Matrix solution and matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method

Abstract

The present invention relates to the field of analysis and detection technology, and in particular to a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method. The matrix solution includes 3-hydroxy-2-picolinic acid, diammonium hydrogen citrate, acetonitrile, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution and a solvent; the final concentration of tris(hydroxymethyl)aminomethane in the matrix solution is 0.01mol / L to 1mol / L. The matrix solution of the present invention can meet the microliter grade sample preparation, form a uniform and dense matrix layer, is conducive to the adsorption of nucleic acid samples, and compensates for the situation that the signal peak intensity is weak or the peak is missing due to the degradation of nucleic acids, greatly improves the collection success rate of mass spectrometry signals, and can make the collection success rate of mass spectrometry signals reach 100%.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method. Background Art

[0002] In the 1980s, the emergence of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) brought revolutionary breakthroughs to the biological and medical fields. Biomacromolecules such as nucleic acids and proteins can also be analyzed and studied using mass spectrometers, which greatly promoted the development of genomics and proteomics. In 2014, the U.S. Food and Drug Administration (FDA) approved MALDI-TOF MS for clinical nucleic acid testing. Nucleic acid mass spectrometry combines biotechnology and mass spectrometry technology. After the sample to be tested undergoes multiple PCR-specific amplification designed for polymorphic sites and single-base extension of SNP sites, the reaction product forms a co-crystal with a specific matrix. Then, in the mass spectrometer, the co-crystal absorbs the incident laser and provides energy to the nucleic acid sample for desorption and ionization, generating ions with different mass-to-charge ratios. The molecular weight of different types of ions in the sample is determined by the mass analyzer. Among them, the specific matrix is ​​the key to nucleic acid mass spectrometry analysis.

[0003] At present, the matrix commonly used for nucleic acid mass spectrometry analysis includes organic substances such as 3-hydroxy-2-pyridine carboxylic acid (3-HPA) and 2,4,6-trihydroxyacetophenone (2,4,6-THAP). Its crystals are thick rod-shaped and scattered on the target. When the nucleic acid is adsorbed on the uneven matrix layer, it will cause problems such as large quality deviation and low resolution of the test results, thereby affecting the site multiplicity of nucleic acid mass spectrometry detection. We know that the uniformity of crystallization is the factor that affects the success rate of acquisition the most. This uneven crystallization seriously affects the stability and repeatability of experimental results. However, if the uneven crystallization is filled by increasing the amount of matrix, the excessive matrix will lead to excessive absorption of laser energy, resulting in the fragmentation of nucleic acid fragments; if the uneven matrix crystallization is avoided by reducing the amount of matrix to avoid the impact on nucleic acid mass spectrometry analysis, then nanoliter-scale sample preparation is required. Nanoliter-scale sample preparation requires a special nanoliter spotter to pre-prepare the matrix and sample on a disposable chip target plate. The sample preparation process is limited by the spotter and specific consumables, and also greatly increases the cost of use, hindering the widespread application of MALDI-TOF MS, an emerging technology, in nucleic acid detection.

[0004] In addition, by improving the uniformity of crystallization, the success rate of nucleic acid mass spectrometry signal acquisition can be greatly improved. However, since nucleic acids may be degraded when bound to the matrix, low signal intensity or missing signal peaks may still occur during signal acquisition, ultimately affecting the stability and repeatability of the experimental results. Summary of the invention

[0005] Based on this, the present invention provides a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method. The matrix solution can meet the microliter-level sample preparation, form a uniform and dense matrix layer, is conducive to the adsorption of nucleic acid samples, and can make up for the weak signal peak intensity or peak loss caused by nucleic acid degradation, greatly improve the collection success rate of mass spectrometry signals, and can make the collection success rate of mass spectrometry signals reach 100%.

[0006] The technical solutions provided by the present invention include the following:

[0007] A matrix solution comprising 3-hydroxy-2-pyridinecarboxylic acid, diammonium hydrogen citrate, acetonitrile, tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution and a solvent;

[0008] The final concentration of the tris(hydroxymethyl)aminomethane in the matrix solution is 0.01 mol / L to 1 mol / L.

[0009] In one embodiment, the final concentration of tris(hydroxymethyl)aminomethane in the matrix solution is 0.015 mol / L to 0.9 mol / L.

[0010] In one embodiment, the pH value of the tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution is 7.0-9.2.

[0011] In one embodiment, the final concentration of the 3-hydroxy-2-pyridinecarboxylic acid in the matrix solution is 0.01 g / mL to 0.5 g / mL; and / or

[0012] The final concentration of diammonium citrate in the matrix solution is 0.05 mol / L to 2 mol / L; and / or

[0013] The volume fraction of the acetonitrile in the matrix solution is 5% to 50%.

[0014] In one embodiment, the final concentration of the 3-hydroxy-2-pyridinecarboxylic acid in the matrix solution is 0.02 g / mL to 0.45 g / mL; and / or

[0015] The final concentration of diammonium citrate in the matrix solution is 0.1 mol / L to 1.8 mol / L; and / or

[0016] The volume fraction of the acetonitrile in the matrix solution is 8% to 46%.

[0017] In one embodiment, the solvent is water.

[0018] A matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method comprises the following steps:

[0019] The matrix solution is spotted on the target plate and dried to form a matrix layer;

[0020] Spotting a sample solution to be tested on the matrix layer and drying it to form a sample layer;

[0021] Perform mass spectrometry analysis.

[0022] In one embodiment, the sample to be detected is nucleic acid.

[0023] In one embodiment, the volume ratio of the matrix solution to the sample to be detected is (0.5-2.5): (0.5-2.5).

[0024] In one embodiment, the target plate is a stainless steel target plate.

[0025] Compared with the traditional solution, the present invention has the following beneficial effects:

[0026] The matrix solution of the invention can be applied to a stainless steel target plate conventionally used in MALDI-TOF MS to form a uniform and dense matrix layer. The addition of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution can also make up for the situation that the signal peak intensity is weak or the peak is missing due to the degradation of nucleic acid, and the acquisition success rate of nucleic acid mass spectrum signals is greatly improved. Conventional random automatic targeting can be realized on a mass spectrometer, the mass spectrum signal is strong and has no missing peaks, the acquisition success rate of the mass spectrum signal can reach 100%, and the mass deviation of the detection result can be less than 300 ppm, the mass resolution can reach more than 1000, and the experimental result has good stability and repeatability.

[0027] In addition, the matrix solution of the present invention can meet the requirements of microliter-scale sample preparation, avoid the use of limited sample preparation instruments and consumables (such as nanoliter-scale sample preparation), reduce costs, lower the threshold for use of nucleic acid mass spectrometry platforms, and is friendly to groups such as scientific research institutions and reagent development companies that do not perform large-scale sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a diagram showing the crystallization of the matrix layer of Example 1;

[0029] Figure 2 This is the nucleic acid mass spectrum collected in Example 1;

[0030] Figure 3 This is a schematic diagram of the mass spectrometry acquisition success rate of Example 1;

[0031] Figure 4 is a diagram showing the crystallization of the matrix layer of Example 2;

[0032] Figure 5 This is the nucleic acid mass spectrum collected in Example 2;

[0033] Figure 6 This is a schematic diagram of the mass spectrum acquisition success rate of Example 2;

[0034] Figure 7 is a diagram showing the crystallization of the matrix layer of Example 3;

[0035] Figure 8 This is the nucleic acid mass spectrum collected in Example 3;

[0036] Fig. 9 This is a schematic diagram of the mass spectrum acquisition success rate of Example 3;

[0037] Fig.10 This is the nucleic acid mass spectrum collected in Comparative Example 1;

[0038] Fig.11 Schematic diagram of the mass spectrum acquisition success rate of Comparative Example 1;

[0039] Fig.12 This is the nucleic acid mass spectrum collected in Comparative Example 2;

[0040] Fig.13 Schematic diagram of the mass spectrometry acquisition success rate of Comparative Example 2. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0043] the term

[0044] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0045] In the present invention, the optional scope of the terms "and / or", "or / and", and "and / or" used includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0046] In the present invention, "one or several" refers to any one, any two or any two or more of the listed items. Among them, "several" refers to any two or any two or more.

[0047] In the present invention, "combinations thereof", "any combinations thereof", "any combination thereof" and the like used include all suitable combinations of any two or more of the listed items.

[0048] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0049] In the present invention, “preferred” is only used to describe an implementation method or example with better effects, and it should be understood that it does not constitute a limitation on the protection scope of the present invention.

[0050] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0051] In the present invention, reference to a numerical range includes both endpoints of the numerical range unless otherwise specified.

[0052] In the present invention, when it comes to percentage content, unless otherwise specified, for solid-liquid mixing and solid-solid mixing, it refers to mass percentage, and for liquid-liquid mixing, it refers to volume percentage.

[0053] In the present invention, when it comes to percentage concentration, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0054] In the present invention, when it comes to temperature parameters, unless otherwise specified, both constant temperature treatment and treatment within a certain temperature range are permitted. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0055] Currently, the matrices commonly used in nucleic acid mass spectrometry analysis include organic substances such as 3-hydroxy-2-pyridinecarboxylic acid (3-HPA) and 2,4,6-trihydroxyacetophenone (2,4,6-THAP). Their crystals are thick rod-shaped and scattered on the target. When nucleic acids are adsorbed on the uneven matrix layer, it will cause problems such as large quality deviation and low resolution of the test results, which will in turn affect the site multiplicity of nucleic acid mass spectrometry detection.

[0056] We know that the uniformity of crystallization is the factor that most affects the success rate of collection. This uneven crystallization seriously affects the stability and repeatability of experimental results.

[0057] However, if the amount of matrix is ​​increased to fill the uneven crystallization, the excess matrix will lead to excessive absorption of laser energy, resulting in the fragmentation of nucleic acid fragments; if the amount of matrix is ​​reduced to avoid uneven matrix crystallization and avoid the impact on nucleic acid mass spectrometry analysis, then nanoliter-scale sample preparation is required. Nanoliter-scale sample preparation requires very small amounts of matrix and sample preparation, and the experimental operation is completely dependent on a special nanoliter spotter and disposable chip target plate. However, the disposable chip target plate and nanoliter spotter are expensive, and the cost of the whole machine is high, which raises the threshold for the use of nucleic acid mass spectrometry platforms. It is not friendly to groups that do not analyze large quantities of samples, such as scientific research institutions and reagent development companies, and hinders the widespread application of MALDI-TOF MS, an emerging technology, in nucleic acid detection.

[0058] In addition, by improving the uniformity of crystallization, the success rate of nucleic acid mass spectrometry signal acquisition can be greatly improved. However, since nucleic acids may be degraded when bound to the matrix, low signal intensity or missing signal peaks may still occur during signal acquisition, ultimately affecting the stability and repeatability of the experimental results.

[0059] Based on this, the present invention provides a matrix solution, and the technical solution is as follows:

[0060] A matrix solution comprising 3-hydroxy-2-pyridinecarboxylic acid, diammonium hydrogen citrate, acetonitrile, tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution and a solvent;

[0061] The final concentration of the tris(hydroxymethyl)aminomethane in the matrix solution is 0.01 mol / L to 1 mol / L.

[0062] The matrix solution of the present invention is based on 3-hydroxy-2-pyridine carboxylic acid, and diammonium hydrogen citrate and acetonitrile are added, which is conducive to forming a uniform and dense matrix layer, and is conducive to the adsorption of nucleic acid samples. At the same time, the addition of trishydroxymethylaminomethane-hydrochloric acid buffer solution can also make up for the situation that the signal peak intensity is weak or the peak is missing due to the degradation of nucleic acid, and the acquisition success rate of nucleic acid mass spectrum signals is greatly improved, and conventional random automatic targeting can be realized on the mass spectrometer, the mass spectrum signal is strong, and there is no missing peak, the acquisition success rate of the mass spectrum signal can reach 100%, and the mass deviation of the detection result can be less than 300ppm, the mass resolution can reach more than 1000, and the experimental result has good stability and repeatability.

[0063] Among them, Tris (hydroxymethyl) aminomethane (generally referred to as Tris) is an organic compound with the molecular formula (HOCH 2 ) 3 CNH 2 Tris-HCl buffer solution can also be expressed as Tris-HCl solution.

[0064] It is understandable that the mother solution of the tris-hydrochloric acid buffer solution is prepared in advance, and then the mother solution is diluted and mixed with other components to prepare the matrix solution. Due to the particularity of the matrix solution storage, it is freshly prepared for each experiment.

[0065] In one embodiment, in the mother liquor of the tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution, the concentration of tris(hydroxymethylaminomethane) is 1 mol / L, the mother liquor is 100 mL in total, and the method for preparing the mother liquor of the tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution comprises the following steps:

[0066] Weigh 121.1 g of tris(hydroxymethyl)aminomethane, place in a 100 mL beaker, add about 80 mL of deionized water, stir thoroughly to dissolve; add concentrated HCl, and adjust the amount of concentrated HCl according to the required pH value; make the solution dilute to 100 mL, sterilize at high temperature, and store at room temperature.

[0067] Optionally, the pH value of the Tris-hydrochloric acid buffer solution is 7.0 to 9.2. Within this range, the buffer solution is stable and effective.

[0068] The mother solution is diluted to prepare a matrix solution. Specifically, the final concentration of the tris(hydroxymethyl)aminomethane in the matrix solution includes but is not limited to 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, 0.55 mol / L, 0.6 mol / L, 0.65 mol / L, 0.7 mol / L, 0.75 mol / L, 0.8 mol / L, 0.85 mol / L, 0.9 mol / L, 0.95 mol / L, and 1 mol / L. Preferably, the final concentration of the tris(hydroxymethyl)aminomethane in the matrix solution is 0.015 mol / L to 0.9 mol / L. More preferably, the final concentration of the tris(hydroxymethyl)aminomethane in the matrix solution is 0.015 mol / L to 0.8 mol / L. More preferably, the final concentration of the tris(hydroxymethyl)aminomethane in the matrix solution is 0.015 mol / L to 0.6 mol / L. More preferably, the final concentration of the tris(hydroxymethyl)aminomethane in the matrix solution is 0.015 mol / L to 0.45 mol / L.

[0069] Optionally, diammonium citrate can also be prepared in advance as diammonium citrate solution. In one embodiment, 100 mL of 4 mol / L diammonium hydrogen citrate aqueous solution is prepared, diluted, and mixed with other components to prepare the matrix solution.

[0070] Optionally, the diammonium citrate aqueous solution is diluted, specifically, the final concentration of the diammonium citrate in the matrix solution includes but is not limited to 0.05mol / L, 0.1mol / L, 0.5mol / L, 1mol / L, 1.5mol / L, 2mol / L. Preferably, the final concentration of the diammonium citrate in the matrix solution is 0.05mol / L to 2mol / L. More preferably, the final concentration of the diammonium citrate in the matrix solution is 0.1mol / L to 1.8mol / L. More preferably, the final concentration of the diammonium citrate in the matrix solution is 0.2mol / L to 1.6mol / L. More preferably, the final concentration of the diammonium citrate in the matrix solution is 0.4mol / L to 1.4mol / L.

[0071] Acetonitrile is also added to the above-mentioned matrix solution. It can be understood that the volume fraction of the acetonitrile in the matrix solution includes but is not limited to 5%, 10%, 20%, 30%, 40%, and 50%. Preferably, the volume fraction of the acetonitrile in the matrix solution is 5% to 50%. More preferably, the volume fraction of the acetonitrile in the matrix solution is 8% to 46%. More preferably, the volume fraction of the acetonitrile in the matrix solution is 13% to 40%. More preferably, the volume fraction of the acetonitrile in the matrix solution is 17% to 37%.

[0072] 3-hydroxy-2-pyridinecarboxylic acid is also added to the above matrix solution. It can be understood that the final concentration of the 3-hydroxy-2-pyridinecarboxylic acid in the matrix solution includes but is not limited to 0.01g / mL, 0.05g / mL, 0.1g / mL, 0.2g / mL, 0.3g / mL, 0.4g / mL, and 0.5g / mL. Preferably, the final concentration of the 3-hydroxy-2-pyridinecarboxylic acid in the matrix solution is 0.01g / mL to 0.5g / mL. More preferably, the final concentration of the 3-hydroxy-2-pyridinecarboxylic acid in the matrix solution is 0.02g / mL to 0.45g / mL. More preferably, the final concentration of the 3-hydroxy-2-pyridinecarboxylic acid in the matrix solution is 0.03g / mL to 0.38g / mL. More preferably, the final concentration of the 3-hydroxy-2-pyridinecarboxylic acid in the matrix solution is 0.04g / mL to 0.35g / mL.

[0073] In one embodiment, the method for preparing the above-mentioned matrix solution comprises the following steps:

[0074] Dilute 100 mL of 4 mol / L diammonium hydrogen citrate solution and 100 mL of 1 mol / L tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution with a pH of 7.0 to 9.2 to prepare 1 mL of a matrix dissolving solution, wherein the matrix dissolving solution contains diammonium hydrogen citrate with a final concentration of 0.05 mol / L to 2 mol / L, tris(hydroxymethylaminomethane) with a final concentration of 0.01 mol / L to 1 mol / L, and acetonitrile with a volume fraction of 5% to 50%, add water to make up to 1 mL, and set aside;

[0075] Add 0.01 g to 0.5 g of 3-hydroxy-2-pyridinecarboxylic acid to the above-mentioned reserve solution, dissolve until clear, and centrifuge to obtain a matrix solution.

[0076] The matrix solution is prepared and used immediately. By adjusting the components, it can ensure that the matrix solution is precipitated evenly and densely at the entire target location, forming a co-crystal with the subsequently adsorbed nucleic acid sample; it can also make up for the weak signal peak intensity or peak loss caused by nucleic acid degradation, greatly improving the success rate of nucleic acid mass spectrometry signal acquisition.

[0077] The above-mentioned matrix solution can meet the requirements of microliter-scale sample preparation, avoid the use of limited sample preparation instruments and consumables (such as nanoliter-scale sample preparation), reduce costs, lower the threshold for using nucleic acid mass spectrometry platforms, and is friendly to scientific research institutions, reagent development companies and other groups that do not perform large-scale sample analysis.

[0078] The present invention also provides a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method, and the technical scheme is as follows:

[0079] Pipette 0.5 μL to 2.5 μL of the prepared matrix solution, spot it on the target center of the stainless steel target plate, dry it, and wait for uniform and dense needle-shaped crystals to precipitate to form a matrix layer;

[0080] Pipette 0.5 μL to 2.5 μL of the nucleic acid sample solution to be detected onto the matrix layer, dry it, and wait for the co-crystallization at the target site to be completed to form a sample layer;

[0081] Perform mass spectrometry analysis.

[0082] It can be understood that the above detection method can adopt microliter-level sample preparation, or flexibly select manual sample preparation according to the application scenario. The sample preparation process is not limited by the instrument, which reduces the cost.

[0083] In addition, stainless steel target plates can also be recycled multiple times, greatly reducing the cost of the entire machine and consumables.

[0084] It can be understood that the method of mass spectrometry analysis is a routine method in the art.

[0085] The matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method of the present invention can avoid the use of nanoliter spotters and disposable chip target plates due to the improvement of the components of the matrix solution. The adsorption and detection of nucleic acid samples can also be completed by using microliter-level sample preparation and stainless steel target plates. Users can choose the sample preparation method according to their respective application needs, thereby expanding the application scenarios of MALDI-TOF MS on a large scale, and providing strong technical support for the application of MALDI-TOF MS in clinical medical diagnosis. A uniform, dense, and flat matrix layer can be obtained, which is conducive to the adsorption of nucleic acid samples, and nucleic acid samples are not easily degraded. The nucleic acid mass spectrometry signal acquisition success rate is high, up to 100%, the mass deviation of the detection results is less than 300ppm, and the mass resolution can reach more than 1000. The experimental results have good stability and repeatability.

[0086] The following is further described in conjunction with specific examples and comparative examples. The raw materials involved in the following specific examples, unless otherwise specified, can all be sourced from commercial sources. The instruments used, unless otherwise specified, can all be sourced from commercial sources. The processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0087] Example 1

[0088] This embodiment provides a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method, the steps are as follows:

[0089] 1. Prepare the mother liquor of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution

[0090] Weigh 121.1 g of tris(hydroxymethyl)aminomethane, place it in a 100 mL beaker, add about 80 mL of deionized water, and stir thoroughly to dissolve; use concentrated HCl to adjust the pH value of the buffer solution to 9.0, then make the solution dilute to 100 mL, sterilize at high temperature and store at room temperature to obtain a mother solution of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a concentration of 1 mol / L.

[0091] 2. Prepare diammonium citric acid solution

[0092] Weigh 90.472 g of diammonium citric acid, place it in a 100 mL beaker, add water to fully dissolve it, then add water to make the volume up to 100 mL to obtain a diammonium citric acid solution with a concentration of 4 mol / L.

[0093] 3. Prepare the matrix solution

[0094] The mother liquor and diammonium citrate solution were diluted with acetonitrile and water to prepare 1 mL of the matrix solution. Specifically, a certain volume of the mother liquor, a certain volume of the diammonium citrate solution, a certain volume of acetonitrile and water were taken, and water was added to make the volume 1 mL, so that the concentration of trishydroxymethylaminomethane in the matrix solution was 0.025 mol / L, the concentration of diammonium citrate was 0.4 mol / L, and the volume fraction of acetonitrile was 30%, and the matrix solution was obtained.

[0095] Add 0.05 g of 3-hydroxy-2-pyridinecarboxylic acid to the above matrix solution, dissolve until clear, and centrifuge to obtain a matrix solution.

[0096] 4. Prepare the sample solution to be tested

[0097] Prepare 1 mL of the nucleic acid sample solution to be tested and set aside.

[0098] 1) Composition of nucleic acid samples to be tested

[0099] The nucleic acid sample solution to be tested is composed of 8 single-stranded nucleotide sequences P1 to P8, and their molecular weights are: P1-3052.1, P2-3655.5, P3-5523.7, P4-6511.3, P5-8100.3, P6-9006.9, P7-9022.9, P8-9938.5;

[0100] 2) Preparation of 8 single-stranded nucleotide sequences P1 to P8

[0101] Add the corresponding deionized water according to the label on each tube of the 8 single-stranded nucleotide sequence synthesized dry powders to ensure that the concentration of each tube of single-stranded nucleotide sequence is 100 μM;

[0102] 3) Preparation of nucleic acid samples to be tested

[0103] The corresponding nucleic acid samples were prepared according to the required amount. In the nucleic acid sample solution, the final concentration of P1 was 3 μM, the final concentration of P2 was 0.5 μM, the final concentration of P3 was 6 μM, the final concentration of P4 was 1 μM, the final concentration of P5 was 13 μM, the final concentration of P6 was 7 μM, the final concentration of P7 was 7 μM, and the final concentration of P8 was 9 μM.

[0104] 5. Order.

[0105] Pipette 1.75 μL of the prepared matrix solution and spot it on the target center of the stainless steel target plate, dry it, and wait for uniform and dense needle-shaped crystals to precipitate to form a matrix layer. Figure 1 As shown;

[0106] Pipette 1 μL of the nucleic acid sample solution to be detected and spot it on the matrix layer, dry it, and wait for the co-crystallization at the target position to be completed to form a sample layer;

[0107] 6. Mass spectrometry

[0108] The dried co-crystallized sample was placed in a Maldi-TOF (model: CMI-1600) for mass spectrometry analysis. The mass spectrum collected is shown in Figure 2 As shown, from Figure 2 It can be seen that there is no missing signal peak in the mass spectrum, and the signal peak intensity is high. Figure 3 As shown, from Figure 3 It can be seen that the mass spectrum acquisition success rate of this embodiment can reach 100%.

[0109] Example 2

[0110] This embodiment provides a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method. The main difference from Embodiment 1 is that the contents of the components in the matrix solution are different. The steps are as follows:

[0111] 1. Prepare the mother liquor of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution

[0112] Weigh 121.1 g of tris(hydroxymethyl)aminomethane, place it in a 100 mL beaker, add about 80 mL of deionized water, and stir thoroughly to dissolve; use concentrated HCl to adjust the pH value of the buffer solution to 9.0, then make the solution dilute to 100 mL, sterilize at high temperature and store at room temperature to obtain a mother solution of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a concentration of 1 mol / L.

[0113] 2. Prepare diammonium citric acid solution

[0114] Weigh 90.472 g of diammonium citric acid, place it in a 100 mL beaker, add water to fully dissolve it, then add water to make the volume up to 100 mL to obtain a diammonium citric acid solution with a concentration of 4 mol / L.

[0115] 3. Prepare the matrix solution

[0116] The mother liquor and diammonium citrate solution were diluted with acetonitrile and water to prepare 1 mL of the matrix solution. Specifically, a certain volume of the mother liquor, a certain volume of the diammonium citrate solution, a certain volume of acetonitrile and water were taken, and water was added to make the volume 1 mL, so that the concentration of trishydroxymethylaminomethane in the matrix solution was 0.04 mol / L, the concentration of diammonium citrate was 0.5 mol / L, and the volume fraction of acetonitrile was 20%, and the matrix solution was obtained.

[0117] Add 0.04 g of 3-hydroxy-2-pyridinecarboxylic acid to the above matrix solution, dissolve until clear, and centrifuge to obtain a matrix solution.

[0118] 4. Prepare the sample solution to be tested

[0119] Prepare 1 mL of the nucleic acid sample solution to be detected in the same manner as in Example 1 for later use.

[0120] 5. Order.

[0121] Pipette 1.5 μL of the prepared matrix solution and spot it on the target center of the stainless steel target plate, dry it, and wait for uniform and dense needle-shaped crystals to precipitate to form a matrix layer. Figure 4 As shown;

[0122] 1 μL of the nucleic acid sample solution to be detected is applied onto the matrix layer, dried, and the target position is subjected to co-crystallization to form a sample layer;

[0123] 6. Mass spectrometry

[0124] The dried co-crystallized sample was placed in a Maldi-TOF (model: CMI-1600) for mass spectrometry analysis. The mass spectrum collected is shown in Figure 5 As shown, from Figure 5 It can be seen that there is no missing signal peak in the mass spectrum, and the signal peak intensity is high. Figure 6 As shown, from Figure 6 It can be seen that the mass spectrum acquisition success rate of this embodiment can reach 100%.

[0125] Example 3

[0126] This embodiment provides a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method. The main difference from Embodiment 1 is that the contents of the components in the matrix solution are different. The steps are as follows:

[0127] 1. Prepare the mother liquor of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution

[0128] Weigh 121.1 g of tris(hydroxymethyl)aminomethane, place it in a 100 mL beaker, add about 80 mL of deionized water, and stir thoroughly to dissolve; use concentrated HCl to adjust the pH value of the buffer solution to 9.0, then make the solution dilute to 100 mL, sterilize at high temperature and store at room temperature to obtain a mother solution of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a concentration of 1 mol / L.

[0129] 2. Prepare diammonium citric acid solution

[0130] Weigh 90.472 g of diammonium citric acid, place it in a 100 mL beaker, add water to fully dissolve it, then add water to make the volume up to 100 mL to obtain a diammonium citric acid solution with a concentration of 4 mol / L.

[0131] 3. Prepare the matrix solution

[0132] The mother liquor and diammonium citrate solution were diluted with acetonitrile and water to prepare 1 mL of the matrix solution. Specifically, a certain volume of the mother liquor, a certain volume of the diammonium citrate solution, a certain volume of acetonitrile and water were taken, and water was added to make the volume 1 mL, so that the concentration of trishydroxymethylaminomethane in the matrix solution was 0.35 mol / L, the concentration of diammonium citrate was 0.75 mol / L, and the volume fraction of acetonitrile was 25%, and the matrix solution was obtained.

[0133] Add 0.35 g of 3-hydroxy-2-pyridinecarboxylic acid to the above matrix solution, dissolve until clear, and centrifuge to obtain a matrix solution.

[0134] 4. Prepare the sample solution to be tested

[0135] Prepare 1 mL of the nucleic acid sample solution to be detected in the same manner as in Example 1 for later use.

[0136] 5. Order.

[0137] Pipette 2 μL of the prepared matrix solution and spot it on the target center of the stainless steel target plate, dry it, and wait for uniform and dense needle-shaped crystals to precipitate to form a matrix layer. Figure 7 As shown;

[0138] 1 μL of the nucleic acid sample solution to be detected is applied onto the matrix layer, dried, and the target position is subjected to co-crystallization to form a sample layer;

[0139] 6. Mass spectrometry

[0140] The dried co-crystallized sample was placed in a Maldi-TOF (model: CMI-1600) for mass spectrometry analysis. The mass spectrum collected is shown in Figure 8 As shown, from Figure 8 It can be seen that there is no missing signal peak in the mass spectrum, and the signal peak intensity is high. Fig. 9 As shown, from Fig. 9 It can be seen that the mass spectrum acquisition success rate of this embodiment can reach 100%.

[0141] Comparative Example 1

[0142] This comparative example provides a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method. The main difference from Example 1 is that the contents of the components in the matrix solution are different. The steps are as follows:

[0143] 1. Prepare the mother liquor of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution

[0144] Weigh 121.1 g of tris(hydroxymethyl)aminomethane, place it in a 100 mL beaker, add about 80 mL of deionized water, and stir thoroughly to dissolve; use concentrated HCl to adjust the pH value of the buffer solution to 9.0, then make the solution dilute to 100 mL, sterilize at high temperature and store at room temperature to obtain a mother solution of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a concentration of 1 mol / L.

[0145] 2. Prepare diammonium citric acid solution

[0146] Weigh 90.472 g of diammonium citric acid, place it in a 100 mL beaker, add water to fully dissolve it, then add water to make the volume up to 100 mL to obtain a diammonium citric acid solution with a concentration of 4 mol / L.

[0147] 3. Prepare the matrix solution

[0148] The mother liquor and diammonium citrate solution were diluted with acetonitrile and water to prepare 1 mL of the matrix solution. Specifically, a certain volume of the mother liquor, a certain volume of the diammonium citrate solution, a certain volume of acetonitrile and water were taken, and water was added to make the volume 1 mL, so that the concentration of trishydroxymethylaminomethane in the matrix solution was 0.005 mol / L, the concentration of diammonium citrate was 0.4 mol / L, and the volume fraction of acetonitrile was 30%, and the matrix solution was obtained.

[0149] Add 0.05 g of 3-hydroxy-2-pyridinecarboxylic acid to the above matrix solution, dissolve until clear, and centrifuge to obtain a matrix solution.

[0150] 4. Prepare the sample solution to be tested

[0151] Prepare 1 mL of the nucleic acid sample solution to be detected in the same manner as in Example 1 for later use.

[0152] 5. Order.

[0153] Pipette 1.75 μL of the prepared matrix solution and spot it on the target center of the stainless steel target plate, dry it, and wait for uniform and dense needle-shaped crystals to precipitate to form a matrix layer;

[0154] Pipette 1 μL of the nucleic acid sample solution to be detected and spot it on the matrix layer, dry it, and wait for the co-crystallization at the target position to be completed to form a sample layer;

[0155] 6. Mass spectrometry

[0156] The dried co-crystallized sample was placed in a Maldi-TOF (model: CMI-1600) for mass spectrometry analysis. The mass spectrum collected is shown in Fig.10 As shown, from Fig.10It can be seen that the signal peak intensity is generally low, and the double peak around m / z9000 is missing, and only a single peak can be detected. Fig.11 As shown, from Fig.11 It can be seen that the mass spectrum acquisition success rate of this embodiment is 11.4%.

[0157] Comparative Example 2

[0158] This comparative example provides a matrix solution and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection method, which is mainly different from Example 1 in that no tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution is added to the matrix solution, and the steps are as follows:

[0159] 1. Prepare diammonium citric acid solution

[0160] Weigh 90.472 g of diammonium citric acid, place it in a 100 mL beaker, add water to fully dissolve it, then add water to make the volume up to 100 mL to obtain a diammonium citric acid solution with a concentration of 4 mol / L.

[0161] 2. Prepare the matrix solution

[0162] The above diammonium citrate solution was diluted with acetonitrile and water to prepare 1 mL of the matrix solution. Specifically, a certain volume of the above diammonium citrate solution, a certain volume of acetonitrile and water were taken, and water was added to make the volume 1 mL, so that the concentration of diammonium citrate in the matrix solution was 0.4 mol / L, and the volume fraction of acetonitrile was 30%, and the matrix solution was obtained.

[0163] Add 0.05 g of 3-hydroxy-2-pyridinecarboxylic acid to the above matrix solution, dissolve until clear, and centrifuge to obtain a matrix solution.

[0164] 3. Prepare the sample solution to be tested

[0165] Prepare 1 mL of nucleic acid sample solution to be tested in the same manner as in Example 1.

[0166] 4. Order.

[0167] Pipette 1.75 μL of the prepared matrix solution and spot it on the target center of the stainless steel target plate, dry it, and wait for uniform and dense needle-shaped crystals to precipitate to form a matrix layer;

[0168] 1 μL of the nucleic acid sample solution to be detected is applied onto the matrix layer, dried, and the target position is subjected to co-crystallization to form a sample layer;

[0169] 5. Mass spectrometry detection

[0170] The dried co-crystallized sample was placed in a Maldi-TOF (model: CMI-1600) for mass spectrometry analysis. The mass spectrum collected is shown in Fig.12 As shown, from Fig.12 It can be seen that the signal peak intensity is generally low, and the double peak around m / z9000 is missing, and only a single peak can be detected. Fig.13 As shown, from Fig.13 It can be seen that the mass spectrum acquisition success rate of this embodiment is 12.5%.

[0171] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0172] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A detection method of matrix-assisted laser desorption ionization time-of-flight mass spectrometer, characterized in that: The following steps are involved: A matrix solution is spotted on a stainless steel target plate and dried to form a matrix layer, wherein the matrix solution consists of 3-hydroxy-2-pyridinecarboxylic acid, diammonium hydrogen citrate, acetonitrile, tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution with a pH value of 9.0 and water, wherein in 1 mL of the matrix solution, the concentration of tris(hydroxymethylaminomethane) is 0.025 mol / L, the concentration of diammonium hydrogen citrate is 0.4 mol / L, the volume fraction of acetonitrile is 30%, and the mass of 3-hydroxy-2-pyridinecarboxylic acid is 0.05 g; or 1 In mL of the matrix solution, the concentration of tris(hydroxymethyl)aminomethane is 0.04 mol / L, the concentration of diammonium citrate is 0.5 mol / L, the volume fraction of acetonitrile is 20%, and the mass of 3-hydroxy-2-pyridinecarboxylic acid is 0.04 g; or in 1 mL of the matrix solution, the concentration of tris(hydroxymethyl)aminomethane is 0.35 mol / L, the concentration of diammonium citrate is 0.75 mol / L, the volume fraction of acetonitrile is 25%, and the mass of 3-hydroxy-2-pyridinecarboxylic acid is 0.35 g; The sample solution to be tested is spotted on the matrix layer and dried to form a sample layer. The sample to be tested is composed of 8 single-stranded nucleotide sequences P1 to P8, and the molecular weights thereof are: P1-3052.1, P2-3655.5, P3-5523.7, P4-6511.3, P5-8100.3, P6-9006.9, P7-9022.9, P8-9938.5; Perform mass spectrometry analysis.

2. The detection method according to claim 1, characterized in that: The volume ratio of the matrix solution to the sample to be detected is (0.5-2.5): (0.5-2.5).

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