CN117368300A - Reagent combination of nucleic acid mass spectrum matrix and preparation method and application thereof - Google Patents
Reagent combination of nucleic acid mass spectrum matrix and preparation method and application thereof Download PDFInfo
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- 239000011159 matrix material Substances 0.000 title claims abstract description 102
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 69
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 69
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 69
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 64
- 238000001819 mass spectrum Methods 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 62
- 150000007524 organic acids Chemical class 0.000 claims abstract description 41
- 150000003863 ammonium salts Chemical class 0.000 claims abstract description 36
- 239000000654 additive Substances 0.000 claims abstract description 22
- 230000000996 additive effect Effects 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 72
- 239000000243 solution Substances 0.000 claims description 71
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 60
- NKOPQOSBROLOFP-UHFFFAOYSA-N 6-methyl-3-sulfanylidene-2h-1,2,4-triazin-5-one Chemical compound CC1=NNC(=S)NC1=O NKOPQOSBROLOFP-UHFFFAOYSA-N 0.000 claims description 45
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 40
- 239000011668 ascorbic acid Substances 0.000 claims description 30
- 229960005070 ascorbic acid Drugs 0.000 claims description 30
- 235000010323 ascorbic acid Nutrition 0.000 claims description 30
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 27
- 239000002105 nanoparticle Substances 0.000 claims description 23
- 239000011347 resin Substances 0.000 claims description 23
- 229920005989 resin Polymers 0.000 claims description 23
- BRARRAHGNDUELT-UHFFFAOYSA-N 3-hydroxypicolinic acid Chemical compound OC(=O)C1=NC=CC=C1O BRARRAHGNDUELT-UHFFFAOYSA-N 0.000 claims description 19
- 239000013543 active substance Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000002086 nanomaterial Substances 0.000 claims description 10
- -1 organic acid compound Chemical class 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 9
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 7
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 7
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 7
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 7
- 239000012266 salt solution Substances 0.000 claims description 7
- 238000009210 therapy by ultrasound Methods 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- VBIXEXWLHSRNKB-UHFFFAOYSA-N ammonium oxalate Chemical compound [NH4+].[NH4+].[O-]C(=O)C([O-])=O VBIXEXWLHSRNKB-UHFFFAOYSA-N 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 229910021485 fumed silica Inorganic materials 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 15
- 230000036267 drug metabolism Effects 0.000 abstract description 9
- 230000009471 action Effects 0.000 abstract description 8
- 230000000694 effects Effects 0.000 description 21
- 239000002904 solvent Substances 0.000 description 20
- 150000002500 ions Chemical class 0.000 description 19
- 239000000203 mixture Substances 0.000 description 15
- 239000012491 analyte Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
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- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 7
- 238000000746 purification Methods 0.000 description 6
- 229940093915 gynecological organic acid Drugs 0.000 description 5
- 235000005985 organic acids Nutrition 0.000 description 5
- 238000010257 thawing Methods 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000007711 solidification Methods 0.000 description 4
- 230000008023 solidification Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229940123457 Free radical scavenger Drugs 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000002516 radical scavenger Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 239000012445 acidic reagent Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000002288 cocrystallisation Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
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- 239000003093 cationic surfactant Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- BITYAPCSNKJESK-UHFFFAOYSA-N potassiosodium Chemical compound [Na].[K] BITYAPCSNKJESK-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- General Physics & Mathematics (AREA)
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- Pathology (AREA)
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the technical field of mass spectrometry, in particular to a reagent combination of a nucleic acid mass spectrometry matrix, a preparation method and application thereof. The invention provides a reagent combination comprising an organic acid solution, ammonium salt and an additive, a nucleic acid mass spectrometry matrix containing the reagent combination and a preparation method thereof, and application of the reagent combination and the nucleic acid mass spectrometry matrix in human drug metabolism and action target polygene joint detection. The nucleic acid mass spectrum matrix solution can effectively reduce the generation of an addition peak at the position of +88Da after a main peak in a mass spectrum analysis result, improves the accuracy of judging the mass spectrum result, has low and stable base line, obviously improves the uniformity of matrix spots, increases the adhesiveness between the matrix spots and a substrate, and is suitable for popularization and application.
Description
Technical Field
The invention relates to the technical field of mass spectrometry, in particular to a reagent combination of a nucleic acid mass spectrometry matrix, a preparation method and application thereof.
Background
In recent years, molecular diagnosis technology is continuously developed, and mainly high-throughput sequencing, PCR reaction, mass spectrometry and the like are available. Compared with other methods, the mass spectrometry has the characteristics of high detection speed, capability of multi-gene multi-site detection, low detection cost and the like. Mass spectrometry uses matrix-assisted laser desorption ionization (MALDI) to volatilize and ionize analytes, and according to the difference in mass of ions themselves, the acceleration speed is different, and particles with small mass arrive at a detector earlier to obtain a corresponding mass spectrum for analysis. The method is widely applied to macromolecular analytes such as proteins, and is gradually applied to the field of nucleic acids in recent years. However, since the molecular weight of nucleic acids is greatly different from that of macromolecules such as proteins, mass spectrometry chips used for mass spectrometry are often not commonly used, and thus a nucleic acid mass spectrometry chip matrix solution prepared from an organic acid capable of forming a homogeneous co-crystal with nucleic acids should be selected. The matrix solution, after solidification, may form co-crystals with the nucleic acid analyte, which may transfer appropriate energy to aid in ionization of the analyte and to protect the analyte from cleavage.
There are a variety of matrix formulations suitable for nucleic acid mass spectrometry, most of which consist of organic acids and ammonium salts. The matrix formulated by such conventional matrix formulations is prone to analyte fragments during analysis of the nucleic acid sample, adduct formation, mass spectrum addition peaks, and significant shifts can be caused by the impact of interpretation of experimental results, particularly low quality analytes. Reducing the generation of mass spectrum addition peaks by altering the formulation of the nucleic acid matrix is the main direction of research in recent years.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a reagent combination of a nucleic acid mass spectrum matrix, a preparation method and application thereof.
The invention provides a reagent combination comprising an organic acid solution, an ammonium salt and an additive, wherein:
the organic acid solution comprises acetonitrile and an organic acid compound comprising 3-hydroxy-2-picolinic acid and/or 6-aza-2-thiothymine;
the ammonium salt comprises diammonium citrate and/or ammonium oxalate;
the additive comprises an active agent, a nanomaterial and ascorbic acid, wherein the active agent comprises cetyl trimethyl ammonium bromide and/or carboxymethyl cellulose, and the nanomaterial comprises mesoporous nano particles and/or fumed silica particles.
Compared with the prior art, the invention adopts two organic acids, namely 3-hydroxy-2-picolinic acid (3-HPA) and 6-aza-2-thiothymine (ATT), which are more suitable for substances with molecular weight such as nucleic acid, can better form co-crystallization with the nucleic acid and provide ionization energy. The ammonium salt is used as a secondary matrix component in a matrix formula, and the ionization of an analyte can be more efficiently completed by matching with an organic acid, a mass spectrogram obtained by a matrix solution only containing the organic acid only has a peak diagram, but the shape of the peak has no clear limit, and after a proper amount of ammonium salt is added, the mass spectrogram shows clear peaks. The additive can be divided into an active agent for reducing adducts, a nano material for increasing the uniformity of matrix spots and a free radical scavenger, and a cationic surfactant Cetyl Trimethyl Ammonium Bromide (CTAB) is easy to resolve, is suitable for acidic analytes, can inhibit related ions in matrixes prepared from organic acid and ammonium salt, can reduce matrix fragment generation in the matrix desorption process, has certain viscosity, is favorable for forming stable round spots in the drying process of the matrixes, and can not introduce an addition peak. In some embodiments, the additive further comprises a nanomaterial. Specific nanomaterials can be classified as mesoporous nanoparticles or fumed silica particles. The nanomaterial can improve the homogeneity of the matrix speckle crystallization. The free radical scavenger ascorbic acid has reducibility, can capture free ions to stabilize, and reduces the generation of addition peaks which can influence interpretation. And because the ascorbic acid can resist oxidation, the ascorbic acid has the effect of prolonging the effective period of the chip. Meanwhile, due to the mutual coordination of the components in the reagent combination, the accuracy of a mass spectrometry result is further improved, and the generation of an addition peak is reduced, so that a more accurate technical effect is obtained.
In some embodiments, the organic acid solution comprises the following components in concentration: 50vol% acetonitrile, 200-800 mM 3-hydroxy-2-picolinic acid and 100-700 mM 6-aza-2-thiothymine, wherein the concentration ratio of the 3-hydroxy-2-picolinic acid to the 6-aza-2-thiothymine is (1-6): 1, a step of;
the concentration of the components in the ammonium salt is as follows: 50-200 mM diammonium citrate;
the concentration of each component in the additive is as follows: 10-150 mM hexadecyl trimethyl ammonium bromide, 0.1-10 mg/mL mesoporous nano particles and 100-300 mM ascorbic acid.
In the above components and concentration ranges, the components among the reagent combinations are more tightly matched, the compatibility effect is better, the effective period of the chip is longer, the mass spectrometry result is more accurate, fewer addition peaks are generated, and therefore more accurate technical effects are obtained.
In some embodiments, the organic acid solution comprises the following components in concentration: 50vol% acetonitrile in water, 450mM 3-hydroxy-2-picolinic acid and 150mM 6-aza-2-thiothymine;
the concentration of the components in the ammonium salt is as follows: 200mM diammonium citrate;
the concentration of each component in the additive is as follows: 100mM cetyltrimethylammonium bromide, 0.1mg/mL mesoporous nanoparticles and 150mM ascorbic acid.
Experiments show that in the concentration range, the components of the reagent combination are most tightly matched, the compatibility is highest, the mass spectrometry analysis result is most accurate, and the least addition peak is generated, so that more accurate technical effects are obtained.
In other embodiments, the working concentration of each component in the organic acid solution is: 50vol% acetonitrile in water, 200 to 600mM 3-hydroxy-2-picolinic acid and 10 to 20mM 6-aza-2-thiothymine;
the working concentration of the diammonium citrate is 10-25 mM;
the working concentration of each component in the additive is as follows: 0.2-1.8 mM hexadecyl trimethyl ammonium bromide, 0.002-0.02 mg/mL mesoporous nano particles and 10-25 mM ascorbic acid.
In the ranges of the components and the working concentration, the mass spectrometry analysis result participated by the reagent combination is more accurate, so that more accurate technical effects are obtained.
In other embodiments, the working concentration of each component in the organic acid solution is: 50vol% acetonitrile in water, 293mM 3-hydroxy-2-picolinic acid and 19.5mM 6-aza-2-thiothymine;
the working concentration of the diammonium citrate is 18mM;
the working concentration of each component in the additive is as follows: 1.6mM cetyltrimethylammonium bromide, 0.01mg/mL mesoporous nanoparticles and 14mM ascorbic acid.
Experiments show that under the above components and working concentrations, the mass spectrometry analysis result participated by the reagent combination is the most accurate, so that more accurate technical effects are obtained.
The invention provides application of the reagent combination in preparing a matrix of nucleic acid mass spectrum.
The invention provides a matrix for nucleic acid mass spectrometry, comprising the combination of reagents.
The invention provides a preparation method of a matrix of the nucleic acid mass spectrum, which comprises the following steps:
mixing the aqueous solution of 3-hydroxy-2-picolinic acid, 6-aza-2-thiothymine and acetonitrile, performing ultrasonic treatment and purifying by resin to obtain the organic acid solution;
mixing the diammonium citrate with water, performing ultrasonic treatment and purifying by resin to obtain an ammonium salt solution;
mixing the cetyl trimethyl ammonium bromide with water, performing ultrasonic treatment and heating to obtain a cetyl trimethyl ammonium bromide solution;
and respectively taking the organic acid solution, the ammonium salt solution, the cetyltrimethylammonium bromide solution, the mesoporous nano particles and the ascorbic acid, mixing and drying to obtain the matrix of the nucleic acid mass spectrum.
In some embodiments, the organic acid solution is purified using a hydrogen-type resin and the ammonium salt solution is purified using an ammonium-type resin.
The invention provides a nucleic acid mass spectrometry method, which is characterized in that mass spectrometry is carried out after sampling a matrix of the nucleic acid mass spectrometry or a matrix of the nucleic acid mass spectrometry prepared by the preparation method.
The invention provides a reagent combination comprising an organic acid solution, ammonium salt and an additive, a nucleic acid mass spectrometry matrix containing the reagent combination and a preparation method thereof, and application of the reagent combination and the nucleic acid mass spectrometry matrix in human drug metabolism and action target polygene joint detection. The nucleic acid mass spectrum matrix solution can effectively reduce the generation of an addition peak at the position of +88Da after a main peak in a mass spectrum analysis result, improves the accuracy of judging the mass spectrum result, has low and stable base line, obviously improves the uniformity of matrix spots, increases the adhesiveness between the matrix spots and a substrate, and is suitable for popularization and application. Compared with the prior art, the organic acid adopted in the nucleic acid mass spectrum matrix is more suitable for substances with molecular weight such as nucleic acid, and can better form co-crystallization with the nucleic acid and provide ionization energy; the diammonium citrate is adopted, so that ionization of an analyte can be more efficiently finished by matching with organic acid, and a mass spectrum shows a clear peak; cetyl trimethyl ammonium bromide is adopted as an active agent, is suitable for an acidic analyte, can inhibit related ions in a matrix prepared from organic acid and ammonium salt, and can reduce matrix fragment generation in the matrix desorption process; mesoporous nano particles are adopted, so that the homogeneity of matrix spot crystallization is further improved, and the homogeneity of matrix solution is improved; the ascorbic acid is adopted, so that free ions can be captured to stabilize the ascorbic acid, the generation of an addition peak which can influence interpretation is reduced, and the ascorbic acid can resist oxidation, so that the ascorbic acid has the effect of prolonging the effective period of a chip.
Drawings
FIG. 1 shows the peak pattern results when two matrix components, 3-hydroxy-2-picolinic acid (3-HPA) and 6-aza-2-thiothymine (ATT) reagents and mixtures thereof, were used for nucleic acid mass spectrometry in example 1, respectively, wherein the X-axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 2 shows the results of the mass spectrum peak plot of the ratio of 3-HPA to ATT concentration in example 1, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 3 shows the results of mass spectra peak diagrams of the concentration studies of 3-HPA and ATT in example 1, in which the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 4 shows the results of mass spectra peak diagrams of different solvent and concentration studies of 3-HPA in example 1, wherein the X-axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 5 shows the peak pattern results of example 2 in which diammonium citrate and ammonium oxalate were used for nucleic acid mass spectrometry, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 6 shows the results of mass spectrometry analysis of nucleic acid by diammonium citrate concentration in example 2, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 7 shows the peak pattern results of 3-HPA, ATT and ammonium salt (diammonium citrate) resins of example 2 before and after purification for nucleic acid mass spectrometry, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 8 shows a mass spectrum generated in example 3 using 9pv in a nucleic acid mass spectrometry reference to verify that no active agent, additive CTAB or carboxymethyl cellulose was added to the matrix formulation, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 9 shows the effect of different solvents of the active agent CTAB in example 3 on nucleic acid mass spectrometry analysis, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 10 shows the effect of different preparation methods of the active agent CTAB in example 3 on the mass spectrometry analysis of nucleic acids, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 11 shows the effect of different concentrations of the active agent CTAB in example 3 on nucleic acid mass spectrometry analysis, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 12 shows the effect of adding nanoparticles to the matrix solution in example 3 on the results of mass spectrometry analysis of nucleic acids, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 13 shows the effect of ascorbic acid addition to the matrix solution of example 3 on the mass spectrometry analysis of nucleic acids, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 14 shows the results of validation of reagent samples using a combined human drug metabolism and action target polygene detection reagent (time-of-flight mass spectrometry) after solidification of the matrix solution determined in the effect example into matrix spots, wherein the X axis: relative molecular weight, Y axis: ion peak intensity;
FIG. 15 shows a graph of mean lines of parameters of experimental groups, showing the validation of reagent samples using a multi-gene joint detection reagent (time-of-flight mass spectrometry) for human drug metabolism and action targets after solidification of the matrix solution determined in the effect example into matrix spots.
Detailed Description
The invention provides a reagent combination of a nucleic acid mass spectrum matrix, a preparation method and application thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The invention discloses a novel matrix formulation suitable for nucleic acid mass spectrometry based on a basic matrix solution formulation of an organic acid and an ammonium salt. The present invention provides a method of preparing a matrix formulation for mass spectrometry. The matrix prepared by the formula has more uniform matrix spots after solidification, the spots are firmly combined with the substrate, the occurrence of addition peaks in an analysis substance spectrogram can be reduced to the greatest extent, the resolution of target peaks is high, and the detection limit is low. The invention mainly researches additives for reducing the addition peak, and matches with a basic matrix detected by nucleic acid mass spectrum to obtain a new matrix formula. Firstly, determining the types of basic matrix organic acid and ammonium salt, and then obtaining a basic nucleic acid mass spectrum matrix formula through different concentration ratios of the basic matrix organic acid and the ammonium salt. When the basic formula is used for analyte analysis, the matrix is found to be non-uniform, the analytical mass spectrum is not highly reproducible and contains adduct peaks due to adducts. Subsequently, several active agents were selected to reduce adduct formation by tuning to different concentrations. Several nano materials are selected for verification in the aspect of improving the uniformity of the matrix. In the actual sample detection, a new addition peak is found to appear, and a free radical scavenger is selected for experiment. And finally, preparing a chip by using the new matrix formula, and verifying an actual sample. All mass spectra were obtained by the same spot application, dispensing onto a substrate under defined dry conditions, introducing the same amount of analyte for mass spectrometry.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples.
Example 1 screening of nucleic acid Mass Spectrometry for organic acids in matrix
1. Screening of organic acids
In the following examples, peak patterns when two matrix components, 3-hydroxy-2-picolinic acid (3-HPA) and 6-aza-2-thiothymine (ATT) reagents and mixtures of the two, were used for nucleic acid mass spectrometry, respectively, are shown. The required solutions are: as shown in table 1.
TABLE 1
| Reagent name | Reagent concentration | Solvent(s) |
| 3-HPA | 450mM | 50% acetonitrile |
| ATT | 150mM | 50% acetonitrile |
| Citric acid diammonium salt | 200mM | ddH 2 O |
The component solutions of the matrix solution were purified using the corresponding resins at a concentration of 1.5mg/mL.
After the prepared matrix solution is uniformly mixed, the semi-automatic sample application instrument developed by the company is used for sample application. Setting the distribution base mass of the semi-automatic sample application instrument to be 0.5 mu L/point, placing the sample application instrument into an ultra-clean workbench, and solidifying at room temperature.
Preparing a sample, thawing the sample, uniformly mixing, and distributing the sample by using a semi-automatic sample application instrument. Setting the sample distribution of the semi-automatic sample application instrument to be 0.08 mu L/point, placing the sample into an ultra-clean workbench, drying the sample at room temperature, and pushing the sample into a mass spectrometer for analysis. In this experimental condition, the working concentrations of the components were: 50vol% acetonitrile in water, 293mM 3-hydroxy-2-picolinic acid and 19.5mM 6-aza-2-thiothymine;
the working concentration of the diammonium citrate was 18mM, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the matrix formulation in which 3-HPA is mixed with ATT works best.
2. Investigation of the concentration ratio of 3-HPA to ATT
The organic acid reagent groups were used for nucleic acid mass spectrometry according to table 2, in which molar concentration ratios were formulated by controlling the addition volumes of the organic acid reagents, and mass spectra peak diagrams are shown in fig. 2.
TABLE 2
As can be seen from FIG. 2, 3-HPA: the optimum ratio of ATT is 3:1.
3. 3-HPA and ATT concentration exploration
The organic acid reagent groups were used for nucleic acid mass spectrometry according to table 3, and the mass spectrum peak diagram is shown in fig. 3.
TABLE 3 Table 3
As can be seen from fig. 3, 3-HPA was determined: 450mM; ATT: a150 mM combination works best, under which conditions the working concentration of 3-HPA is 293mM and the working concentration of ATT is 19.5mM.
4. Investigation of different solvents and concentrations of 3-HPA
The different solvents and concentration groupings of 3-HPA were used for nucleic acid mass spectrometry according to Table 4, wherein 3-HPA concentration 450mM, ATT concentration 150mM, diammonium citrate concentration 200mM, CTAB concentration 100mM, and the mass spectrum peak diagram as shown in FIG. 4.
TABLE 4 Table 4
| Grouping | Solvent(s) |
| 1 | Methanol |
| 2 | 50% acetonitrile |
| 3 | 30% acetonitrile |
As can be seen from FIG. 4, the optimal solvents for 3-HPA are water and acetonitrile, and the acetonitrile concentration in the solvents is 50%.
Example 2 in-matrix ammonium salt screening of nucleic acid Mass Spectrometry
1. Comparative experiment of diammonium citrate and ammonium oxalate in ammonium salt
According to the two matrix components 3-hydroxy-2-picolinic acid (3-HPA) and 6-aza-2-thiothymine (ATT) selected in example 1 as in Table 5, two ammonium salts as in Table 5 were added respectively, showing peak patterns when they were used for nucleic acid mass spectrometry, and mass spectra peak patterns are shown in FIG. 5.
TABLE 5
| Reagent name | Reagent concentration | Solvent(s) |
| 3-HPA | 450mM | 50% acetonitrile |
| ATT | 150mM | 50% acetonitrile |
| Citric acid diammonium salt | 200mM | ddH 2 O |
| Ammonium oxalate | 200mM | ddH 2 O |
As can be seen from fig. 5, diammonium citrate is more suitable as the ammonium salt component in the matrix formulation.
2. Effect of diammonium citrate concentration in ammonium salts on nucleic acid Mass Spectrometry
The 3-HPA concentration 450mM and the ATT concentration 150mM were used for the nucleic acid mass spectrometry according to the different concentration groupings of diammonium citrate in Table 6, and the results are shown in FIG. 6.
TABLE 6
| Grouping | Concentration of diammonium citrate | Solvent(s) |
| 1 | 200mM | ddH 2 O |
| 2 | 100mM | ddH 2 O |
| 3 | 50mM | ddH 2 O |
| 4 | 0mM | ddH 2 O |
FIG. 6 shows a mass spectrum of the results of the use of 9pv in a nucleic acid mass spectrometry reference to verify the presence of organic acids (3-HPA and ATT) and ammonium salts (diammonium citrate). In FIG. 6, the base composition of the matrix solution is determined and the presence of an additional peak at +88Da after the main peak is identified (peak height about 40% of the height of the parent peak at 8265 Da). Meanwhile, as is clear from the results of FIG. 6, the optimal concentration of diammonium citrate is 200mM, and the optimal working concentration is 18mM.
3. Investigation of purification conditions of 3-HPA, ATT and ammonium salt (diammonium citrate)
The effect on the mass spectrum results was investigated for analyte analysis of 3-HPA, ATT and diammonium citrate before and after resin purification. In the preparation process of 3-HPA, ATT and ammonium salt, solvent is added into a reagent tube containing reagent powder, and the mixture is uniformly mixed and subjected to ultrasonic treatment, so that the reagent is more fully dissolved. Wherein, the resin purification group is to add the obtained reagent solution, such as organic acid solution, into hydrogen type resin (1.5 mg/mL), and add ammonium salt solution into ammonium type resin (1.5 mg/mL), and fully mix and purify. Standing for 5min, and matching with a disposable filter membrane (0.22 μm) by using a disposable syringe to obtain a purified matrix component solution; the reagent solution was not subjected to the resin purification process in the resin unpurified group. The mass spectra generated before and after purification of the organic acid and ammonium salt using the resin (sample nucleic acid purification reagent) were verified using 9pv in the nucleic acid mass spectrometry reference, and the results are shown in fig. 7.
As shown in fig. 7, it was demonstrated that when the matrix component reagent was purified using a resin, the obtained matrix spots were used for analyte analysis, and the mass spectrum obtained had few addition peaks, and the baseline was low and stable.
Example 3 matrix additive screening for nucleic acid Mass Spectrometry
1. Adding active agent into matrix solution
In the following experiments, the results of peak patterns in the case of using the experimental group with or without the active agent, with CTAB added or carboxymethyl cellulose added for nucleic acid mass spectrometry were compared. The required solutions are shown in Table 7, and the component solutions of the matrix solution, except CTAB and carboxymethyl cellulose, are purified by using corresponding resins with a resin concentration of 1.5mg/mL.
After the prepared matrix solution is uniformly mixed, the semi-automatic sample application instrument developed by the company is used for sample application. Setting the distribution base mass of the semi-automatic sample application instrument to be 0.5 mu L/point, placing the sample application instrument into an ultra-clean workbench, and solidifying at room temperature.
Preparing a sample, thawing the sample, uniformly mixing, and distributing the sample by using a semi-automatic sample application instrument. Setting the semi-automatic sample application instrument to distribute the sample to 0.08 mu L/point, placing the sample into an ultra-clean workbench, drying the sample at room temperature, and pushing the sample into a mass spectrometer for analysis. The results are shown in FIG. 8.
TABLE 7
| Reagent name | Reagent concentration | Solvent(s) |
| 3-HPA | 450mM | 50% acetonitrile |
| ATT | 150mM | 50% acetonitrile |
| Citric acid diammonium salt | 200mM | ddH 2 O |
| CTAB or carboxymethyl cellulose | 100mM | ddH 2 O |
As can be seen from fig. 8, the addition peak of the mass spectrum obtained by adding the additive CTAB to the existing purified base matrix solution is significantly reduced, the baseline is stable, and the baseline impurities of the carboxymethyl cellulose group are more.
2. Comparative experiments on different solvents, preparation methods and concentrations of CTAB as an active agent
In the following experiments, the results of peak patterns of different solvents, preparation methods, and different concentrations of CTAB for nucleic acid mass spectrometry were compared. The required solutions are shown in table 8. The results of the mass spectrum peak diagrams of different solvents are shown in fig. 9, the results of the mass spectrum peak diagrams of different preparation methods are shown in fig. 10, and the results of the mass spectrum peak diagrams of different concentrations are shown in fig. 11.
TABLE 8
| Reagent name | Reagent concentration | Solvent(s) |
| 3-HPA | 450mM | 50% acetonitrile |
| ATT | 150mM | 50% acetonitrile |
| Citric acid diammonium salt | 200mM | ddH 2 O |
| CTAB | 50/100/150mM | ddH 2 O/pyridine |
As can be seen from FIGS. 9 to 11, the CTAB solution was prepared by adding CTAB powder to a predetermined amount of pure water as the best solvent, and the CTAB powder was prepared at an optimum CTAB concentration of 100mM and an optimum working concentration of 1.6mM under ultrasonic heating.
3. Nanoparticle addition to the matrix solution
The effect of adding mesoporous nano particles into a matrix solution is verified by using a polygene combined detection reagent (time-of-flight mass spectrometry) of human drug metabolism and action targets.
In the following experiments, the same sample was selected and subjected to experimental verification at the same time. Matrix homogeneity analysis was performed using mass spectrogram analysis software Typer. The color and mass spectrum of each hole site represents the score of the base particle and the average value of each parameter. It is known that the dark green hole site score is higher than the light green score, which is assessed based on the average of 9 different positions of the base particle, and can represent the homogeneity of the base particle (FIG. 12).
The pretreatment reagents for PCR reaction and the main components of the pretreatment reagents are shown in Table 9, and the solutions required for nucleic acid mass spectrometry are shown in Table 10.
TABLE 9
Table 10
| Reagent name | Reagent concentration | Solvent(s) |
| 3-HPA | 450mM | 50% acetonitrile |
| ATT | 150mM | 50% acetonitrile |
| Citric acid diammonium salt | 100mM | ddH 2 O |
| CTAB | 100mM | ddH 2 O |
| Mesoporous nanoparticles or fumed silica particles | 0.1mg/mL | ddH 2 O |
The component solutions of the matrix solution except CTAB are purified by using corresponding resin with the concentration of 1.5mg/mL.
After the prepared matrix solution is uniformly mixed, the semi-automatic sample application instrument developed by the company is used for sample application. Setting the distribution base mass of the semi-automatic sample application instrument to be 0.5 mu L/point, placing the sample application instrument into an ultra-clean workbench, and solidifying at room temperature.
Preparing a sample, thawing the sample, uniformly mixing, carrying out sample rotation, and setting a mass spectrometer for automatic sample feeding and automatic mass spectrometry.
FIG. 12 shows a mass spectrum generated after addition of mesoporous nanoparticles or fumed silica particles using a human drug metabolism and action target polygene joint detection reagent (time-of-flight mass spectrometry). As can be seen from fig. 12, the nanomaterial improvement matrix uniformity was identified and mesoporous nanoparticles improved uniformity exhibited better, wherein the optimal working concentration of mesoporous nanoparticles was 0.01mg/mL.
4. Adding ascorbic acid to a matrix solution
The effect of ascorbic acid added to the matrix solution was verified using a human drug metabolism and effect target polygene combined detection reagent (time-of-flight mass spectrometry). The pretreatment reagents for PCR reaction and the main components of the pretreatment reagents are shown in Table 9, and the solutions required for nucleic acid mass spectrometry are shown in Table 11.
TABLE 11
| Reagent name | Reagent concentration | Solvent(s) |
| 3-HPA | 450mM | 50% acetonitrile |
| ATT | 150mM | 50% acetonitrile |
| Citric acid diammonium salt | 200mM | ddH 2 O |
| Ascorbic acid | 150mM | ddH 2 O |
| CTAB | 100mM | ddH 2 O |
| Mesoporous nanoparticles | 0.1mg/mL | ddH 2 O |
(1) Preparing related solutions according to the table, and mixing proportionally
(2) The component solutions of the matrix solution were purified using the corresponding resins at a concentration of 1.5mg/mL (organic acid and ammonium salt fractions were purified, the remaining additives were not required).
(3) After the prepared matrix solution is uniformly mixed, the semi-automatic sample application instrument developed by the company is used for sample application. Setting the distribution base mass of the semi-automatic sample application instrument to be 0.5 mu L/point, placing the sample application instrument into an ultra-clean workbench, and solidifying at room temperature.
(4) Preparing a sample, thawing the sample, uniformly mixing, and distributing the sample by using a semi-automatic sample application instrument. The semi-automatic spotter was set to dispense the sample at 0.08 μl/spot, placed on an ultra clean bench, dried at room temperature, and pushed into the mass spectrometer for analysis, where the working concentration of ascorbic acid was 14mM.
In the following experiments, the ratio of the sum peaks of five sites to the major peak height was selected and repeated X times to obtain specific data parameters for the addition of no ascorbic acid adduct and specific data parameters for the addition of ascorbic acid-reduced adduct as shown in table 12.
Table 12
As can be seen from table 12, the addition of ascorbic acid (VC) to the matrix solution reduced the peak height ratio of the sodium potassium adduct peaks, further demonstrating that ascorbic acid reduced the generation of adduct peaks that affected the interpretation of the results. As can be seen from FIG. 13, the +22Da adduct peaks at the 5 sites of the experimental group (Na + ) Peak of adduct with +38Da (K) + ) The peak height average ratio of (c) is significantly reduced. Ascorbic acid can be used as an additive to reduce the peak of the addition.
Effect example nucleic acid mass spectrometry matrix solution effect verification
In the following examples, the verification of clinical samples using chips prepared using the matrix solution prepared by the present method is shown, wherein the pretreatment reagents for PCR reaction and the main components of the pretreatment reagents are shown in Table 9, and the matrix solution components for nucleic acid mass spectrometry are shown in Table 13.
TABLE 13
| Reagent name | Reagent concentration | Solvent(s) |
| 3-HPA | 450mM | 50% acetonitrile |
| ATT | 150mM | 50% acetonitrile |
| Citric acid diammonium salt | 200mM | ddH 2 O |
| Ascorbic acid | 150mM | ddH 2 O |
| CTAB | 100mM | ddH 2 O |
| Mesoporous nanoparticles | 0.1mg/mL | ddH 2 O |
The component solutions of the matrix solution except CTAB are purified by using corresponding resin with the concentration of 1.5mg/mL.
After the prepared matrix solution is uniformly mixed, the semi-automatic sample application instrument developed by the company is used for sample application. Setting the distribution base mass of the semi-automatic sample application instrument to be 0.5 mu L/point, placing the sample application instrument into an ultra-clean workbench, and solidifying at room temperature.
The chip prepared by the invention is used for verifying human drug metabolism and action target polygene joint detection reagent (time-of-flight mass spectrometry) samples: preparing a sample, thawing the sample, uniformly mixing the sample, carrying out sample rotation, setting a mass spectrometer for automatic sample feeding, carrying out automatic mass spectrometry, repeating the two experiments for 9 times to obtain specific clinical sample data parameters (the parameters comprise peak area, peak height, resolution and signal to noise ratio) as shown in a table 14, after the determined matrix solution is solidified into matrix spots, randomly selecting two mass spectrograms for verification of reagent samples by using a multi-gene combined detection reagent (time-of-flight mass spectrometry) for human drug metabolism and action targets, and displaying (Test 1 and Test 2) as shown in fig. 14, wherein the mass of the rest mass spectrograms is the same as that of fig. 14, and the average value line graph of each parameter of the experiment group is shown in fig. 15.
TABLE 14
According to the analysis of table 14 in combination with fig. 14 and 15, it can be seen that the nucleic acid mass spectrum chip prepared by the matrix formulation can be used in clinical sample detection. From fig. 14, the mass spectrum has a good peak shape and a stable base line. From the peak areas, peak heights, signal to noise, and the average of the resolution multiple tests in fig. 15 and table 14, the mass spectrum results were good and stable.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A reagent combination comprising an organic acid solution, an ammonium salt, and an additive, wherein:
the organic acid solution comprises acetonitrile and an organic acid compound comprising 3-hydroxy-2-picolinic acid and/or 6-aza-2-thiothymine;
the ammonium salt comprises diammonium citrate and/or ammonium oxalate;
the additive comprises an active agent, a nanomaterial and ascorbic acid, wherein the active agent comprises cetyl trimethyl ammonium bromide and/or carboxymethyl cellulose, and the nanomaterial comprises mesoporous nano particles and/or fumed silica particles.
2. The combination of claim 1, wherein,
the concentration of each component in the organic acid solution is as follows: 50vol% acetonitrile, 200-800 mM 3-hydroxy-2-picolinic acid and 100-700 mM 6-aza-2-thiothymine, wherein the concentration ratio of the 3-hydroxy-2-picolinic acid to the 6-aza-2-thiothymine is (1-6): 1, a step of;
the concentration of the components in the ammonium salt is as follows: 50-200 mM diammonium citrate;
the concentration of each component in the additive is as follows: 10-150 mM hexadecyl trimethyl ammonium bromide, 0.1-10 mg/mL mesoporous nano particles and 100-300 mM ascorbic acid.
3. The combination of claim 2, wherein,
the concentration of each component in the organic acid solution is as follows: 50vol% acetonitrile in water, 450mM 3-hydroxy-2-picolinic acid and 150mM 6-aza-2-thiothymine;
the concentration of the components in the ammonium salt is as follows: 200mM diammonium citrate;
the concentration of each component in the additive is as follows: 100mM cetyltrimethylammonium bromide, 0.1mg/mL mesoporous nanoparticles and 150mM ascorbic acid.
4. The reagent combination according to claim 1 to 3,
the working concentration of each component in the organic acid solution is as follows: 50vol% acetonitrile in water, 200 to 600mM 3-hydroxy-2-picolinic acid and 10 to 20mM 6-aza-2-thiothymine;
the working concentration of the diammonium citrate is 10-25 mM;
the working concentration of each component in the additive is as follows: 0.2-1.8 mM hexadecyl trimethyl ammonium bromide, 0.002-0.02 mg/mL mesoporous nano particles and 10-25 mM ascorbic acid.
5. The combination of claim 4, wherein,
the working concentration of each component in the organic acid solution is as follows: 50vol% acetonitrile in water, 293mM 3-hydroxy-2-picolinic acid and 19.5mM 6-aza-2-thiothymine;
the working concentration of the diammonium citrate is 18mM;
the working concentration of each component in the additive is as follows: 1.6mM cetyltrimethylammonium bromide, 0.01mg/mL mesoporous nanoparticles and 14mM ascorbic acid.
6. Use of a combination of reagents according to any one of claims 1 to 5 for the preparation of a matrix for nucleic acid mass spectrometry.
7. A matrix for nucleic acid mass spectrometry comprising the reagent combination according to any one of claims 1 to 5.
8. The method for preparing a matrix for mass spectrometry of nucleic acids according to claim 7, comprising:
mixing the aqueous solution of 3-hydroxy-2-picolinic acid, 6-aza-2-thiothymine and acetonitrile, performing ultrasonic treatment and purifying by resin to obtain the organic acid solution;
mixing the diammonium citrate with water, performing ultrasonic treatment and purifying by resin to obtain an ammonium salt solution;
mixing the cetyl trimethyl ammonium bromide with water, performing ultrasonic treatment and heating to obtain a cetyl trimethyl ammonium bromide solution;
and respectively taking the organic acid solution, the ammonium salt solution, the cetyltrimethylammonium bromide solution, the mesoporous nano particles and the ascorbic acid, mixing and drying to obtain the matrix of the nucleic acid mass spectrum.
9. The method according to claim 8, wherein the organic acid solution is purified using a hydrogen-type resin and the ammonium salt solution is purified using an ammonium-type resin.
10. A method for analyzing a nucleic acid mass spectrum, comprising sampling a substrate according to claim 7 or a substrate according to claim 8 or 9, and performing mass spectrometry.
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