CN116660359A - Composition for mass spectrometry analysis, chip and application thereof - Google Patents

Abstract

The invention belongs to the technical field of mass spectrometry, and specifically discloses a composition for mass spectrometry, a chip and applications thereof. The composition includes component A and component B, and component A is used as a matrix material in mass spectrometry. The compound, component B is at least one selected from the following compounds: crown ether, azacrown ether, thia crown ether and their derivatives, tautomers or analogues; the chip includes the composition. The composition provided by the invention can form plump, smooth and uniform crystals on the chip, thereby avoiding or minimizing the formation of crystallized coffee rings, and will not damage the ionization, mass analysis or detection of sample ions; based on this, the present invention Also provided is an improved chip for mass spectrometry analysis, mass spectrometry analysis method and mass spectrometry analyzer, using the composition to replace the traditional mass spectrometry matrix, solving the problems of uneven crystallization of mass spectrometry matrix, poor mass spectrometry analysis effect, low efficiency, etc., and easy to implement , good economy.

Description

Composition, chip and application thereof for mass spectrometry analysis

Technical Field

The present invention relates to the technical field of mass spectrometry analysis, and in particular to a composition for mass spectrometry analysis, a chip and applications thereof.

Background Art

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) breaks the tradition that mass spectrometry can only analyze small molecules, and allows biomacromolecules such as nucleic acids and proteins to be studied by mass spectrometry. In the past decade, nucleic acid mass spectrometry technology based on MALDI-TOF MS has become one of the mainstream means of nucleic acid detection in multiple departments such as obstetrics and gynecology, pediatrics, genetics, cardiovascular medicine, oncology, and laboratory as a high-throughput, high-sensitivity, accurate and convenient technology. It is widely used in the fields of genetic disease screening, cancer precision analysis, drug genomics detection, pathogen detection, etc. In MALDI detection, the mixed solution of the sample and the matrix needs to be deposited on the sample target together, dried and crystallized naturally at room temperature, and then the sample target is sent to the mass spectrometer for detection. Through the preparation of hydrophilic and hydrophobic patterns, it can achieve simultaneous detection of 96 or 384 people or even more people on a substrate material, greatly improving its detection throughput and detection content. In this process, the size of the sample deposition area on the sample target and the uniformity of crystallization, as well as the uniformity of co-crystallization of the sample and the matrix on the sample target, will directly affect the results of mass spectrometry detection. Therefore, using appropriate methods to control the properties of the sample target surface is crucial for accurately controlling the matrix crystal deposition area and improving the uniformity of co-crystallization of the sample and the matrix.

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

When you spill a drop of coffee on the table, if you ignore it, you will see a ring pattern with dark outside and light inside the next day. This is the famous "coffee ring effect". Physicists have uncovered the secret behind the "coffee ring effect". The liquid at the edge of the coffee drop evaporates faster than the liquid in the middle, so the liquid in the middle of the drop will flow to the edge, and at the same time drive the particles in it to move to the edge. The liquid evaporates and the particles are left behind, forming a coffee ring. The coffee ring has little impact on daily life and can be wiped off. However, in the processing of inkjet printing and biochips, the "coffee ring effect" will affect the performance of the final product. For example, many papers reported that the coffee ring effect will cause uneven distribution of matrix and analyte, thereby affecting the analysis of MALDI mass spectrometry (Coffee-ring effects in laser desorption/ionization massspectrometry; e-MALDI: an electrowetting-enhanced drop drying method for MALDImass spectrometry; A review on suppression and utilization of the coffee-ring effect). Current studies have shown that the coffee ring effect of the matrix on the mass spectrometry chip can be suppressed by controlling the evaporation rate of the matrix through electro-wetting or acoustic waves. However, the process precision, difficulty and cost of the existing approaches are high, and the final effect is limited.

Therefore, developing a simple and economical technology that can reduce or minimize the formation of matrix crystallized coffee rings and applying it to mass spectrometry analysis to improve the effect and efficiency of mass spectrometry analysis is of great significance for the development and improvement of mass spectrometry analysis and its chip field.

Summary of the invention

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a composition, a chip and applications thereof for mass spectrometry analysis, which are used to solve the problems of uneven crystallization of matrix materials, poor mass spectrometry analysis effect and low efficiency in the prior art, and high process precision, high difficulty, high cost and limited effect in suppressing the coffee ring effect of the matrix on the mass spectrometry chip.

To achieve the above-mentioned object and other related objects, the first aspect of the present invention provides a composition for mass spectrometry analysis, the composition comprising component A and component B, wherein component A is a compound used as a matrix material in mass spectrometry analysis, and component B is selected from at least one of the following compounds: crown ethers, azacrown ethers, thiocrown ethers and their derivatives, tautomers or analogues.

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

Furthermore, the composition also includes a solvent. In the composition, the concentration of component A is 0.1 to 1 mol/L, and the concentration of component B is 0.00004 to 0.4 mol/L.

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

Further, the component A also includes 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), anthracene (DIT), sinapinic 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-isoquinolol, 3-aminoquinoline, 2,6-dihydroxyacetophenone, glycerol or nitroaniline.

Furthermore, the solvent is selected from acetonitrile aqueous solution.

A second aspect of the present invention provides a chip for mass spectrometry analysis, the chip comprising the composition according to any one of claims 1 to 5.

Furthermore, the chip also includes a substrate, and the composition is deposited on the substrate and forms crystals after drying.

Furthermore, the substrate includes a substrate, and the substrate is made of at least one of the following materials:

Silicon, silica, glass, nylon, resin, cross-linked dextran, agarose, cellulose, magnetic beads, magnetic beads, metals, alloys, plastics, polymers.

The third aspect of the present invention provides a mass spectrometry analysis method, which uses the chip described in the second aspect, and the sample and the composition are co-crystallized on the chip.

A fourth aspect of the present invention provides a mass spectrometer, comprising the chip described in the second aspect.

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

The composition provided by the present invention can form full, flat and uniform crystals on the chip, thereby avoiding or minimizing the formation of crystallized coffee rings, and will not damage the ionization, mass analysis or detection of sample ions. It can replace traditional mass spectrometry matrix materials, and effectively solve the problems of uneven crystallization of mass spectrometry matrix, poor mass spectrometry analysis effect, low efficiency, etc. Moreover, compared with other existing technical means that can inhibit the coffee ring effect of mass spectrometry matrix on mass spectrometry chip, the use of the composition of the present invention is simpler, easier to implement and more economical. Based on this, the present invention also provides an improved chip for mass spectrometry analysis, a mass spectrometry analysis method and a mass spectrometer, which are beneficial to improving the mass spectrometry detection effect and play an important role in the field of mass spectrometry detection and its chip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a process of crystallization of a composition on a substrate according to an embodiment of the present invention.

FIG. 2 is a microscope photograph showing crystals of a composition according to an embodiment of the present invention.

FIG. 3 shows microscopic photographs of crystals of the standard matrix and the new matrix in Example 2 of the present invention.

FIG. 4 shows the mass spectrometry analysis results obtained by detecting the 17 loci of the deafness gene using a mass spectrometry chip made from a standard matrix in Example 2 of the present invention.

FIG5 shows the mass spectrometry analysis results obtained by detecting the 17th locus of the deafness gene using a mass spectrometry chip prepared from a new matrix in Example 2 of the present invention.

FIG. 6 is a microscope photograph showing the crystals of matrix 0 to 7 in Example 3 of the present invention.

FIG. 7 is a microscope photograph showing the crystallization of matrix 8 to 31 in Example 4 of the present invention.

FIG. 8 is a microscopic photograph showing the crystallization of matrix 32 to 36 in Example 5 of the present invention.

DETAILED DESCRIPTION

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

In order to solve the problems of uneven crystallization of traditional mass spectrometry matrices, poor mass spectrometry analysis effect, low efficiency, etc., an embodiment of the present invention provides a composition for mass spectrometry analysis, wherein the composition comprises component A and component B, wherein component A is a compound used as a matrix material in mass spectrometry analysis, and component B is selected from at least one of the following compounds: crown ethers, azacrown ethers, thiocrown ethers and their derivatives, tautomers or analogues.

In some embodiments, the component A includes 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), 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), sinapinic 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-isoquinolinol, 3-aminoquinoline, 2,6-dihydroxyacetophenone, glycerol and nitroaniline. Component A in the composition of the embodiment of the present invention is equivalent to a traditional mass spectrometry matrix, including but not limited to the compounds listed above.

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 ether.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, dicyclohexanoyl-24-crown-8-ether, dicyclohexane-18-crown-6, aza-12-crown-4, 1-aza-15-crown-5, aza-18-crown-6, [1,10]-diaza-18-crown-6, [4',4" (5")]-di-tert-butyldibenzo-18-crown-6, [4,13]-dithiabenzo-18-crown-6, [7,10]-dithiabenzo-18-crown-6. The embodiments of the present invention list specific types of crown ethers, azacrown ethers, thiacrown ethers and their derivatives, tautomers or analogs, including but not limited to these.

Crown ethers are macrocyclic polyethers containing multiple -oxy-methylene structural units in the molecule. Common crown ethers include 15-crown-5 and 18-crown-6. Since crown ethers are macromolecular cyclic compounds with a large internal space, the hole structure of crown ethers has a selective effect on ions and can react with positive ions, especially alkali metal ions, to bring inorganic substances into organic substances, and can be used as catalysts in organic reactions. For example: 12-crown-4 complexes with lithium ions; 18-crown-6 complexes with sodium and potassium ions; 15-crown-5 complexes with sodium ions. Studies have shown that in the application of intact cell matrix-assisted laser desorption/time-of-flight mass spectrometry (ICM-TOFMS), crown ethers can be used for sample pretreatment to remove metal ion adducts, reduce the interference of metal ions on the spectral quality, and improve the spectral resolution and signal-to-noise ratio (Effects _of ion mode and matrix additives in the identification of bacteria by intact cell mass spectrometry); in MALDI mass spectrometry nucleic acid analysis, crown ethers (2-hydroxymethyl[15]crown-5, 2-hydroxymethyl[18]crown-5) can be used as desalting materials to remove sodium ion interference in samples (Using sol-gel/crown ether hybrid materials as desalting substrates formatrix-assisted laser desorption/ionization analysis of oligonucleotides); and other studies have shown that dibenzo-18-crown-6 can be used for solid phase extraction of phosphopeptides (Highly selective enrichment of phosphopeptides using Poly(dibenzo-18-crown-6) as a solid-phase extraction material). However, previous technologies have only focused on the application of crown ethers and metal ions in the field of sample purification and extraction, and no one has disclosed that crown ethers can be used as additives in mass spectrometry analysis to promote uniform crystallization of the matrix.

In the embodiment of the present invention, component B as an additive can promote the uniform crystallization of component A (traditional mass spectrometry matrix material), thereby avoiding or minimizing the formation of crystallized coffee rings.

In some embodiments, in the composition, the molar content of component B accounts for 0.01-30% of the total molar content of component A, preferably 0.01-20%, more preferably 0.01-15%, and most preferably 0.1-10%, 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%.

In another embodiment, the composition further comprises a solvent, and in the composition, the concentration of component A is 0.1 to 1 mol/L, and the concentration of component B is 0.00004 to 0.4 mol/L, preferably 0.0004 to 0.12 mol/L, more preferably 0.0004 to 0.0.08 mol/L, and most preferably 0.0004 to 0.06 mol/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, 1 mol/L; the concentration of 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, 0.4 mol/L.

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

One embodiment of the present invention provides a chip for mass spectrometry analysis, wherein the chip includes the composition described in the above embodiment.

In some embodiments, the chip further comprises a substrate, the composition is deposited on the substrate, and forms crystals after drying. The composition can be dried by natural drying, air drying or oven drying.

In some embodiments, the substrate includes a substrate, which can be selected from any material suitable for mass spectrometry or made of such material, including but not limited to at least one of the following materials: silicon, silicon dioxide, glass, nylon, resin, cross-linked dextran, agarose, cellulose, magnetic beads, magnetic beads (Dynabead), metals, alloys, plastics, and high molecular polymers. Among them, glass includes but is not limited to ordinary glass and controlled pore glass (CPG); resins include but are not limited to Wang resins and Merrifield resins; metals include but are not limited to steel, gold, silver, stainless steel, aluminum, copper, etc.; alloy refers to a solid product with metallic properties obtained by mixing and melting one metal with another or several metals or non-metals, cooling and solidifying, that is, it is divided into metal-metal alloys and metal-non-metal alloys. The non-metal in the metal-non-metal alloy suitable for the chip substrate of the embodiment of the present application can be silicon; plastics are usually made of polymers, such as polyethylene, polypropylene, polystyrene, polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), and resin glass (Plexiglas); high molecular polymers include but are not limited to polyamide, polyester, polytetrafluoroethylene, Teflon, polyvinylidene fluoride (PVDF), and cycloolefin polymers.

In some embodiments, the substrate includes a substrate and a coating coated on the substrate, and the coating is made of at least one of the following materials: gold, fluorocarbon polymer, photoresist, dimethyldichlorosilane, wherein the fluorocarbon polymer includes but is not limited to fluorinated ethylene-propylene and polytetrafluoroethylene.

In some embodiments, a sample area is provided on the substrate, and the sample area may be a circular area, preferably with a diameter of 20 to 3000 microns, or may be a square, rectangular or polygonal area, with a side/length/width of 20 to 3000 microns.

A specific embodiment of the present invention prepares a mass spectrometry analysis chip, and its preparation process includes the following steps:

S1, preparing a mixed solution 1 containing 300 mM 3-hydroxypicolinic acid (3-HPA) and 25 mM diammonium citrate (DAC) using a 30% acetonitrile aqueous solution;

S2, prepare 0.75M 18-crown-6-ether solution with 30% acetonitrile aqueous solution;

S3, add 10 μl of 0.75 M 18-crown-6-ether solution to 1000 μl of mixed solution 1, mix well, and obtain mixed solution 2;

S4. Use a pipette or a dispensing machine to transfer 0.3 μl of mixed solution 2 onto the substrate, dry it naturally at room temperature, and observe it under a microscope after crystallization.

As shown in FIG1 , after the mixed solution 2 is dropped onto the substrate, it is naturally dried and crystallized at room temperature. As can be seen from the microscope photograph of FIG2 , the composition for mass spectrometry analysis provided by the present invention can form full, flat and uniform crystals on the substrate, and will not form or can minimize the formation of crystalline coffee rings.

One embodiment of the present invention provides a mass spectrometry analysis method, wherein the method uses the chip described in the above embodiment, and the sample and the composition are co-crystallized on the chip.

An embodiment of the present invention provides a mass spectrometer, which includes the chip described in the above embodiment.

In some embodiments, the mass spectrometer further comprises an ionization source, and the ionization source includes but is 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), and thermospray ionization (TSP). In other words, the composition for mass spectrometry, the chip, and the mass spectrometry method provided in the embodiments of the present invention can be used in combination with any ionization source and have versatility.

In some embodiments, the mass spectrometer further comprises a mass analyzer, which includes but is not limited to a quadrupole time-of-flight (TOF) analyzer, a sector magnetic field, a Fourier transform, and a quadrupole ion trap, and the mass analyzers can be used alone or in series. That is to say, the mass spectrometry composition, chip, and mass spectrometry method provided in the embodiments of the present invention can be used in conjunction with any mass analyzer and have universality.

The following specific examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values exemplified below.

Example 1

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

(1) An extraction kit, comprising: a lysis solution, a washing solution I, a washing solution II, an elution solution, a proteinase K, and a magnetic bead solution;

(2) Enrichment of reaction components: enrichment of reaction solution, enrichment of enzyme solution;

(3) Shrimp alkaline phosphatase (SAP enzyme) reaction components: SAP reaction solution, SAP enzyme solution;

(4) Extension reaction components: extension reaction solution, extension enzyme solution.

The above extraction kit, enrichment reaction components, SAP enzyme reaction components, and extension reaction components are all from Zhongyuan Huiji Biotechnology Co., Ltd.

The steps for nucleic acid mass spectrometry detection are as follows:

(1) DNA extraction from samples: DNA was extracted using the components of the Zhongyuan Huiji extraction kit, and the instrument was the Zhongyuan Huiji fully automatic nucleic acid extraction instrument EXM6000.

(2) Multiplex PCR reaction:

Table 1. PCR reaction system

Reagent name Volume (μL) Amplification reaction solution 10 Amplification enzyme solution 5 DNA (DNA extracted as above) 5

Prepare the multiplex PCR reaction system according to Table 1. The reaction volume can be reduced in proportion according to the experimental requirements so that high-throughput detection can be performed through the 384 PCR plate. The prepared multiplex PCR reaction system is amplified on a PCR instrument. The PCR amplification reaction is shown in Table 2:

Table 2. PCR amplification reaction

(3) After the PCR amplification is completed, a SAP reaction is performed, which includes digestion at 37°C for 30-40 min and inactivation at 65°C for 5 min. The reaction volume can be reduced proportionally according to the experimental requirements so that high-throughput detection can be performed using a 384 PCR plate.

(4) After digestion, perform an extension reaction. Prepare an extension reaction system, add 7 μl per well to the SAP reaction product. The extension reaction settings are shown in Table 3.

Table 3. Single base extension reaction

(5) Resin desalting treatment: Add 20-40 mg of resin and 40 μl of ddH ₂ O to each reaction well. The volume of resin and ddH ₂ O can be reduced in proportion to the experimental requirements in order to achieve high-throughput detection through the 384 PCR plate. The above resin is from Zhongyuan Huiji Biotechnology Co., Ltd. Cover the reaction tube (if using a 384 PCR plate, seal it with a sealing film), shake it upside down for 5-15 minutes, and then centrifuge it briefly.

(6) After desalting, the system was tested on a machine. The mass spectrometer was an EXS8000 time-of-flight mass spectrometer system from Zhongyuan Huiji Biotechnology Co., Ltd.

Example 2

This embodiment prepares different matrices and uses silicon as the substrate. The sample area on the substrate is a circular area (800 microns in diameter). Furthermore, this embodiment uses matrix-assisted laser desorption ionization (MALDI) mass spectrometry and the SNP detection kit mentioned in Example 1 to detect the deafness gene.

The specific preparation process of the mass spectrometry chip in this embodiment is as follows:

S1. Prepare a mixed solution 1 containing 300 mM 3-hydroxypicolinic acid (3-HPA) and 25 mM diammonium citrate (DAC) using 30% acetonitrile aqueous solution as a standard matrix.

S2. Prepare 0.75M 18-crown-6-ether solution with 30% acetonitrile aqueous solution.

S3. Take 10 μl of 0.75 M 18-crown-6-ether solution and add it to 1000 μl of mixed solution 1, mix well, and obtain mixed solution 2 as a new matrix.

s4. Use a pipette or dispenser to transfer 0.3 μl of standard matrix or new matrix to the substrate sample area and let it dry naturally.

The mass spectrometer used in this example is the EXS8000 time-of-flight mass spectrometer system of Zhongyuan Huiji Biotechnology Co., Ltd.

1. Crystallization Detection

The crystallization effects of the standard matrix and the new matrix on the mass spectrometry chip were observed under a microscope, and the results are shown in Figure 3. As shown in Figure 3, the standard matrix prepared in this example forms coffee rings after crystallization, while the new matrix presents full, flat and uniform crystals on the substrate.

2. Detection Application

Two mass spectrometry chips were prepared using this embodiment to detect 17 loci of the deafness gene, and the 17 loci are specifically:

Four SNP mutation sites on the GJB2 gene: rs80338943 (235delC), rs750188782 (176_191del16), rs111033204 (299_300delAT), rs80338939 (35delG);

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

Eleven 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), and rs111033313 (IVS7-2A＞G).

The specific operation of the detection process is as follows:

S1. Extraction of sample DNA;

S2, multiplex PCR reaction;

S3, phosphatase digestion treatment;

S4, single base extension reaction

S5, resin desalination treatment;

S6. Use a pipette or spotter to transfer the reaction product to the detection chip, and use the MALDI-tofEXS8000 time-of-flight mass spectrometry system of Zhongyuan Huiji Biotechnology Co., Ltd. for detection.

Among them, the primers for the multiplex PCR reaction are shown in Table 4:

Table 4. Primers for multiplex PCR reactions

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

Table 5. Single base extension reaction primers

The 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 analysis results of the 17 loci of the deafness gene detected by the mass spectrometry chip made of the standard matrix and the new matrix are shown in Figure 4 and Figure 5, respectively. By comparison, the mass spectrometry analysis results obtained by the mass spectrometry chip made of the new matrix have high resolution and can accurately detect the SNP typing of the deafness-related susceptibility gene.

The mass spectrometry acquisition success rates of the two matrices are shown in Table 7:

Table 7. Cumulative success rate summary

Mass spectrometry analysis of standard matrix Mass spectrometry analysis 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, it can be seen that the mass spectrometry chip made of the new matrix can obtain more high-quality acquisition spectra with the same number of mass spectrometry acquisitions, and the mass spectrometry acquisition becomes more efficient. The reason is that the new matrix can crystallize fully, flatly and evenly on the substrate.

Example 3

In this embodiment, eight different matrices (matrices 0 to 7) were prepared, and eight different mass spectrometry chips were prepared using copper as the substrate. The sample area on the substrate was a circular area (1000 micrometers in diameter). The specific preparation process was as follows:

S1. A mixed solution containing 270 mM 3-hydroxypicolinic acid (3-HPA), 100 mM 2,5-dihydroxybenzoic acid (DHB) and 30 mM diammonium citrate (DAC) was prepared with 40% acetonitrile aqueous solution as matrix 0.

S2. Dissolve 0.04 mM aza-12-crown ether-4 in matrix 0 and mix well to prepare matrix 1.

S3. Dissolve 0.4 mM aza-12-crown ether-4 in matrix 0 and mix well to prepare matrix 2.

S4. Dissolve 4 mM aza-12-crown ether-4 in matrix 0 and mix well to prepare matrix 3.

S5. Dissolve 40 mM aza-12-crown ether-4 in matrix 0 and mix well to prepare matrix 4.

S6. Dissolve 60 mM aza-12-crown ether-4 in matrix 0 and mix well to prepare matrix 5.

S6. Dissolve 80 mM aza-12-crown ether-4 in matrix 0 and mix well to prepare matrix 6.

S7. Dissolve 120 mM aza-12-crown ether-4 in matrix 0 and mix well to prepare matrix 7.

S8. Use a pipette or dispenser to transfer 0.4 μl of matrix 0-7 to the substrate sample area and let it dry naturally.

The crystallization effects of substrates 0 to 7 on the mass spectrometry chip were observed under a microscope, and the results are shown in FIG6 . As can be seen from FIG6 , the substrate 0 (standard substrate) prepared in this embodiment forms coffee rings after crystallization, and the substrates 2/3/4/5 (new substrates) show full, smooth and uniform crystals on the substrate, the substrate 6 has the second best effect, and the substrate 1/7 (new substrate) has a slightly worse crystallization effect, which indicates that the content of component B accounts for 0.01 to 30% of the total content of component A, preferably 0.01 to 20%, more preferably 0.01 to 15%, and most preferably 0.1 to 10%. It is best to control the content of component B to 0.1 to 10% of the total content of component A.

Example 4

In this embodiment, twenty-four different matrices (matrices 8 to 31) were prepared, and twenty-four different mass spectrometry chips were prepared using silicon as the substrate, and the sample area on the substrate was a circular area (500 microns in diameter); further, this embodiment used matrix-assisted laser desorption ionization (MALDI) mass spectrometry and the SNP detection kit mentioned in Example 1 to detect the deafness gene.

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

S1. A mixed solution A containing 500 mM 3-hydroxypicolinic acid (3-HPA) and 35 mM ammonium oxalate was prepared using a 30% acetonitrile aqueous solution as the matrix 8.

S2. Prepare 100 mM solution B from component B in Table 8 with water.

S3. Take 10 μl of 100 mM solution B and add it to 1000 μl of mixed solution A, mix well, and obtain mixed solution C. In this way, matrix 9 to 31 are prepared as new matrix.

S4. Use a pipette or a dispensing machine to transfer 0.1 μl of matrix 8-31 to the substrate sample area and let it dry naturally.

The mass spectrometer used in this example is the EXS8000 time-of-flight mass spectrometer system of Zhongyuan Huiji Biotechnology Co., Ltd.

Table 8. Matrix 9 to 31 and its corresponding component B

1. Crystallization Detection

The crystallization effects of matrix 8 (standard matrix) and matrix 9-31 (new matrix) on the mass spectrometry chip were observed under a microscope, and the results are shown in Figure 7. As can be seen from Figure 7, matrix 8 prepared in this embodiment forms coffee rings after crystallization, while matrix 9-31 presents full, flat and uniform crystals on the substrate.

2. Detection Application

The mass spectrometry chip prepared in this example was used to detect 17 loci of the deafness gene. The mass spectrometry chip prepared from matrix 8 (standard matrix) and matrixes 9 to 31 was used to detect 17 loci of the deafness gene (see Example 2). The mass spectrometry analysis results and the average success rate of mass spectrometry analysis acquisition were shown in Table 9:

Table 9. Summary of the accuracy of mass spectrometry analysis results and the average success rate of mass spectrometry analysis acquisition corresponding to matrices 8 to 31

From the above, it can be seen that the mass spectrometry chip prepared using matrix 9-31 can obtain more high-quality collection spectra under the same number of mass spectrometry collection times, and the replacement of component B has little effect on the collection efficiency, indicating that component B of the present invention can be selected from crown ethers, azacrown ethers, thiocrown ethers and their derivatives, tautomers or analogues. The use of the new matrix of the present invention makes mass spectrometry collection more efficient, because the new matrix can be fully, flatly and evenly crystallized on the substrate.

Example 5

In this embodiment, five different matrices (matrices 32 to 36) are prepared, and five different mass spectrometry chips are prepared using glass as the substrate. The sample area on the substrate is a circular area (1200 microns in diameter); further, this embodiment uses matrix-assisted laser desorption ionization (MALDI) mass spectrometry and the SNP detection kit mentioned in Example 1 to detect the deafness gene.

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

S1. A mixed solution containing 1000 mM 3-hydroxypicolinic acid (3-HPA) and 15 mM 15-crown-5-ether was prepared using a 10% acetonitrile aqueous solution as the matrix 32.

S2. A mixed solution containing 1000 mM 3-hydroxypicolinic acid (3-HPA) and 15 mM 15-crown-5-ether was prepared using a 20% acetonitrile aqueous solution as the matrix 33.

S3. A mixed solution containing 1000 mM 3-hydroxypicolinic acid (3-HPA) and 15 mM 15-crown-5-ether was prepared using 50% acetonitrile aqueous solution as the matrix 34.

S4. A mixed solution containing 1000 mM 3-hydroxypicolinic acid (3-HPA) and 15 mM 15-crown-5-ether was prepared using 60% acetonitrile aqueous solution as the matrix 35.

S5. A mixed solution containing 1000 mM 3-hydroxypicolinic acid (3-HPA) and 15 mM 15-crown-5-ether was prepared using 80% acetonitrile aqueous solution as the matrix 36.

S6. Use a pipette or a dispensing machine to transfer 0.1 μl of matrix 32-36 to the substrate sample area and let it dry naturally.

The mass spectrometer used in this example is the EXS8000 time-of-flight mass spectrometer system of Zhongyuan Huiji Biotechnology Co., Ltd.

1. Crystallization Detection

The crystallization effects of the matrices 32 to 36 on the mass spectrometry chip were observed under a microscope, and the results are shown in Figure 8. As can be seen from Figure 8, the matrices 33 to 34 prepared in this embodiment present full, flat, and uniform crystals on the substrate, the effect of the matrix 35 is second, and the crystallization effects of the other matrices (matrices 32 and 36) are slightly worse.

2. Detection Application

Five mass spectrometry chips were prepared using this embodiment to detect 17 loci of the deafness gene, and the mass spectrometry acquisition success rates of the five matrices are shown in Table 10. As can be seen from Table 10, the average success rates of mass spectrometry acquisition of matrices 33 and 34 are higher than those of the other three.

Table 10. Cumulative success rate summary

As can be seen from the above, the concentration of the solvent used to prepare the matrix, ie, the acetonitrile aqueous solution, is 10 to 80%, preferably 10 to 60%, and most preferably 20 to 50%.

Example 6

In this embodiment, different matrices are prepared, and different mass spectrometry chips are prepared using resin glass as the substrate. The sample area on the substrate is a circular area (300 microns in diameter). Furthermore, in this embodiment, matrix-assisted laser desorption ionization (MALDI) mass spectrometry and the SNP detection kit mentioned in Example 1 are used to detect the deafness gene.

The specific preparation process of the mass spectrometry chip in this embodiment is as follows:

S1. Prepare 100 mM solution A of component A in Table 11 with 30% acetonitrile aqueous solution as the standard matrix.

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

S3. Take 100 μl of 4M 1-aza-15-crown-5 solution and add it to 1000 μl of solution A, mix well, and obtain mixed solution C. In this way, matrix 37-68 is prepared as a new matrix.

S4. Use a pipette or a dispensing machine to transfer 0.3 μl of the standard matrix or new matrix (i.e., matrix 37-68) to the substrate sample area and allow to dry naturally.

The mass spectrometer used in this example is the EXS8000 time-of-flight mass spectrometer system of Zhongyuan Huiji Biotechnology Co., Ltd.

Table 11. Matrix 37-68 and its corresponding component A

Detection Application

The mass spectrometry chip prepared in this example was used to detect 17 loci of the deafness gene, and the mass spectrometry chip prepared using matrices 37 to 68 was used to detect 17 loci of the deafness gene (see Example 2). The average success rate of mass spectrometry analysis acquisition is shown in Table 12.

Table 12. Average success rate of mass spectrometry acquisition corresponding to matrices 37 to 68

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

As can be seen from Table 12, when component A is 3-hydroxypicolinic acid and 2,5-dihydroxybenzoic acid, the collection success rate is high. The collection efficiency of 3-hydroxypicolinic acid and 2,5-dihydroxybenzoic acid shared with other added components will be improved to a certain extent, but the added components do not have a decisive influence on the collection efficiency, and the added components are separately prepared as solution A, and the collection efficiency is low, indicating that 3-hydroxypicolinic acid or 2,5-dihydroxybenzoic acid has a decisive influence on the collection efficiency. 3-hydroxypicolinic acid or 2,5-dihydroxybenzoic acid must be added to solution A, and other added components can be added by those skilled in the art according to demand. At the same time, it can be seen from the experimental data that the molar content of the core components 3-HPA and/or DHB in component A is preferably 90-100%.

The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims (10)

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.