the composition of said compound according to the invention and at least one solvent for the compound. The solvent is suitably an organic solvent, such as an alcohol, an alkenone (ketone or aldehyde), an ether, a cyano-substituted alkane, an alkyl-acetate. The organic solvent is preferably based on a Ci-Cs-alkyl chain, more preferably C(C3 alkyl. Preferably, the polarity of the organic solvent is not too low, which enables appropriate solubility of the matrix material and dispersability of the analyte. Furthermore, tin adequate polarity enables that the solvent is miscible with any antisolvent, if present. For instance, the solvent may have a polarity as expressed by means of a polarity index P’ of at least 2.0, more preferably at least 3.0, or even at least 3.5 or at least 4.0. This polarity index P’ is defined by L.R. Snyder (see L.R. Snyder, “Classification of the Solvent Properties of Common Liquids”, J. Chromatogr. Sci. 1978, 16, 223-234). More particularly, the solvent has a boiling point below 90°C or preferably below 85°C at atmospheric pressure. In a most preferred embodiment according to the invention, the solvent is chosen from the group consisting of acetone, acetonitrile, dichloromethane, ethanol, ethyl acetate, /.w-propanol, methanol, 2-methoxyethanol, n-propanol.
In a further embodiment according to the invention, the composition further comprising a cat-ion scavenger. Suitable cat-ion scavengers are for instance phosphates, such as monoammonium phosphate and diammonium phosphate, 18-Crown-6-ether and other crownethers, L-serine, tri- and tetraacetic acids, such as nitrilotriacetic acid and ethylene diamine tetraacetic acid (EDTA). All these are examples of compounds that are able to form coordination complexes with metal ions. It is generally preferable in the development of compositions of MALDI matrix materials not to add a variety of additives. Any such additive may interact with the analyte and lead to masking of peaks or rather to the creation of additional peaks in the spectrum which cannot be distinguished from the analyte. However, a cat-ion scavenger is deemed useful, in order to catch any cationic species that may be detrimental for the MALDI mass spectrometry.
In again a further embodiment, the composition further comprises an antisolvent. This is deemed particularly relevant for a version of MALDI mass spectrometry, in which the sample is obtained by evaporation of a droplet in a flow path. One specific version hereof is single-cell MALDI, wherein droplet formation and/or selection are controlled to provide one cell (i.e. microorganism) per droplet. Notwithstanding this preferred use, it is not excluded that an antisolvent may also be used for any other type of MALDI mass spectrometry, such as the provision of samples onto a plate, followed by laser ionisation of a sample that is provided with a correct crystalline layer of matrix material. As a consequence of the presence of an aqueous anti-solvent, the matrix material will reach its saturation limit in the composition more quickly, particularly due to evaporation of the solvent. The organic solvent is more particularly chosen such that it is more volatile than the aqueous solvent.
The antisolvent is more particularly aqueous. In one embodiment, the aqueous antisolvent is pure water. In another and preferred embodiment, the antisolvent is acidified water, for instance to a pH in the range of 0-5, preferably 1-4. It is believed that the presence of acid during evaporation may contribute to extraction of proteins from the micro-organism and therewith to cocrystallisation of the matrix material and the extracted proteins. The aqueous antisolvent may be a buffer that is compatible with mass spectrometry as known to the skilled person. Suitably, the salt concentration in the buffer is at most 1 mM. Such buffer salts contain more preferably compounds that may be decomposed and become volatile, so as to evaporate and prevent that the salts are incorporated into the crystal. As known to the skilled person, the incorporation of conventional salts, such as alkali salts into crystals, will render the MALDI mass spectrometry measurement useless.
Suitably, the matrix material has a solubility in the solvent of at least 0.1 mg/ml, preferably at least 0.5 mg/ml, more preferably at least 1 mg/ml. The solubility is herein defined at room temperature as the intrinsic solubility. This is typically defined in silico. It is formally defined as the solubility in a state wherein a molecule is not dissociated. Preferably, the said solubility limit is furthermore met in experimental conditions at pH2 at 25°C. More preferably, the matrix material has a limited solubility in the antisolvent, for instance of at least 0.0005 mg/ml, such as at least 0.001 mg/ml, up to 0.01 mg/ml.
Preferably, the aqueous antisolvent is present in excess quantities, relative to the solvent. In experiments leading to the invention (wherein the composition with matrix material and biological analyte was dispensed in the form of droplets each comprising a single cell), mass ratios between the solvent and water in the range of 0.03 (1:33) to 0.33 (1:3) have been found suitable. The ratio is among others dependent on the flow path available for evaporation and crystallisation and also on the temperature and other physical conditions at which the evaporation occurs. Preferably, the mass ratio is in the range of 0.05 (1:20) up to 0.2 (1:5), by further preference up to 0.125 (1:8), such as 1:, for instance 10% (acidified) water and 90% ethanol. While it is deemed practical to carry out evaporation and the preceding droplet generation at room temperature, it is not excluded to vary this temperature. A suitable temperature is for instance in a range of 15 to 50 °C, preferably in the range of 20 to 40 °C.
As specified above, it may well be that a mixture of matrix materials is used. It will then be understood that this mixture is present in the composition.
The third object is achieved in the use of the said compounds as a matrix material in MALDI mass spectrometry. As is apparent from the experimental results, excellent results have been obtained by means of the invention. While the experiments are obtained by means of dispensing droplets, it is deemed that equally good results can be achieved wherein the matrix material is crystallized onto the analyte in an alternative manner, such as from a plate.
In an embodiment according to the invention, the matrix material co-crystallizes on the analyte after dispensing droplets of a composition of the matrix material and the analyte. The cocrystallisation means that proteins from the analyte, such as ribosomal proteins, crystallize simultaneously with the matrix material and are therewith built into the crystal. Upon laser ablation, these parts will be liberated. This is deemed to enhance the signal-to-noise ratio. In a further embodiment according to the invention, the droplets further contain the analyte in particulate form, and wherein preferably each droplet contains at most 5 analyte particles, preferably at most 3 analyte particles and more preferably 1 analyte particle. This results in a fewparticle or even single-particle MALDI. A preferred number is one, although another limited number, for instance up to 10 cellular analytes, suitably 1-5, such as 2 or 3, is also feasible. By limiting the number of analytes per sample, it becomes more easily to identify an analyte; i.e. there will not be any ambiguity as from which analyte within the sample any portion of the resulting spectrum originates. The single-particle MALDI is effective not merely because it is ascertained that all relevant peaks in the mass spectrogram will originate from the same single particle. It is additionally effective, as the analysis can be repeated easily, for instance 100-500 times, therewith allowing to obtain sufficient signal.
In yet a further embodiment according to the invention, the analyte sample is chosen from peptides and proteins with a molar mass in the range of 2000-20,000 Dalton, such as 2500 -15,000 Dalton. Although the matrix material is primarily intended for micro-organisms, it is not excluded that it is also used for analysis of isolated peptides and proteins. This is also suitable to build up a database of mass spectra. Such mass spectra may then be used as a reference for interpretation of an unknown analyte.
A fourth object is achieved in a method of identifying comprising the steps of: (1) providing a test composition comprising the analyte sample, a matrix material, a solvent, an anti-solvent, wherein the test composition is a suspension of the analyte sample; (2) generating a beam of droplets from the test composition, wherein the droplets being ejected into a flow path with length sufficient to achieve evaporation of the solvent and crystallise the matrix material on the analyte sample, therewith obtaining test samples; (3) ionizing at least some of the test samples in the flow path to obtain ionised components; (4) detecting the ionised components by a detector; and (5) identifying the analyte sample on the basis of the detected ionised components.
In accordance with the invention, the matrix material is chosen from the group of cyano substituted cinnamic acid compounds according to formula (I) and/or cyano substituted cinnamic sulfonic acid compounds according to formula (II) and/or cyano substituted cinnamic phosphonate compound according to formula (III)
Figure NL2022038B1_D0002
formula Ill wherein R1, Rla and/or Rlb is chosen from group of: hydrogen; and R2, R3, R4, R' and R6 are independently chosen from the group of: hydrogen, hydroxyl, C] - C15 alkoxy, chloride, bromide, fluoride, Ci - C; alkyl, and wherein at least R2 is a Ci - C15 alkoxy.
The inventors have observed in investigations leading to the present invention that 4-alkoxysubstituted α-cyano cinnamic acid compounds and the corresponding sulfonic acids, phosphonic acids surprisingly turn out to have signal-to-noise ratio that exceeds that of CHCA by far, and is highly reproducible. Excellent spectra have been obtained based on averaging of 100 - 1000 individual single cell measurements, which include individual peaks up to a mass of 20,000 g/mol. Moreover, it was found that these compounds turn out to crystallize onto the analyte in an appropriate manner, therewith rendering them not only suitable for use from a MALDI plate, but also and particularly in MALDI methods, wherein the analyte is provided into a solution of the matrix material and wherein droplets of the solution with analyte are dried under crystallisation. The inventors see as preliminary, non-binding explanation that the combination of the alkoxysubstitution and the free acid lead to appropriate crystallisation behaviour while the free acid group aids in binding to the analyte surface. As a result, more analyte ions are released from the crystal during the ablation phase that occurs upon irradiation of the crystallized sample, typically with laser light, and wherein the protonated fragments are formed that will be visible in the mass spectrum.
For sake of clarity, it is observed that any embodiment or implementation discussed hereinabove and hereinafter in relation to one aspect of the invention, is also applicable to other aspects of the invention.
BRIEF INTRODUCTION OF THE FIGURES
These and other aspects of the invention will be further elucidated with reference to the Figures, wherein:
Fig. 1 shows a schematic representation of a device for MALDI mass spectrometry with a preferred pre-treatment for a liquid composition;
Fig. 2 shows a schematic representation of the particle flow' path and mass spectrometer within the device of Fig. 1;
Fig. 3 shows a single particle MALDI spectrum using the matrix materials CHCA, aCyano-4-propoxycinnamic acid (CPCA), (E)-2-cyano-3-(naphthalen-2-yl)acrylic acid ) (NpCCA); and
Fig. 4 shows a single particle MALDI spectrum using the matrix material CPCA.
DETAILED DISCUSSION OF ILLUSTRATED EMBODIMENTS
The figures are not drawn to scale. Equal reference numerals in different figures refer to equal or corresponding features.
Figure 1 shows a schematic representation of a first embodiment of a device for MALDI mass spectrometry. Fig. 2 shows in more detail the portion 200 of the device, hereinafter also referred to as a flight path unit 200. MALDI mass spectrometry is particularly suitable for identification of biological material. One preferred type of biological material is micro-organisms such as bacteria, fungi, viruses and pollen. Other types of biological material that can be identified with MALDI include for instance blood cells, peptides. One specific form of MALDI is single particle MALDI, wherein a single test sample such as a droplet contains one or a limited number of individual biological organisms. The limited number is for instance at most 10, preferably at most 5, w'ith further preference 1 - 3. It is however most preferred that the single particle MALDI is carried out such that there is one micro-organism per test sample.
The device comprises a sample receiver 10, conduits 11, a first mixing unit 12, a second mixing unit 14, and a flight path unit 200. The flight path unit comprises a drying chamber 15, an ionization chamber 191 and a time-of-flight tube 194. A droplet is ejected by any droplet ejector 16, such as for instance based on a piezoelectric resonator or inkjet printing device. The droplet follows a droplet beam 24 that extends from the drying chamber 15 into the time-of-flight tube 194. Upon drying the droplet beam 24 is actually converted into a particle beam 192. Upon ionization by radiation from a pulse laser 18, the particle beam 192 is converted into an ion beam
195. The mass spectrometer - not shown - measures the ions of the ion beam 195 and creates spectra on the basis thereof. According to one embodiment of the invention, use is made of a sensor 20, 22 for determining a morphology parameter so as to select particles that are ionized by a laser pulse of the pulse laser 18.
The first mixing unit 12 comprises a first mixer 120, a container 122 for solvent and/or antisolvent, such as water, and a detector 124. Rather than one container 122, two separate containers may be present. Sample material that is for instance obtained from a patient, is diluted with the solvent and/or antisolvent in the first mixer 120. Detector 124 is suitably an optical detector configured to detect light scattered from individual micro-organisms when the microorganisms flow through a measurement beam. From a count of micro-organisms that are detected on average per unit of time interval, the density may be determined. Such detector 124 is known per se and is for instance a cytometer or flow cytometer. Particle detector 124 is shown coupled to a control input of first mixer 120. The control mechanism is arranged to increase the amount of solvent and/or antisolvent, until the measured density has dropped to or below a predefined density. Preferably both are added in a predefined ratio. A liquid circulation circuit may be used to circulate the composition until the desired density has been achieved. The second mixing unit 14 comprises a second mixer 14 and a matrix material reservoir 142. Matrix material reservoir 142 is coupled to the second mixer 140. The second mixer 14 is configured to mix the matrix material into the test composition obtained from the first mixing unit 12.
The droplet generator 16 may be provided with means for evaluation whether a droplet contains a single micro-organism or any other number of micro-organisms. Such a detecting means may be arranged to view the suspension in a channel prior to ejection by a nozzle. The generator 16 may further be provided with means for directing an ejected droplet to a first position or to a second position depending on information obtained from the detecting means. The first position is then a target position, i.e. a flow path towards the position where a laser source may eject radiation on the particle so as to ionize it. The second position is a waste position. The directing means are configured for deflection of the droplet or a motorized stage configured for directing the nozzle. Such a device is known per se from EP2577254B1, and is included herein by reference.
In operation, a stream of liquid, containing analyte from sample receiver 10, a solvent and antisolvent from first mixing unit 12 and matrix material from the second mixing unit 14, is separated into sections that each result in a small liquid drop launched in flight through chamber 15. During flight through the drying chamber 15, the matrix material in a liquid drop crystallizes on the analyte, typically a micro-organism, while the drop dries in flight, resulting in a dried particle, which is also referred to as the test sample. Typically, the drop is launched with a diameter in the range of 30 - 60 pm. The dried particle has an aerodynamic diameter of less than 3.0 pm in a first embodiment, wherein the test sample contains a single bacteria. If the dried particle crystallizes in accordance with the invention, rather than in amorphous form as in the prior art, the aerodynamic diameter of the dried particle in the first embodiment is even smaller, typically in the order of 1 - 2 pm. Because of the small size of the droplets, only little time during flight is needed to prepare the drops for ionization. Subsequently, a laser pulse is fired at the dried particle from pulse laser 18. This results in ionization of material from the test sample. The ionized material is detected in mass spectrometer. The processor that is coupled thereto processes the obtained data to generate a spectrum or data set that can be compared with known data sets. Such known data sets are typically stored in a library.
Sensing of droplets is achieved by means of determining a morphology parameter. In the present example, as discussed hereinafter, the sensor senses the aerodynamic diameter of a particle, and/or the standard deviation thereof. This is achieved by means of a first and a second detection channel 20, 22, each comprising a light source and a detector. The light source of the first detection channel 20 may be of any type, such as a source of visible light and a source of ultraviolet radiation. The light source of the second detection channel 22 is most preferably a source of visible light, such as for instance a light emitting diode of any suitable wavelength. The light detector is a photomultiplier in one embodiment.
While the first detection channel 20 could make use of a laser device with a wavelength in the UVrange, such as 266 nm, this requires the use of a fluorescence detector. However, fluorescence has a lower sensitivity requires a more sensitive detector. Moreover, the fluorescence detector needs at least two detection channels, one for the fluorescence and one for the scattering of visible light, including filters. Moreover two lasers are required, of which the UV-laser requires a high power. All in all, this constitutes a costly and complex detector that can be avoided w'hen using visible light. With two detection channels of visible light, a single laser and a beamsplitter is sufficient.
EXAMPLES
Example 1 a-cyano-4-propoxycinnamic acid was synthesised starting from 4-propoxybenzaldehyde using a Knoevenagel condensation. The starting material 4-propoxybenzaldehyde (1 equivalent) and cyanoacetic acid (1.05 equivalent) were dissolved in toluene before piperidinium (0.015 equivalent) was added to catalyse the reaction. The reaction mixture was refluxed for 5 hours. The during the reaction formed water was separated using a Dean-Stark trap. The crude product was purified by repeated recrystallization from methanol/water. A yield of around 80% was obtained. The resulting compound was observed with MALDI mass spectrometry. A molecular weight of 231 g/mol was found, corresponding to the propyl ether.
Example 2:
Solutions were prepared of the matrix materials -cyano-4-hydroxycinnamic acid (CHCA), aCyano-4-propoxycinnamic acid (CPCA), (E)-2-cyano-3-(naphthalen-2-yl)acrylic acid ) (NpCCA) in EtOH : H2O (25 : 75). The matrix material CPCA was prepared as specified above, the matrix materials NpCCA and CHCA were obtained from Sigma Aldrich. The matrix materials were present in a concentration of 0,1 mg/mL. Water (0.1% TFA in water) was added as an antisolvent. The solution was maintained at room temperature.
The thus created solution was added into the reservoir of a single cell MALDI mass spectrometry device as under developed in Biosparq. The device comprises an inkjet printing device, followed by a drying chamber (Figures 1 and 2). Droplets of 30 pm - 70 pm in diameter were dispensed from the inkjet printing device.
Figure 3 shows an averaged spectrum of 100 MALDI droplets containing 0.2 pM insuline, which has a molecular weight of 5807 g/mol. The MALDI particles were formed with the above mentioned matrix materials. A peak in the intensity between around 5700 m/z and around 6000 m/z is shown. This peak can be assigned to insulin. It becomes clear that CPCA has a higher intensity compared to CHCA and NpCCA. Furthermore, it is noted that the peak to baseline is better when CPCA is used as matrix material.
Figure 4 shows an averaged spectrum of 500 MALDI-particles containing Escherichia coli. The matrix material used to perform this spectrum was CPCA. It becomes clear that CPCA is a suitable matrix material to analyse samples with a molecular weight above 2000 g/mol, which are thus suitable for micro-organisms. The spectrum forms a fingerprint for the specific bacterial species. The main peaks can be assigned to ribosomal proteins. The signal intensity is sufficient for identification. The location of the peaks was shown to be reproducible.
Thus, in summary, the invention relates to 4-alkoxy-a-cyano substituted cinnamic acid compounds and corresponding sulfuric acid and phosphonic acids are used as matrix material for use in MALDI Mass Spectrometry, wherein the analytes are micro-organisms such as bacteria, fungi, virus and pollen. The matrix material is also suitable for a sample pre-treatment wherein a dispersion or solution of the analyte and the matrix material is dried on the fly without deposition on a plate. Such a pre-treatment enables a so-called single cell MALDI matrix procedure. The preferred 4-alkoxy substituent is Cl-C5-alkoxy, by further preference C2-C4 alkoxy. Mixtures of materials with different C2-C4-alkoxy substituents are feasible. The compounds are suitably part of a composition further comprising a solvent and a preferably acidified aqueous carrier, also known as anti-solvent.

Claims (18)

I. Samenstelling voor gebruik als matrixmateriaal in MALDI massaspectrometrie omvattende ten minste één verbinding uit de groep van nitrilgesubstitueerde kaneelzuurverbinding volgens formule (I) en/of nitrilgesubstitueerde kaneelzurige sulfonzuurverbinding volgens formule (Π) en/of nitrilgesubstitueerde kaneelzurige fosfanaatverbinding volgens formule (III),I. Composition for use as a matrix material in MALDI mass spectrometry comprising at least one compound from the group of nitrile-substituted cinnamic acid compound of formula (I) and / or nitrile-substituted cinnamic sulfonic acid compound of formula (Π) and / or nitrile-substituted cinnamic phosphanate compound of formula (III),
Figure NL2022038B1_C0001
Figure NL2022038B1_C0001
Figure NL2022038B1_C0002
Figure NL2022038B1_C0002
 formule I formule IIformula I formula II
Figure NL2022038B1_C0003
Figure NL2022038B1_C0003
 formule III waarinformula III wherein - R1. R‘:1 en/of R‘b gekozen zijn uit waterstof;- R 1 . R ' : 1 and / or R' b are selected from hydrogen; - R2, R', R4, R3 en R° onafhankelijk van elkaar gekozen zijn uit de groep van waterstof, hydroxyl, C( - C!5 alkoxy, chloride, bromide, fluoride, C]-C3 alkyl;- R 2, R ', R 4, R 3 and R ° are independently selected from the group of hydrogen, hydroxyl, C (- C 5 alkoxy, chloride, bromide, fluoride, C] -C 3 alkyl;! - ten minste R‘ is een C: - C15 alkoxy-group, en ten minste één oplosmiddel voor de verbinding gekozen is uit de groep bestaande uit acelon. dichloormethaan, ethanol, ethylacetaat, iso-propanol en methanol, waarbij de samenstelling verder omvat een waterig antisolvent voor het matrixmateriaal.at least R 'is a C 1 -C 15 alkoxy group, and at least one solvent for the compound is selected from the group consisting of acelon. dichloromethane, ethanol, ethyl acetate, isopropanol and methanol, the composition further comprising an aqueous antisolvent to the matrix material. 2. Samenstelling volgens conclusie 1, waarbij het waterige antisolvent aangezuurd is.The composition of claim 1, wherein the aqueous anti-solvent is acidified. 3. Samenstelling volgens conclusies 1 of 2, verder een kationenvanger omvat.The composition of claims 1 or 2, further comprising a cation scavenger. 4. Samenstelling voigens één van de voorgaande conclusies 1 tot en met 3, waarbij een verdere 4alkoxy. α-nitrilgesubstitueerde kaneel zuurverbinding volgens de formule (I), (II) of (III) aanwezig is ais een verder matrixmateriaal, waarbij R1, R'a en/of Rl0 gekozen zijn uit de groep van waterstof; waarbij R2 C( - C5 -aikoxy is en verschilt van het - eerste- matrixmateriaal· en R’-R onafhankelijk van elkaar gekozen zijn uit de groep van: waterstof, hydroxyl, Cj - Cs alkoxy, chloride, bromide, fluoride, Ci - C3 alkyl, waarbij bij voorkeur R'-R> waterstof is.The composition according to any of the preceding claims 1 to 3, wherein a further 4-alkoxy. α-nitrile substituted cinnamic acid compound of the formula (I), (II) or (III) is present as a further matrix material, wherein R 1 , R 1 a and / or R 10 are selected from the group of hydrogen; wherein R 2 C (- C 5 -aikoxy, and is different from the - first-matrix material · R and R 'are independently selected from the group consisting of: hydrogen, hydroxy, C - C alkoxy, chloride, bromide, fluoride C 1 -C 3 alkyl, wherein preferably R 1 -R > is hydrogen. 5. Samenstelling voigens één van de voorgaande conciusies I tot en met 4, waarbij de Cj - Cl5 aikoxy is a Ci - C5 aikoxy, bijvoorbeeld methoxy, ethoxy, propoxy, iso-propoxy, butoxy, isobutoxy, tert-butoxy, CH3(CH2)aO, bij voorkeur waarbij de alkoxy is methoxy, ethoxy, propoxy, iso-propoxy, met meer voorkeur waarbij de aikoxy is propoxy.The composition according to any one of the preceding claims I to 4, wherein the C 1 -C 15 aikoxy is a C 1 -C 5 aikoxy, for example methoxy, ethoxy, propoxy, iso-propoxy, butoxy, isobutoxy, tert-butoxy, CH 3 (CH 2 ) aO, preferably where the alkoxy is methoxy, ethoxy, propoxy, iso-propoxy, more preferably where the alkoxy is propoxy. 6. Samenstelling volgens één van de voorgaande conclusies I tot en met 5, waarbij R3 - Rb is waterstof.The composition according to any of the preceding claims I to 5, wherein R 3 - R b is hydrogen. 7. Samenstelling voigens één van de voorgaande conciusies I tot en met 6, waarbij één van R' - R° gekozen is uit hydroxyl, Cj - C3 alkoxy, chloride, bromide, fluoride. Ci - C3 alkyl, en de overige van R3 - Rb waterstof zijn.A composition according to any one of the preceding claims I to 6, wherein one of R 1 - R 0 is selected from hydroxyl, C 1 - C 3 alkoxy, chloride, bromide, fluoride. C 1 -C 3 alkyl, and the rest of R 3 -R b are hydrogen. 8. Samenstelling volgens één van de voorgaande conclusies 1 tot en met 7, waarbij de verbinding een nitriigesubstitueerd kaneelzuur voigens formule (I) is.The composition of any one of claims 1 to 7, wherein the compound is a nitri substituted cinnamic acid according to formula (I). 9. Gebruik van de samenstelling volgens één van de conciusies 1-8 ais matrixmateriaal voor MALDI massaspectrometrie van een analiet, waarbij het analietmonster een microorganisme is, waarbij het matrixmateriaal co-kristalliseert op het analiet na het afgeven van druppels van een samenstelling van het matrixmateriaal en het analiet.Use of the composition according to any one of claims 1-8 as matrix material for MALDI mass spectrometry of an analyte, the analyte sample being a microorganism, the matrix material co-crystallizing on the analyte after dispensing drops of a composition of the matrix material and the analyte. 10. Gebruik volgens conclusie 9, waarbij de druppels voorts het analiet in deeltjesvorm bevatten, en waarbij bij voorkeur elke druppel ten hoogste 5 analietdeeitjes bevat, bij verdere voorkeur ten hoogste 3 analietdeeitjes en met de grootste voorkeur één analietdeeltje per druppel. 11 Use according to claim 9, wherein the drops further contain the analyte in particulate form, and preferably each drop contains at most 5 analyte eggs, further preferably at most 3 analyte eggs and most preferably one analyte particle per drop. 11 11. Werkwijze voor het analyseren van een analietmonster, waarbij het analietmonster een microorganisme is, omvattende:A method of analyzing an analyte sample, wherein the analyte sample is a microorganism, comprising: - Het verschaffen van een testsamenstelling omvattende het analietmonster, een matrixmateriaal, een oplosmiddel, een anti-solvent, w aarbij de testsamenstelling een suspensie van het analietmonster is;- Providing a test composition comprising the analyte sample, a matrix material, a solvent, an anti-solvent, the test composition being a suspension of the analyte sample; - Het genereren van een straal druppels van de testsamenstelling, waarbij de druppels losgelaten worden in een stroompad met voldoende lengte om verdamping van het oplosmiddel en kristallisatie van het matrixmateriaal op het analietmonster te bewerkstellingen, daarmee testmonsters verkrijgend;- Generating a jet of drops from the test composition, releasing the drops in a flow path of sufficient length to effect evaporation of the solvent and crystallization of the matrix material on the analyte sample, thereby obtaining test samples; - Het ioniseren van ten minste sommige van de testmonsters in het stroompad om zo geïoniseerde componenten te verkrijgen;- Ionizing at least some of the test samples in the flow path to obtain ionized components; - Het detecteren van de geïoniseerde componenten in een detector; en- Detecting the ionized components in a detector; and - Het identificeren van het analietmonster op basis van de gedetecteerde geïoniseerde componenten, waarbij het matrixmateriaal gekozen is uit de groep van nitrilgesubstitueerde kaneelzuurverbindingen volgens formule (I) en/of nitrilgesubstitueerde kaneelzurige sulfonzuurverbindingen volgens formule (II) en/of nitrilgesubstitueerde kaneelzurige fosfonaatverbindingen volgens formule (III)- Identifying the analyte sample based on the detected ionized components, wherein the matrix material is selected from the group of nitrile-substituted cinnamic acid compounds of formula (I) and / or nitrile-substituted cinnamic sulfonic acid compounds of formula (II) and / or nitrile-substituted cinnamic acid phosphonate compounds of formula ( III)
Figure NL2022038B1_C0004
Figure NL2022038B1_C0004
Figure NL2022038B1_C0005
Figure NL2022038B1_C0005
 formule I formule IIformula I formula II
Figure NL2022038B1_C0006
Figure NL2022038B1_C0006
 formule III waarinformula III wherein - R' , R'a en/of R'b gekozen zijn uit waterstof;- R ', R' a and / or R ' b are selected from hydrogen; - R2, R’, R4, R; en R6 onafhankelijk van elkaar gekozen zijn uit de groep van waterstof, hydroxyl,, C} - C45 alkoxy, chloride, bromide, fluoride, Ci-C:. alkyl;- R 2 , R ', R 4 , R ; and R 6 is selected independently from each other selected from the group of hydrogen, hydroxyl, C ,,} - C45 alkoxy, chloride, bromide, fluoride, Ci-C:. alkyl; - ten minste R is een C; - CU alkoxy-group.- at least R is a C; - CU alkoxy group. 12. Werkwijze volgens conclusie 11, waarbij het antisolvent is een waterig antisolvent is, waarbij het oplosmiddel een hogere vluchtigheid heeft dan het antisolvent en waarbij het antisolvent in een ovennaat aanwezig is ten opzichte van het oplosmiddel.The method of claim 11, wherein the antisolvent is an aqueous antisolvent, the solvent having a higher volatility than the antisolvent, and the antisolvent being in an oven vent relative to the solvent. 13. Werkwijze volgens één van de conclusies 11-12. waarbij de druppels een doorsnede in het bereik van 20 - 70 pm hebben, bij voorkeur 30 - 60 pm. waarbij het verschaffen van de testsamenstelling als druppels met de voorgeschreven druppeldoorsnede kristallisatie van het matrixmateriaal op het the analietmonster na de druppelvorming faciliteert.A method according to any one of claims 11-12. the drops having a diameter in the range of 20-70 µm, preferably 30-60 µm. wherein providing the test composition as droplets with the prescribed droplet diameter facilitates crystallization of the matrix material on the analyte sample after the droplet formation. 14. Werkwijze volgens conclusie 13, waarbij de kristallisatie tot een niet-bolvormige deeltjesvorm van het testmonster leidt.The method of claim 13, wherein the crystallization results in a non-spherical particle shape of the test sample. 15. Werkwijze volgens één van de conclusies 11-14. waarbij het matrixmateriaal bij kamertemperatuur een intrinsieke oplosbaarheid in het antisolvent heeft van ten hoogste 0,1 mg/ml, bij voorkeur ten hoogste 0,01 mg/ml en bij verdere voorkeur ten hoogste 0,001 mg/ml.A method according to any one of claims 11-14. wherein the matrix material at room temperature has an intrinsic solubility in the antisolvent of at most 0.1 mg / ml, preferably at most 0.01 mg / ml, and further preferably at most 0.001 mg / ml. 16. Werkwijze volgens één van de conclusies 11-15, waarbij het oplosmiddel en het antisolvent aanwezig zijn in een gewichtsverhouding in het bereik van 0,03 (1:33) tot 0,33 (1:3), bij voorkeur van 0,05 (1:20) tot 0.25 (1:4).A method according to any one of claims 11-15, wherein the solvent and the antisolvent are present in a weight ratio ranging from 0.03 (1:33) to 0.33 (1: 3), preferably from 0, 05 (1:20) to 0.25 (1: 4). 17. Werkwijze volgens conclusie 11-16, waarbij het Ci - Ci$ alkoxy een Cj-C$-alkoxy-groep is, bij voorkeur Cj-Ca-alkoxy, bij verdere voorkeur propoxy of iso-propoxy, en met de grootste voorkeur de alkoxy-groep propoxy is.A process according to claims 11-16, wherein the C 1 -C 1 alkoxy is a C 1 -C 8 alkoxy group, preferably C 1 -C 7 alkoxy, further preferably propoxy or iso-propoxy, and most preferably the alkoxy group is propoxy. 18. Werkwij ze volgens één van de conclusies 11-17, waarbij de detector deel van een time-offlight massaspectrometer is.The method of any one of claims 11-17, wherein the detector is part of a time-offlight mass spectrometer.
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Effective date: 20210702

PD Change of ownership

Owner name: DEEM CONSULTING B.V.; NL

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Effective date: 20211217

MM Lapsed because of non-payment of the annual fee

Effective date: 20211201