CN116660359A

CN116660359A - Composition for mass spectrometry, chip and application thereof

Info

Publication number: CN116660359A
Application number: CN202310579602.6A
Authority: CN
Inventors: 叶彬彬; 马玉珍
Original assignee: Zhongyuan Huiji Biotechnology Co Ltd
Current assignee: Zhongyuan Huiji Biotechnology Co Ltd
Priority date: 2023-05-19
Filing date: 2023-05-19
Publication date: 2023-08-29

Abstract

The application belongs to the technical field of mass spectrometry, and particularly discloses a composition for mass spectrometry, a chip and application thereof, wherein the composition comprises a component A and a component B, the component A is a compound used as a matrix material in mass spectrometry, and the component B is at least one of the following compounds: crown ethers, aza crown ethers, thiacrown ethers, and derivatives, tautomers or analogues thereof; the chip comprises the composition. The composition provided by the application can form full, flat and uniform crystals on a chip, so that the formation of crystalline coffee rings is avoided or reduced to the greatest extent, and ionization, mass analysis or detection of sample ions are not damaged; based on the above, the application also provides an improved chip for mass spectrometry, a mass spectrometry method and a mass spectrometer, wherein the composition is used for replacing the traditional mass spectrometry matrix, so that the problems of nonuniform mass spectrometry matrix crystallization, poor mass spectrometry effect, low efficiency and the like are solved, and the chip is easy to implement and has good economy.

Description

Composition for mass spectrometry, chip and application thereof

Technical Field

The application relates to the technical field of mass spectrometry, in particular to a composition and a chip for mass spectrometry and application thereof.

Background

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) breaks through the tradition that the mass spectrometry can only analyze small molecular substances, so that biological macromolecules such as nucleic acid, protein and the like can be studied by mass spectrometry. In recent decades, a nucleic acid mass spectrometry technology based on MALDI-TOF MS has become one of the main stream means for performing nucleic acid detection in a plurality of departments such as gynaecology and obstetrics, pediatrics, genetics, cardiovascular medicine, oncology, clinical laboratory and the like as a technology with high flux, high sensitivity, accuracy and convenience, and is widely applied to the fields of genetic disease screening, cancer accurate analysis, pharmacogenomics detection, pathogen detection and the like. In MALDI detection, the mixed solution of a sample and a matrix is required to be deposited on a sample target together, naturally dried and crystallized at room temperature, and then the sample target is sent into a mass spectrometer for detection, and the detection can be realized on a piece of substrate material through the preparation of a hydrophilic-hydrophobic pattern, so that the detection flux and the detection content of the sample target are greatly improved. In the process, the size of the deposition area of the sample on the sample target and the uniformity of crystallization, the uniformity of co-crystallization of the sample and the matrix on the sample target directly influence the mass spectrum detection result. Therefore, the property of the target surface of the sample is regulated by a proper mode, which is important to accurately regulating the deposition area of the matrix crystallization and improving the uniformity of the co-crystallization of the sample and the matrix.

In certain forms of mass spectrometry, such as MALDI mass spectrometry, matrix materials are used to separate analytes from each other, absorb energy imparted by laser photons, and transfer energy to analyte molecules, thereby desorbing and ionizing them. Once the analyte is ionized, the ion mass can be measured using a mass spectrometer such as a time of flight (TOF) analyzer. The choice of matrix material for mass spectrometry typically depends on the type of biomolecule being analyzed. For example, when nucleic acids are analyzed by mass spectrometry, a commonly used matrix material is 3-hydroxypicolinic acid (3-HPA), and another matrix material for promoting ionization of sample analytes is 2, 5-dihydroxybenzoic acid (DHB); alpha-cyano-4-hydroxycinnamic acid (a-CHCA) is widely used in matrix-assisted laser time-of-flight mass spectrometry for ionizing protein and peptide analytes.

When drinking coffee, a drop is sprayed on a table, and if the user does not take the coffee, the next day can see an annular pattern with a dark color and a light color, which is the famous 'coffee ring effect'. Physicists have revealed a secret behind the "coffee ring effect", and the liquid at the edge of the coffee droplets evaporates faster than in the middle, so that the liquid in the middle of the droplets flows to the edge, and simultaneously drives particles therein to move to the edge, and the liquid evaporates particles to leave behind, thereby forming the coffee ring. The coffee ring has little influence on daily life, and can be wiped off. However, during processing such as ink jet printing and biochips, the "coffee ring effect" affects the properties of the final product. For example, several documents report that the Coffee ring effect can cause uneven distribution of matrix and analyte, thereby affecting analysis of MALDI mass spectrometry (Coffee-ring effects in laser desorption/ionization mass spectrometry; e-MALDI: an electrowetting-enhanced drop drying method for MALDI mass spectrometry; A review on suppression and utilization of the Coffee-ring effect). At present, researches show that the evaporation speed of a matrix can be controlled through electric wetting or sound waves, so that the coffee ring effect of the matrix on a mass spectrum chip is inhibited, but the process accuracy, difficulty and cost of the existing approach are high, and the final harvesting effect is limited.

Therefore, a simple and economical technology capable of reducing or minimizing formation of matrix crystallization coffee ring is developed, and is applied to mass spectrometry, so that the mass spectrometry effect and efficiency are improved, and the method has very important significance for development and improvement of mass spectrometry and chip fields thereof.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present application aims to provide a composition and a chip for mass spectrometry and an application thereof, which are used for solving the problems of uneven crystallization of a matrix material, poor mass spectrometry effect, low efficiency, high process precision, high difficulty, high cost, limited effect and the like in the prior art, and can inhibit the coffee ring effect of the matrix on the mass spectrometry chip.

To achieve the above and other related objects, a first aspect of the present application provides a composition for mass spectrometry including a component a which is a compound used as a matrix material in mass spectrometry, and a component B which is at least one selected from the group consisting of: crown ethers, aza crown ethers, thiacrown ethers, derivatives, tautomers or analogues thereof.

Further, the content of the component B in the composition accounts for 0.01-30% of the total mass of the component A.

Further, the composition also comprises a solvent, wherein the concentration of the component A in the composition is 0.1-1 mol/L, and the concentration of the component B in the composition is 0.00004-0.4 mol/L.

Further, the component A comprises 3-hydroxypicolinic acid (3-HPA) and/or 2, 5-dihydroxybenzoic acid (DHB).

Further, the component a further includes one or more of 3-hydroxypicolinic acid (3-HPA), diammonium citrate (DAC), ammonium oxalate, 2, 5-dihydroxybenzoic acid (DHB), α -cyano-4-hydroxycinnamic acid (α -CHCA), 2,4, 6-Trihydroxyacetophenone (THAP), T-2- (3- (4-tert-butylphenyl) -2-methyl-2-propenylidene) malononitrile (DCTB), dithranol (DIT), sinapic Acid (SA), trans-3-indoleacrylic acid (IAA) and 2- (4-hydroxyphenylazo) benzoic acid (HABA), anthranilic acid, nicotinic acid, succinic acid, ferulic acid, caffeic acid, salicylamide, 1-isoquinolyl alcohol, 3-aminoquinoline, 2, 6-dihydroxyacetophenone, glycerol or nitroaniline.

Further, the solvent is selected from the group consisting of aqueous acetonitrile solutions.

In a second aspect the present application provides a chip for mass spectrometry comprising a composition according to any one of claims 1 to 5.

Further, the chip also comprises a substrate, the composition is deposited on the substrate, and crystals are formed after drying.

Further, the substrate comprises a substrate made of at least one of the following materials:

silicon, silica, glass, nylon, resins, cross-linked dextran, agarose, cellulose, magnetic beads, metals, alloys, plastics, and high molecular polymers.

In a third aspect the present application provides a method of mass spectrometry employing a chip as described in the second aspect, the sample being co-crystallised with the composition on the chip.

In a fourth aspect the application provides a mass spectrometer comprising a chip according to the second aspect.

As described above, the composition for mass spectrometry, the chip and the application thereof of the present application have the following advantageous effects:

the composition provided by the application can form full, smooth and uniform crystals on a chip, so that the formation of crystalline coffee rings is avoided or reduced to the greatest extent, ionization, mass analysis or detection of sample ions are not damaged, the traditional mass spectrum matrix material can be replaced, the problems of nonuniform mass spectrum matrix crystallization, poor mass spectrum analysis effect, low efficiency and the like are effectively solved, and compared with other existing technical means capable of inhibiting the coffee ring effect of a mass spectrum matrix on a mass spectrum chip, the composition provided by the application is simpler to use, easy to implement and good in economical efficiency; based on the above, the application also provides an improved chip for mass spectrometry, a mass spectrometry method and a mass spectrometer, which are beneficial to improving the mass spectrometry detection effect and have important functions in the mass spectrometry detection and chip fields thereof.

Drawings

FIG. 1 is a schematic diagram showing a process of forming crystals on a substrate according to an embodiment of the present application.

FIG. 2 shows a photomicrograph of a composition crystallized in accordance with one embodiment of the application.

FIG. 3 shows a photomicrograph of the standard and new matrix crystals of example 2 of the present application.

FIG. 4 is a diagram showing the results of mass spectrometry performed in example 2 according to the present application using a mass spectrometry chip prepared from a standard substrate to detect the 17 site of the deafness gene.

FIG. 5 is a diagram showing the results of mass spectrometry performed in example 2 of the present application using a mass spectrometry chip prepared from a novel matrix to detect the 17 locus of the deafness gene.

FIG. 6 shows a photomicrograph of crystals of substrates 0 to 7 in example 3 of the present application.

FIG. 7 shows a photomicrograph of crystals of substrates 8 to 31 in example 4 of the present application.

FIG. 8 shows a photomicrograph of crystals of substrates 32 to 36 in example 5 of the present application.

Detailed Description

Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application.

In order to solve the problems of non-uniformity of crystallization, poor mass spectrometry effect, low efficiency and the like of the conventional mass spectrometry matrix, an embodiment of the present application provides a composition for mass spectrometry, which comprises a component a and a component B, wherein the component a is a compound used as a matrix material in mass spectrometry, and the component B is at least one compound selected from the following compounds: crown ethers, aza crown ethers, thiacrown ethers, derivatives, tautomers or analogues thereof.

In some embodiments, the component a comprises 3-hydroxypicolinic acid (3-HPA) and/or 2, 5-dihydroxybenzoic acid (DHB); wherein the molar content of 3-hydroxypicolinic acid (3-HPA) and/or 2, 5-dihydroxybenzoic acid (DHB) in the component A is 90-100%.

In some embodiments, the component a further comprises at least one of the following compounds:

3-hydroxypicolinic acid (3-HPA), diammonium citrate (DAC), ammonium oxalate, 2, 5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA), 2,4, 6-Trihydroxyacetophenone (THAP), T-2- (3- (4-tert-butylphenyl) -2-methyl-2-propenylidene) malononitrile (DCTB), dithranol (DIT), sinapic Acid (SA), trans-3-indoleacrylic acid (IAA) and 2- (4-hydroxyphenylazo) benzoic acid (HABA), anthranilic acid, nicotinic acid, succinic acid, ferulic acid, caffeic acid, salicylamide, 1-isoquinolyl alcohol, 3-aminoquinoline, 2, 6-dihydroxyacetophenone, glycerol and nitroaniline. Component a in the compositions of the present embodiments is equivalent to conventional mass spectrometry matrices, including but not limited to the compounds listed previously.

In some embodiments, the component B is selected from at least one of the following compounds: 12-crown-4-ether, 15-crown-5-ether, 18-crown-6-ether, 2-hydroxymethyl-12-crown-4, 2-aminomethyl-15-crown-5, 2-aminomethyl-18-crown-5, benzo-15-crown-5, benzo-18-crown-6-ether, dibenzo-18. Crown-6, dibenzo-21-crown-7, dibenzo-24-crown-8-ether, 4' -carboxybenzo-15. Crown-5, 4' -aminobenzo-15-crown-5, 4' -aminobenzo-18-crown-6, 4' -aminodibenzo-18-crown-6, dicyclohexyl-24-crown-8-ether, dicyclohexyl-18-crown-6, aza-12-crown-4, 1-aza-15-crown-5, aza-18-crown-6, [1, 10] -diaza-18-6 ', 4' -diaza-6, [4', 4' -aminobenzo-18-crown-6, [4' -dibenzo-18-crown-6 ] (1, 10-diaza-6, 13-crown-6, and [ 13-thia-6 ] - [4 ] thio ] benzo-18-crown-6. Specific classes of crown ethers, aza crown ethers, thiacrown ethers and derivatives, tautomers or analogues thereof are exemplified in the examples of the present application, including but not limited to.

Crown ethers are macrocyclic polyethers containing multiple-oxy-methylene-structural units in the molecule. The common crown ether has 15-crown-5 and 18-crown-6, and the crown ether is one kind of macromolecular cyclic compound with great space inside, and has hole structure with ion selective effect, and may be complex reacted with positive ion, especially alkali metal ion, to bring inorganic matter into organic matter and to act as catalyst in organic reaction. For example: complexing 12-crown-4 with lithium ions; complexing 18-crown-6 with sodium and potassium ions; 15-crown-5 complexes with sodium ions. Studies have shown that crown ethers can be used for sample pretreatment in matrix-assisted laser desorption time-of-flight mass spectrometry (intact cell matrix-assisted laser desorption/time-of-flight mass spectrometry (ICM-TOFMS)) applications of intact cells, thereby removing metal ion adducts, reducing interference of metal ions with spectrogram quality, improving spectrogram resolution and signal-to-noise ratio (Effects) _o f ion mode and matrix additives in the identification of bacteria by intact cell mass spectrometry); in MALDI mass spectrometry nucleic acid analysis, crown ether (2-hydroxymethyl [15 ]]Crown-5, 2-hydroxymethyl [18 ]]Crown-5) can be used as desalting material to remove sodium ion interference in the sample (Using sol-gel/crown ether hybrid materials as desalting substrates for matrix-assisted laser desorption/ionization analysis of oligonucleotides); also form of studyMinodibenzo-18-crown-6 can be used for solid phase measurement of phosphopeptides (Highly selective enrichment of phosphopeptides using poly (dibenzo-18-crown-6) as a solid-phase extraction material). However, the prior art only surrounds the application of complexation of crown ether and metal ions in the field of sample purification and extraction, and no disclosure has been made that crown ether can be applied as an additive to mass spectrometry to promote uniform crystallization of a matrix.

In the embodiment of the application, the component B can be used as an additive to promote the uniform crystallization of the component A (traditional mass spectrum matrix material), so that the formation of a crystallized coffee ring is avoided or minimized.

In some embodiments, the molar content of component B in the composition is 0.01 to 30%, preferably 0.01 to 20%, more preferably 0.01 to 15%, most preferably 0.1 to 10%, for example 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% of the total molar content of component a.

In another embodiment, the composition further comprises a solvent, wherein the concentration of component A is from 0.1 to 1mol/L and the concentration of component B is from 0.00004 to 0.4mol/L, preferably from 0.0004 to 0.12mol/L, more preferably from 0.0004 to 0.08mol/L, most preferably from 0.0004 to 0.06mol/L. For example, the concentration of component a is: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mol/L; the concentration of the component B is 0.00004, 0.0004, 0.001, 0.004, 0.0075, 0.04, 0.06, 0.08, 0.1, 0.12, 0.2 and 0.4mol/L.

In some embodiments, the solvent is selected from the group consisting of aqueous acetonitrile; further, the concentration of the acetonitrile aqueous solution is 10 to 80%, preferably 10 to 60%, most preferably 20 to 50%, for example 20%, 25%, 30%, 35%, 40%, 50%.

An embodiment of the present application provides a chip for mass spectrometry comprising a composition as described in the examples above.

In some embodiments, the chip further comprises a substrate, on which the composition is deposited, and upon drying forms crystals. Wherein, the drying mode of the composition can adopt natural airing, air drying or drying.

In some embodiments, the substrate comprises a substrate, which may be selected from any material suitable for mass spectrometry or the like, including but not limited to at least one of the following: silicon, silica, glass, nylon, resins, cross-linked dextran, agarose, cellulose, magnetic beads (Dynabeads), metals, alloys, plastics, high molecular polymers. Among them, glass includes, but is not limited to, common glass, controlled Pore Glass (CPG); resins include, but are not limited to, wang's resin, phillips resin (Merrifield Resins); metals include, but are not limited to, steel, gold, silver, stainless steel, aluminum, copper, and the like; the alloy refers to a solid product with metal properties obtained by mixing and melting one metal with another or a plurality of metals or nonmetal, and cooling and solidifying the mixture, namely the solid product is divided into metal-metal alloy and metal-nonmetal alloy, and nonmetal in the metal-nonmetal alloy which is suitable for the chip substrate of the embodiment of the application can be silicon; plastics are generally made of polymers such as polyethylene, polypropylene, polystyrene, polyvinylchloride (PVC), polymethyl methacrylate (PMMA), plexiglas; high molecular polymers include, but are not limited to, polyamides, polyesters, polytetrafluoroethylene, teflon (Teflon), polyvinylidene difluoride (PVDF)), cyclic olefin polymers.

In some embodiments, the substrate comprises a substrate and a coating applied to the substrate, the coating being made of at least one of the following materials: gold, fluorocarbon polymers including, but not limited to, fluorinated ethylene-propylene, polytetrafluoroethylene, photoresist, dimethyldichlorosilane.

In some embodiments, the substrate is provided with a sample area, which may be a circular area, preferably 20-3000 microns in diameter, or may be square, rectangular or polygonal, with sides having a length/width of 20-3000 microns.

The application provides a mass spectrometry chip prepared by one specific embodiment, which comprises the following steps:

s1, preparing a mixed solution 1 containing 300mM 3-hydroxypicolinic acid (3-HPA) and 25mM diammonium citrate (DAC) by using a 30% acetonitrile aqueous solution;

s2, preparing a 0.75M 18-crown-6-ether solution by using a 30% acetonitrile aqueous solution;

s3, adding 10 microliters of 0.75M 18-crown-6-ether solution into 1000 microliters of the mixed solution 1, and uniformly mixing to obtain a mixed solution 2;

s4, transferring 0.3 microliter of the mixed solution 2 onto the substrate by using a pipette or a dispensing machine, naturally drying at room temperature, and observing under a microscope after crystallization.

As shown in fig. 1, the mixed solution 2 is naturally dried and crystallized at room temperature after being dripped on a substrate; as can be seen from the micrograph of fig. 2, the composition for mass spectrometry provided by the present application can form a full, flat and uniform crystal on a substrate without or with minimal formation of crystalline coffee rings.

In one embodiment, the application provides a method of mass spectrometry using the chip described in the examples above, on which the sample and the composition are co-crystallized.

An embodiment of the present application provides a mass spectrometer comprising a chip as described in the above examples.

In some embodiments, the mass spectrometer further comprises an ionization source including, but not limited to, atmospheric Pressure Chemical Ionization (APCI), chemical Ionization (CI), electron Impact (EI), electrospray ionization (ESI or ES), fast Atom Bombardment (FAB), field desorption/field ionization (FD/FI), matrix-assisted laser desorption ionization (MALDI), thermal spray ionization (TSP). That is, the composition for mass spectrometry, the chip, and the mass spectrometry method provided by the embodiments of the present application can be used in combination with any ionization source, and have versatility.

In some embodiments, the mass analyzer further comprises a mass analyzer including, but not limited to, a quadrupole time of flight (TOF) analyzer, a magnetic sector, a fourier transform, and a quadrupole ion trap, which can be used alone or in series. That is, the composition, chip and method for mass spectrometry provided by the embodiments of the present application can be used in combination with any mass analyzer, and have versatility.

The following specific exemplary examples illustrate the application in detail. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the application, as many insubstantial modifications and variations are within the scope of the application as would be apparent to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.

Example 1

The present embodiment provides a nucleic acid mass spectrometry detection method, using a SNP (single nucleotide polymorphism (single nucleotide polymorphism) detection kit comprising the following components:

(1) The extraction kit comprises the following components: lysate, washing solution I, washing solution II, eluent, proteinase K and magnetic bead solution;

(2) Enriching reaction components: enriching a reaction solution and an enzyme solution;

(3) Shrimp alkaline phosphatase (SAP enzyme) reaction component: SAP reaction liquid and SAP enzyme liquid;

(4) Extension reaction components: and (3) extending the reaction solution and extending the enzyme solution.

The extraction kit, the enrichment reaction component, the SAP enzyme reaction component and the extension reaction component are all from the midget Gibbs Biotechnology Co.

The steps for performing mass spectrometry of nucleic acids are as follows:

(1) Extraction of sample DNA: DNA was extracted using the midrange metaji extraction kit components, instrument: and an EXM6000 is a full-automatic medium-element Shineway nucleic acid extraction instrument.

(2) Multiplex PCR reaction:

TABLE 1 PCR reaction System

Reagent name	Volume (mu L)
		Amplification reaction solution	10
Amplification enzyme solution	5
		DNA (extracted DNA described above)	5

The multiplex PCR reaction system was formulated according to table 1 and the reaction volumes were scaled down according to the experimental requirements to allow high throughput detection through 384PCR plates. The prepared multiplex PCR reaction system is amplified by a PCR instrument, and the PCR amplification reaction is shown in Table 2:

TABLE 2 PCR amplification reaction

(3) SAP reaction is carried out after PCR amplification is finished, the steps comprise digestion at 37 ℃ for 30-40 min and inactivation at 65 ℃ for 5min, and the reaction volume can be reduced according to the experiment requirement in proportion, so that high-throughput detection can be carried out through 384PCR plates.

(4) After digestion is completed, an extension reaction is performed. An extension reaction system was formulated and added to the SAP-via reaction product at 7 μl per well. The elongation reaction settings are shown in Table 3.

TABLE 3 Single base extension reaction

(5) Resin desalination: adding resin 20-40 mg and 40 mu lddH into each reaction hole ₂ O. Resin and ddH can be proportionally reduced according to experimental requirements ₂ O, so that high throughput detection can be performed by 384PCR plates. The above resins were obtained from the midget metaji Biotechnology Co. The reaction tube (sealing film if 384PCR plates are used) is covered, inverted and shaken for 5-15 minutes before centrifugation.

(6) And after desalting is finished, performing on-machine detection, wherein an instrument for mass spectrometry detection is from an EXS8000 flight time mass spectrometry system of Zhongyuan Hui biotechnology Co., ltd.

Example 2

In this example, different matrixes are prepared, silicon is used as a substrate, and a sample area on the substrate is a circular area (with the diameter of 800 micrometers); further, in this example, a Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrum was used to detect the deafness gene together with the SNP detection kit mentioned in example 1.

The mass spectrum chip in this embodiment is specifically prepared as follows:

s1, a mixed solution 1 containing 300mM 3-hydroxypicolinic acid (3-HPA) and 25mM diammonium citrate (DAC) was prepared with 30% acetonitrile aqueous solution as a standard substrate.

S2, preparing 0.75M18-crown-6-ether solution by using 30% acetonitrile water solution.

S3, adding 10 microliters of 0.75M18-crown-6-ether solution into 1000 microliters of the mixed solution 1, and uniformly mixing to obtain a mixed solution 2 serving as a new matrix.

And S4, transferring 0.3 microliter of standard matrix and new matrix into a substrate sample area by using a pipette or a dispenser, and naturally airing.

The mass spectrometer used in this example was an EXS8000 time-of-flight mass spectrometry system from Messaging Biotechnology Co., ltd.

1. Crystallization detection

The crystallization effect of the standard and new matrices on the mass spectrometry chip was observed by a microscope and the results are shown in fig. 3. As can be seen from FIG. 3, the standard matrix prepared in this example crystallized to form coffee rings, while the new matrix exhibited full, flat, uniform crystallization on the substrate.

2. Detection application

The two mass spectrum chips prepared by the embodiment are used for detecting 17 sites of the deafness gene, and the 17 sites are specifically respectively:

4 SNP mutation sites on the GJB2 gene: rs80338943 (235 delC), rs750188782 (176_191del16), rs111033204 (299_300 delAT), rs80338939 (35 delG);

2 SNP mutation sites on the GJB3 gene: rs74315318 (547G > A), rs74315319 (538C > T);

11 SNP mutation sites on the SLC26A4 gene: rs111033220 (1229C > T), rs201562855 (1174A > T), rs111033305 (1226G > A), rs200455203 (1975G > C), rs111033318 (2027T > A), rs121908363 (2162C > T), rs121908362 (2168A > G), rs1057516953 (281C > T), rs111033380 (589G > A), rs192366176 (IVS15+5G > A), rs111033313 (IVS 7-2A > G).

The detection process specifically comprises the following steps:

s1, extracting sample DNA;

s2, multiplex PCR reaction;

s3, carrying out phosphatase digestion treatment;

s4, single base extension reaction

S5, resin desalination treatment;

s6, transferring the reaction product to a detection chip by using a pipette or a sample application instrument, and detecting by using a MALDI-tofEXS8000 time-of-flight mass spectrometry system of Zhongyuan Ji biotechnology Co., ltd.

The primers for multiplex PCR are shown in Table 4:

TABLE 4 multiplex PCR primers

The primers for the single base extension reaction are shown in Table 5:

TABLE 5 Single base extension reaction primers

SNP typing of deafness-related susceptibility genes is shown in Table 6:

TABLE 6 SNP typing of deafness-related susceptibility genes

Site name	Clinical samples
		1975G＞C	G
1174A＞T	A
		1226G＞A	G
2027T＞A	T
		235delC	C
IVS7-2A＞G	A
		538C＞T	C
281C＞T	C
		1229C＞T	C
2162C＞T	C
		299_300delAT	AT
2168A＞G	A
		176_191del16	GCTGCAAGAACGTGTG
589G＞A	G
		IVS15+5G＞A	G
35delG	G
		547G＞A	G

The mass spectrometry results obtained by detecting the 17 locus of the deafness gene by using mass spectrometry chips prepared from the standard matrix and the new matrix are shown in fig. 4 and 5, respectively. Compared with the prior art, the mass spectrum analysis result obtained by utilizing the mass spectrum chip prepared from the novel matrix has high resolution, and can accurately detect SNP typing of the deafness-related susceptibility genes.

The mass spectrometry acquisition success rates for the two matrices are shown in table 7:

TABLE 7 accumulation success Rate summary table

Mass spectrometry of standard matrix	Mass spectrometry of new matrix
		30％	80％
10％	80％
		30％	70％
20％	90％
		60％	80％
20％	80％
		40％	100％
30％	70％
		30％	80％
60％	100％
		10％	70％
50％	70％
		40％	60％
60％	80％
		50％	90％
30％	80％

From the above, the mass spectrum chip prepared by the new matrix can obtain more high-quality acquisition spectra under the same mass spectrum acquisition times, and the mass spectrum acquisition becomes more efficient, because the new matrix can be crystallized fully, smoothly and uniformly on the substrate.

Example 3

Eight different matrixes (matrixes 0 to 7) are prepared in the embodiment, and eight different mass spectrum chips are prepared by taking metal copper as a base material, wherein a sample area on the base material is a circular area (with the diameter of 1000 microns), and the preparation process is as follows:

s1, a mixed solution containing 270mM of 3-hydroxypicolinic acid (3-HPA), 100mM of 2, 5-dihydroxybenzoic acid (DHB) and 30mM of diammonium citrate (DAC) was prepared with a 40% acetonitrile aqueous solution as a substrate 0.

S2, dissolving 0.04mM aza-12-crown ether-4 into the substrate 0, and uniformly mixing to obtain the substrate 1.

S3, dissolving 0.4mM aza-12-crown ether-4 into the substrate 0, and uniformly mixing to obtain a substrate 2.

S4, dissolving 4mM aza-12-crown ether-4 into the matrix 0, and uniformly mixing to obtain the matrix 3.

S5, dissolving 40mM aza-12-crown ether-4 into the matrix 0, and uniformly mixing to obtain the matrix 4.

S6, dissolving 60mM aza-12-crown ether-4 into the matrix 0, and uniformly mixing to obtain the matrix 5.

S6, dissolving 80mM aza-12-crown ether-4 into the matrix 0, and uniformly mixing to obtain the matrix 6.

S7, dissolving 120mM aza-12-crown ether-4 into the matrix 0, and uniformly mixing to obtain the matrix 7.

S8, transferring 0.4 microliter of matrix 0-7 into a substrate sample area by using a pipette or a dispensing machine, and naturally airing.

The crystallization effect of matrices 0 to 7 on the mass spectrometry chip was observed by a microscope, and the results are shown in FIG. 6. As can be seen from FIG. 6, the matrix 0 (standard matrix) prepared in this example forms a coffee ring after crystallization, and the matrix 2/3/4/5 (new matrix) exhibits a full, flat and uniform crystallization on the substrate, and the matrix 6 is slightly inferior in effect, and the crystallization effect of the matrix 1/7 (new matrix) is slightly inferior, which means that the content of component B is 0.01 to 30%, preferably 0.01 to 20%, more preferably 0.01 to 15%, and most preferably 0.1 to 10% of the total content of component A. The content of the component B is optimally controlled to be 0.1-10% of the total content of the component A.

Example 4

Twenty four different matrixes (matrixes 8-31) are prepared in the embodiment, silicon is used as a base material, twenty four different mass spectrum chips are prepared, and a sample area on the base material is a circular area (with the diameter of 500 microns); further, in this example, a Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrum was used to detect the deafness gene together with the SNP detection kit mentioned in example 1.

The specific preparation process of the twenty-four mass spectrum chips in this embodiment is as follows:

s1, a mixed solution A containing 500mM 3-hydroxypicolinic acid (3-HPA) and 35mM ammonium oxalate was prepared with 30% acetonitrile aqueous solution as a substrate 8.

S2, preparing 100mM solution B of component B in table 8 by using water.

S3, adding 10 microliters of 100mM solution B into 1000 microliters of mixed solution A, uniformly mixing to obtain mixed solution C, and preparing the substrates 9-31 as new substrates according to the mode.

S4, transferring 0.1 microliter of matrix 8-31 to a substrate sample area by using a pipette or a dispensing machine, and naturally airing.

TABLE 8 matrices 9-31 and their corresponding components B

1. Crystallization detection

The crystallization effect of matrix 8 (standard matrix) and matrices 9 to 31 (new matrix) on the mass spectrometry chip was observed by a microscope, and the results are shown in fig. 7. As can be seen from FIG. 7, the matrix 8 prepared in this example crystallized to form coffee rings, while the matrices 9-31 exhibited full, flat, uniform crystallization on the substrate.

2. Detection application

The mass spectrum chip prepared by the embodiment is used for detecting 17 sites of the deafness gene, and the mass spectrum analysis result accuracy and the mass spectrum analysis acquisition average success rate obtained by detecting 17 sites of the deafness gene by using the mass spectrum chip prepared by a matrix 8 (standard matrix) and matrixes 9 to 31 (see the embodiment 2) are shown in the table 9:

TABLE 9 accuracy of mass spectrometry results and average success rate of mass spectrometry acquisitions for matrices 8-31

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From the above, the mass spectrum chip prepared by using the matrix 9-31 can obtain more high-quality acquisition spectra under the same mass spectrum acquisition times, and the replacement component B has little influence on the acquisition efficiency, which indicates that the component B can be selected from crown ether, aza crown ether, thiacrown ether and derivatives, tautomers or analogues thereof.

Example 5

Five different matrixes (matrixes 32-36) are prepared in the embodiment, and five different mass spectrum chips are prepared by taking glass as a base material, wherein a sample area on the base material is a circular area (with the diameter of 1200 microns); further, in this example, a Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrum was used to detect the deafness gene together with the SNP detection kit mentioned in example 1.

The specific preparation process of the five mass spectrum chips in this embodiment is as follows:

s1, a mixed solution containing 1000mM of 3-hydroxypicolinic acid (3-HPA) and 15mM of 15-crown-5-ether was prepared with a 10% acetonitrile aqueous solution as a substrate 32.

S2, a mixed solution containing 1000mM of 3-hydroxypicolinic acid (3-HPA) and 15mM of 15-crown-5-ether was prepared with 20% acetonitrile aqueous solution as a substrate 33.

S3, a mixed solution containing 1000mM of 3-hydroxypicolinic acid (3-HPA) and 15mM of 15-crown-5-ether was prepared with 50% acetonitrile aqueous solution as the substrate 34.

S4, a mixed solution containing 1000mM of 3-hydroxypicolinic acid (3-HPA) and 15mM of 15-crown-5-ether was prepared with a 60% acetonitrile aqueous solution as a substrate 35.

S5, a mixed solution containing 1000mM of 3-hydroxypicolinic acid (3-HPA) and 15mM of 15-crown-5-ether was prepared with an aqueous 80% acetonitrile solution as a substrate 36.

S6, transferring 0.1 microliter of the matrix 32-36 to a substrate sample area by using a pipette or a dispensing machine, and naturally airing.

1. Crystallization detection

The crystallization effect of the matrices 32 to 36 on the mass spectrum chip was observed by a microscope, and the results are shown in fig. 8. As can be seen from FIG. 8, the substrates 33 to 34 prepared in this example exhibited full, flat and uniform crystallization on the substrate, the effect of the substrate 35 was inferior, and the crystallization effect of the other substrates (substrates 32, 36) was slightly inferior.

2. Detection application

Five mass spectrum chips prepared by the embodiment are used for detecting 17 sites of the deafness gene, and the mass spectrum analysis acquisition success rates of the five matrixes are shown in table 10. As can be seen from table 10, the mass spectrometry acquisition average success rate of the matrices 33, 34 is higher than the other three.

TABLE 10 accumulation success Rate summary table

From the above, the concentration of the solvent for preparing the matrix, that is, acetonitrile aqueous solution is 10 to 80%, preferably 10 to 60%, and most preferably 20 to 50%.

Example 6

Different matrixes are prepared in the embodiment, resin glass is used as a base material, different mass spectrum chips are prepared, and a sample area on the base material is a circular area (with the diameter of 300 microns); further, in this example, a Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrum was used to detect the deafness gene together with the SNP detection kit mentioned in example 1.

The mass spectrum chip in this embodiment is specifically prepared as follows:

s1, component A in Table 11 was prepared as a 100mM solution A with 30% acetonitrile in water as a standard substrate.

S2, preparing a 4M 1-aza-15-crown-5 solution by using a 30% acetonitrile aqueous solution.

S3, adding 100 microliters of 4M 1-aza-15-crown-5 solution into 1000 microliters of solution A, uniformly mixing to obtain mixed solution C, and preparing the matrix 37-68 serving as a new matrix according to the mode.

S4, transferring 0.3 microliter standard matrix (namely, matrix 37-68) to a substrate sample area by using a pipette or a dispenser, and naturally airing.

TABLE 11 matrices 37-68 and their corresponding component A

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Detection application

The mass spectrum chip prepared in this example was used to detect 17 sites of deafness gene, and the average success rate of mass spectrum analysis and collection obtained by detecting 17 sites of deafness gene (see example 2) using mass spectrum chip prepared by using matrices 37 to 68 is shown in table 12.

TABLE 12 average success rate of mass spectrometry analysis and acquisition for matrices 37-68

Novel matrix	Average success rate of mass spectrometry and collection	Novel matrix	Average success rate of mass spectrometry and collection
				Substrate 37	79％	Substrate 53	23％
Matrix 38	1％	Matrix 54	46％
				Matrix 39	2％	Matrix 55	10％
Matrix 40	75％	Substrate 56	1％
				Matrix 41	0％	Substrate 57	1％
Matrix 42	0％	Matrix 58	0％
				Matrix 43	0％	Matrix 59	0％
Matrix 44	0％	Matrix 60	0％
				Matrix 45	0％	Substrate 61	1％
Matrix 46	0％	Matrix 62	1％
				Matrix 47	80％	Matrix 63	85％
Matrix 48	80％	Matrix 64	84％
				Matrix 49	85％	Substrate 65	84％
Matrix 50	92％	Matrix 66	89％
				Substrate 51	89％	Matrix 67	89％
Matrix 52	11％	Substrate 68	88％

As can be seen from Table 12, the collection success rate is high when the component A is 3-hydroxypicolinic acid and 2, 5-dihydroxybenzoic acid, the collection efficiency of the 3-hydroxypicolinic acid and the 2, 5-dihydroxybenzoic acid together with other additive components is improved to a certain extent, but the additive components do not have a decisive influence on the collection efficiency, and the additive components are independently prepared into a solution A, the collection efficiency is lower, which means that the 3-hydroxypicolinic acid or the 2, 5-dihydroxybenzoic acid has a decisive influence on the collection efficiency, the 3-hydroxypicolinic acid or the 2, 5-dihydroxybenzoic acid must be added into the solution A, and other additive components can be added according to the requirements by a person skilled in the art. Meanwhile, from the experimental data, it is preferable that the molar content of 3-HPA and/or DHB as the core component in component A is 90 to 100%.

The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. A composition for mass spectrometry, characterized in that it comprises a component a, which is a compound used as a matrix material in mass spectrometry, and a component B, which is at least one compound selected from the group consisting of: crown ethers, aza crown ethers, thiacrown ethers, derivatives, tautomers or analogues thereof.

2. The mass spectrometry composition of claim 1, wherein: the content of the component B in the composition is 0.01-30% of the total content of the component A.

3. The mass spectrometry composition of claim 1, wherein: the composition also comprises a solvent, wherein the concentration of the component A in the composition is 0.1-1 mol/L, and the concentration of the component B in the composition is 0.00004-0.4 mol/L.

4. The mass spectrometry composition of claim 1, wherein: the component A comprises 3-hydroxypicolinic acid and/or 2, 5-dihydroxybenzoic acid.

5. The mass spectrometry composition according to claim 4, wherein: the component A also comprises one or more of diammonium citrate, ammonium oxalate, alpha-cyano-4-hydroxycinnamic acid, 2,4, 6-trihydroxyacetophenone, T-2- (3- (4-tert-butylphenyl) -2-methyl-2-propenylidene) malononitrile, dithranol, sinapic acid, trans-3-indoleacrylic acid and 2- (4-hydroxyphenylazo) benzoic acid, anthranilic acid, nicotinic acid, succinic acid, ferulic acid, caffeic acid, salicylamide, 1-isoquinolyl alcohol, 3-aminoquinoline, 2, 6-dihydroxyacetophenone, glycerol or nitroaniline.

6. A composition for mass spectrometry according to claim 3, wherein: the solvent is selected from acetonitrile aqueous solution.

7. A chip for mass spectrometry, characterized in that: the chip comprising the composition of any one of claims 1 to 6.

8. The chip for mass spectrometry according to claim 7, wherein: the chip further comprises a substrate, the composition is deposited on the substrate, and crystals are formed after drying.

9. A method of mass spectrometry, characterized by: the method employs a chip according to any one of claims 7 to 8, on which the sample is co-crystallized with the composition.

10. A mass spectrometer, characterized by: the mass spectrometer comprising the chip of any one of claims 7 to 8.