CN103245717A - High-pressure formed substrate film assisted laser desorption dissociation mass spectrum quantitative analysis method - Google Patents
High-pressure formed substrate film assisted laser desorption dissociation mass spectrum quantitative analysis method Download PDFInfo
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Abstract
A high-pressure formed substrate film assisted laser desorption dissociation mass spectrum quantitative analysis method is disclosed. According to the method, an organic micro-molecular substrate is compacted under high pressure to obtain a film; the film is then adhered to a sample target of a mass spectrometer; a sample is pointed on the film to be naturally dried; the sample undergoes in situ cleaning to remove inorganic salt; and after natural drying, the sample is putted into the mass spectrometer for analysis. By the adoption of the method, size and shape inhomogeneity of crystals obtained by a crystallization process is avoided. The obtained substrate film can have an effect of a hydrophobic reverse-phase stationary phase and adsorbs sample molecules. Inorganic salt is removed by in situ cleaning, and the sample molecules are prevented from spreading on the surface of the sample target so as to raise sensitivity. The method provided by the invention has advantages of stable mass spectrum signal, good reappearance, little metal ion adduct interference, small spectra background, good linear relation between signal intensity and sample amount and feasible quantitative analysis. The method is simple, requires no complex apparatus, complex sample pretreatment or organic solvent, and is green and environmentally friendly.
Description
Technical field
The present invention relates to a kind of high-pressure molding matrix film assist-quantitative analysis method of laser parsing disassociation mass spectrum, relate in particular to even, big or small controllable thickness matrix film preparation method, and the sample quantitative analysis method.Principle of the present invention presses down at constant high pressure based on the organic molecule that can absorb laser energy and shortens film (Compressed Matrix Thin Film into, abbreviate CMTF as), prepared matrix film can be realized the function of commonsense method gained matrix absorption laser energy, again can be as hydrophobic film adsorption sample molecule, and the washing of inorganic salts original position is removed, and eliminate the diffusion of sample on target, the sensitivity of raising method.This method can also be handled solid sample, has improved range of application.
Background technology
It is a kind of analytical technology of widespread use that ground substance assistant laser is resolved the disassociation mass spectrum, this technology utilization can absorb organic molecule matrix and the sample mix of laser energy, and energy passed to sample molecule, and make it vaporization and ionization, detected by mass analyzer again.In this technology, organic molecule matrix and sample molecule mixed mode are crucial, because the reappearance of its direct impact analysis result's accuracy, resolution and experimental result and the ability of quantitative test.
In the existing technology, usually adopt organic solvent elder generation dissolved matrix, allow matrix solution mix with sample solution again, treat solvent evaporates after, sample and substrate molecule formation mixed crystal.The major defect of prior art is to be difficult to form size evenly, the crystal that pattern is controlled, thus the collection of illustrative plates that makes laser obtain in the different scanning time does not have reappearance, does not have quantitative relationship between signal intensity and the sample size.And, because the difference of crystal size and pattern, cause after the laser bombardment sample molecule initial velocity of gained ion different with direction, influence resolution and the mass accuracy of experimental result.
Summary of the invention
The object of the invention aims to provide a kind of high-pressure molding matrix film (CMTF), and auxiliary-laser is resolved the quantitative analysis method of disassociation mass spectrum, be specially organic molecule matrix under high pressure is pressed into film even, big or small controllable thickness, avoid the uncertainty of solvent recrystallization process, the matrix film that obtains can absorb ultraviolet light, can and being washed by original position, remove in inorganic salts as anti-phase hydrophobic layer adsorption sample molecule again.Simultaneously, the hydrophobic surface that this method provides can also avoid sample solution in the diffusion on sample target surface, the sensitivity of raising method.This method need not the organic solvent recrystallization process, can handle solid sample, enlarges range of application.The From Spectral Signal that this method obtains is stable, between signal intensity and the sample size good linear relationship is arranged, and favorable reproducibility is highly sensitive.
Realize technical scheme of the present invention:
A kind of high-pressure molding matrix film is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, this method under high pressure is pressed into film with organic molecule matrix, again film is bonded on the mass spectrometric sample target, sample spot is on film and natural air drying, inorganic salts are removed in the washing of sample original position, put into mass spectrometer analysis behind the natural air drying.
In the technical scheme of the present invention, described organic molecule matrix comprises: alpha-cyano-4-hydroxycinnamic acid (, α-cyano-4-hydroxycinnamic acid, CHCA), 2,5 dihydroxy-benzoic acids (2,5-dihydroxybenzoic acid, DHB).
In the technical scheme of the present invention, described organic molecule matrix under high pressure is pressed into film, is that organic molecule matrix is pressed into film separately, perhaps with after solid sample mixes is pressed into film.
In the technical scheme of the present invention, the method step that matrix is pressed into film separately comprises:
1), take by weighing matrix alpha-cyano-4-hydroxycinnamic acid or 2,5 dihydroxy-benzoic acids, the kind of matrix is decided according to different samples with amount;
2), matrix that step 1) is obtained is levigate with agate mortar, makes its grain size less than 2 μ m;
3), with step 2) the matrix powder that obtains puts into the mould of sheeter, die area 50 * 70mm puts into sheeter again, applies 4800kg pressure, and keeps 1 minute under this pressure;
4), the matrix film of step 3) compacting is taken out, be kept at room temperature and get final product.
In the technical scheme of the present invention, matrix comprises with the method step that is pressed into film after solid sample mixes:
1), take by weighing matrix alpha-cyano-4-hydroxycinnamic acid or 2,5 dihydroxy-benzoic acids, the kind of matrix is decided according to different samples with amount; Take by weighing solid sample;
2), matrix and solid sample that step 1) is obtained are levigate with agate mortar, make its grain size less than 2 μ m, so that matrix film size and thickness that compacting is obtained are even;
3), with step 2) matrix and the solid sample powder that obtain, be that 300: 1 ratios are mixed in matrix and solid sample mol ratio, put into the mould of sheeter, die area 50 * 70mm, put into sheeter again, apply 4800kg pressure, and under this pressure, kept 1 minute;
4), the film of step 3) compacting is taken out, the gained film can directly carry out laser and resolve the disassociation mass spectrophotometry.
In the technical scheme of the present invention, the method that inorganic salts are removed in described sample original position washing is, use pipettor that 10 μ l or more concentration 0.1wt%TFA (trifluoroacetic acid) aqueous solution are covered sample spot fully, kept for 2 seconds, again with the water on the dust-free paper absorption sample, inorganic salts contained in the sample are removed, and repeat to wash 2-3 time.
High-pressure molding matrix film of the present invention is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, analysing protein samples: the enzymolysis liquid of getting a certain amount of albumen, sample is transferred to acidity, pH 1-2, use pipettor with 1 μ l sample spot at the sample target, after waiting for its natural air drying, use pipettor that 10 μ l or more concentration 0.1wt% trifluoroacetic acid aqueous solutions are covered sample spot fully, kept for 2 seconds, again with the water on the dust-free paper absorption sample, inorganic salts contained in the sample are removed, and repeat to wash 2-3 time, put into mass spectrometer analysis behind the natural air drying; Described protein is the α casein.Laser beam is bombarded sample molecule with certain frequency because the sample and the matrix that make according to this method evenly distributes, therefore adopt signal stabilization, the measurer of signal absolute strength and sample has good linear relationship, can carry out quantitative test,
High-pressure molding matrix film of the present invention is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, analysing solid sample: be that 1: 300 ratio is mixed and is prepared into film according to above-mentioned method with solid sample and matrix according to mol ratio, the gained film can directly carry out laser and resolve the disassociation mass spectrophotometry; Described solid sample is the freeze drying dry powder of α casein enzymolysis liquid.
Effect of the present invention and advantage:
1. high-pressure molding matrix film of the present invention is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, and gained matrix film surface is even, and big or small controllable thickness is simple to operate, need not poisonous and harmful reagent, environmental protection.
2. whole process of preparation is simple, control easily, and power consumption is few, steady quality, favorable reproducibility, and be easy to characterize, cost is low, is suitable for carrying out extensive actual production, is convenient to quality control and industrialization.Because it is the analytical technology of widespread use that ground substance assistant laser is resolved the disassociation mass spectrum, so the present invention is with a wide range of applications.
3. compare with existing matrix preparation method, the present invention need not organic reagent, not only environmental protection, and eliminated the uncertainty of recrystallization process.The obtained matrix film of the present invention is even, institute's picked up signal intensity stabilization, and the good linear relation between absolute signal intensity and the sample size can be carried out the sample quantitative test.
4. sample in-situ desalination wash-out is easy, and the metallic ion adduct disturbs little, and analysis speed is fast.
Further the present invention will be described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1-A is the DHB film of embodiment 1 high-pressure molding preparation
Fig. 1-B is the CHCA film of embodiment 1 high-pressure molding preparation
Fig. 1-C is 100 power microscope figure of DHB high-pressure molding film
Fig. 1-D is 100 power microscope figure of CHCA high-pressure molding film
Fig. 1-E is 100 power microscope figure below of the DHB of recrystallization
Fig. 1-F is the 100 power microscope figure of the CHCA of recrystallization
Above picture shows that high-pressure molding matrix film smooth surface is even, and recrystallization obtains the crystal size shape and differs.
Following Fig. 2-Fig. 5 is that embodiment 2 products therefroms carry out sample analysis total ion current figure.
Fig. 2 is DHB recrystallization method gained total ion current figure.
Fig. 3 is DHB high-pressure molding matrix film gained total ion current figure
Fig. 4 is CHCA recrystallization method gained total ion current figure
Fig. 5 is CHCA high-pressure molding matrix film gained total ion current figure
Fig. 2-Fig. 5 shows institute's picked up signal intensity stabilization on the high-pressure molding matrix film, and relative standard deviation is little.
Following Fig. 6-Fig. 9 is embodiment 3 high-pressure molding matrix film in-situ desalination design sketchs
Fig. 6 is that the alpha-casein enzymolysis liquid is at the mass spectrogram of DHB matrix
Among the figure, the arrow indication is sodium and potassium adduct peak.
Fig. 7 is alpha-casein enzymolysis liquid gained mass spectrogram behind in-situ desalination on the DHB matrix
As seen from the figure, sodium peak and potassium peak obviously reduce.
Fig. 8 is that the alpha-casein enzymolysis liquid is at the mass spectrogram of CHCA matrix
The arrow indication is sodium and potassium adduct peak among the figure.
Fig. 9 is alpha-casein enzymolysis liquid gained mass spectrogram behind in-situ desalination on the CHCA matrix
As seen from the figure, sodium peak and potassium peak obviously reduce.
Figure 10 is embodiment 4 high-pressure molding matrix film analysing solid sample mass spectrograms
Among the figure: alpha-casein enzymolysis liquid freezing dry powder sample directly mixes high-pressure molding afterwards with CHCA matrix, carry out mass spectrophotometry then, as seen the mass spectrogram that obtains and traditional analysis method to obtain mass spectrogram consistent.
Following Figure 11-Figure 13 be embodiment 5 high-pressure molding matrix films auxiliary-laser resolves the quantitative test ability of disassociation mass spectroscopy
Figure 11 is the working curve diagram of m/z1337 polypeptide
As seen from the figure, the range of linearity is 1.0 μ g-0.005 μ g, and related coefficient square is respectively 0.7066..
Figure 12 is the working curve diagram of m/z 1759 polypeptide
As seen from the figure,, the range of linearity is 1.0 μ g-0.005 μ g, related coefficient square is 0.8917.
Figure 13 is the working curve diagram of m/z2316 polypeptide
As seen from the figure, the range of linearity is 1.0 μ g-0.005 μ g, related coefficient square branch 0.9629.
Embodiment
The preparation of high-pressure molding matrix film, preparation process are as follows successively:
1), take by weighing a certain amount of matrix with analytical balance, such as 50mg CHCA (α-cyano-4-hydroxycinnamic acid) and 12mg DHB (2,5-dihydroxybenzoic acid), the kind of matrix can be decided according to different samples with amount;
2), matrix that step 1) is obtained is levigate with agate mortar, makes its grain size less than 2 μ m;
3), with step 2) the matrix powder that obtains puts into the grinding tool of sheeter, puts into sheeter again, applies 4800kg pressure, and under this pressure, kept 1 minute;
4), suppress step 3) to such an extent that matrix film (CMTF) takes out, be kept at room temperature.
Wherein, the picture of step 4) gained CHCA film is shown in Fig. 1-A, and the picture of gained DHB film shows gained matrix film smooth surface among the figure shown in Fig. 1-B, and matrix is even.Step 4) gained CHCA film and DHB film after 100 power microscopes amplify respectively shown in Fig. 1-C and Fig. 1-D, with crystallisation gained CHCA film (Fig. 1-E) and DHB film (Fig. 1-F) compare, matrix is evenly distributed, and crystallisation gained crystal grain size and shape inequality.
Embodiment 2
The high-pressure molding matrix film of embodiment 1 preparation is used for sample analysis signal intensity stability
1), the high-pressure molding matrix film with embodiment 1 preparation is bonded on the sample target;
2), alpha-casein enzymolysis liquid sample is transferred to pH 1-2, use pipettor with 1 μ l sample spot different sample spot position on film, and allow its natural air drying;
3), get 10 μ l concentration 0.1wt%TFA aqueous solution and make it cover sample spot, kept for 2 seconds, sop up the globule with dust-free paper, repeat 3 times;
4), the sample target is put into mass spectrometer;
5), use the laser bombardment sample of certain frequency, make testing sample gasification and ionization, and detect its signal intensity;
Wherein, step 5) obtains DHB high-pressure molding matrix film total ion current figure as shown in Figure 3, CHCA high-pressure molding matrix film total ion current figure as shown in Figure 5, compare with DHB recrystallization method gained total ion current figure (Fig. 2) and CHCA recrystallization method gained total ion current figure (Fig. 4), high-pressure molding matrix film method institute picked up signal intensity stabilization, relative standard deviation is little.
Table 1 has been listed the relative standard deviation with distinct methods duplicate measurements institute picked up signal, and visible high-pressure molding matrix film method obviously is better than crystallisation.Crystallisation is because the crystal size inequality, the skewness of crystal grain on target, and inequality causes the signal intensity inequality, even can not bombard sample (as arrow indication position among the figure) when making laser scanning.
Embodiment 3
The in-situ desalination effect of the high-pressure molding matrix film of embodiment 1 preparation
1), the high-pressure molding matrix film with embodiment 1 preparation stands on the sample target;
2), alpha-casein enzymolysis liquid sample is transferred to pH 1-2, use pipettor with 1 μ l sample spot different sample spot position on film, and allow its natural air drying;
3), get 10 μ l concentration 0.1wt%TFA aqueous solution and make it cover sample spot, kept for 2 seconds, sop up the globule with dust-free paper, repeat 3 times;
4), the sample target is put into mass spectrometer;
5), use the laser bombardment sample of certain frequency, make testing sample gasification and ionization, and detect its signal intensity.
Wherein, step 5) gained alpha-casein enzymolysis liquid behind in-situ desalination on the DHB matrix mass spectrogram as shown in Figure 7, mass spectrogram is as shown in Figure 9 behind in-situ desalination on the CHCA matrix for the alpha-casein enzymolysis liquid; With the alpha-casein enzymolysis liquid of not desalination shown in Figure 6 mass spectrogram in DHB matrix, and alpha-casein enzymolysis liquid shown in Figure 8 compares at the mass spectrogram of CHCA matrix, and sodium peak and potassium peak obviously reduce.
The high-pressure molding matrix film analysing solid sample effect of embodiment 1 preparation
1), take by weighing 50mg CHCA and 12mg DHB with analytical balance, the kind of matrix can be decided according to different samples with amount;
2) be that 1: 300 ratio is mixed with alpha-casein enzymolysis liquid freezing dry powder solid sample and matrix according to mol ratio;
3), with step 2) potpourri that obtains is levigate with agate mortar, makes its grain size less than 2 μ m;
4), mix powder that step 3) is obtained puts into the mould of sheeter, die area 50 * 70mm puts into sheeter again, applies 4800kg pressure, and keeps 1 minute under this pressure;
5), suppress step 4) to such an extent that film (CMTF) takes out, be bonded at the sample target, and put into mass spectrometer analysis.
Wherein, step 5) gained mass spectrum as shown in figure 10.As seen, solid sample is by the ionization of gasifying effectively, and feature polypeptide peak signal is strong.
Embodiment 5
The quantitative test ability of the high-pressure molding matrix film of embodiment 1 preparation.
1), the high-pressure molding matrix film with embodiment 1 preparation stands in the sample target;
2), alpha-casein enzymolysis liquid sample is transferred to pH 1-2, use pipettor with 1 μ l sample spot different sample spot position on film, and allowing its natural air drying, the amount that wherein contains alpha-casein in every microlitre sample is respectively 1.0 μ g, 0.5 μ g, 0.05 μ g, 0.005 μ g;
3), get 10 μ l concentration 0.1wt%TFA aqueous solution and make it cover sample spot, kept for 2 seconds, sop up the globule with dust-free paper, repeat 3 times;
4), the sample target is put into mass spectrometer;
5), use the laser bombardment sample of certain frequency, make testing sample gasification and ionization, and detect its signal intensity.
Wherein, the signal intensity of step 5) gained and the relation between the sample size such as Figure 11-shown in Figure 13, visible not homopolypeptide has different response slopes, and signal intensity becomes good linear relationship with sample size.
Claims (8)
1. a high-pressure molding matrix film is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, it is characterized in that, this method under high pressure is pressed into film with organic molecule matrix, again film is bonded on the mass spectrometric sample target, sample spot is on film and natural air drying, inorganic salts are removed in the washing of sample original position, put into mass spectrometer analysis behind the natural air drying.
2. high-pressure molding matrix film according to claim 1 is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, it is characterized in that described organic molecule matrix comprises: alpha-cyano-4-hydroxycinnamic acid or 2,5 dihydroxy-benzoic acids.
3. high-pressure molding matrix film according to claim 1 is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, it is characterized in that, described organic molecule matrix under high pressure is pressed into film, be that organic molecule matrix is pressed into film separately, perhaps with after solid sample mixes be pressed into film.
4. high-pressure molding matrix film according to claim 3 is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, it is characterized in that the method step that matrix is pressed into film separately comprises:
1), take by weighing matrix alpha-cyano-4-hydroxycinnamic acid or 2,5 dihydroxy-benzoic acids, the kind of matrix is decided according to different samples with amount;
2), matrix that step 1) is obtained is levigate with agate mortar, makes its grain size less than 2 μ m;
3), with step 2) the matrix powder that obtains puts into the mould of sheeter, die area 50 * 70mm puts into sheeter again, applies 4800kg pressure, and keeps 1 minute under this pressure;
4), the matrix film of step 3) compacting is taken out, be kept at room temperature and get final product.
High-pressure molding matrix film according to claim 3 auxiliary-laser resolves the quantitative analysis method of disassociation mass spectrum, it is characterized in that, matrix comprises with the method step that is pressed into film after solid sample mixes:
1), take by weighing matrix alpha-cyano-4-hydroxycinnamic acid or 2,5 dihydroxy-benzoic acids, the kind of matrix is decided according to different samples with amount; Take by weighing solid sample;
2), matrix and solid sample that step 1) is obtained are levigate with agate mortar, make its grain size less than 2 μ m;
3), with step 2) matrix and the solid sample powder that obtain, be that 300: 1 ratios are mixed in matrix and solid sample mol ratio, put into the mould of sheeter, die area 50 * 70mm, put into sheeter again, apply 4800kg pressure, and under this pressure, kept 1 minute;
4), the film of step 3) compacting is taken out, the gained film can directly carry out laser and resolve the disassociation mass spectrophotometry.
6. high-pressure molding matrix film according to claim 1 is assisted-quantitative analysis method of laser parsing disassociation mass spectrum, it is characterized in that, the method that inorganic salts are removed in described sample original position washing is, use pipettor that 10 μ l or more 1% trifluoroacetic acid aqueous solutions are covered sample spot fully, kept for 2 seconds, with the water on the dust-free paper absorption sample, inorganic salts contained in the sample are removed, and repeat to wash 2-3 time again.
7. the described high-pressure molding matrix film of claim 1 is assisted-laser parsing disassociation mass spectrum quantitative analysis method analysing protein samples, it is characterized in that: the enzymolysis liquid of getting a certain amount of albumen, sample is transferred to acidity, pH1-2, use pipettor with 1 μ l sample spot at the sample target, after waiting for its natural air drying, use pipettor that 10 μ l or more concentration 0.1wt% trifluoroacetic acid aqueous solutions are covered sample spot fully, kept for 2 seconds, again with the water on the dust-free paper absorption sample, inorganic salts contained in the sample are removed, and repeat to wash 2-3 time, put into mass spectrometer analysis behind the natural air drying; Described protein is the α casein.
8. the described high-pressure molding matrix film of claim 1 is assisted-laser parsing disassociation mass spectrum quantitative analysis method analysing solid sample, it is characterized in that: be that 1: 300 ratio is mixed and is prepared into film in accordance with the method for claim 5 with solid sample and matrix according to mol ratio, the gained film can directly carry out laser and resolve the disassociation mass spectrophotometry; Described solid sample is the freeze drying dry powder of α casein enzymolysis liquid.
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| WO2016116012A1 (en) * | 2015-01-21 | 2016-07-28 | 华中师范大学 | Mass correction kit and correction method for low-mass area of high-resolution mass spectrometer in negative ion mode |
| CN111386463A (en) * | 2018-08-30 | 2020-07-07 | 株式会社Lg化学 | Method for relative quantitative analysis of polymers using MALDI mass spectrometry |
| CN111527391A (en) * | 2018-09-11 | 2020-08-11 | 株式会社Lg化学 | Method for preparing test piece for MALDI mass spectrometry using water-insoluble substance and method for quantitatively analyzing water-insoluble substance using MALDI mass spectrometry |
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| CN105445308A (en) * | 2014-08-28 | 2016-03-30 | 中国科学院上海硅酸盐研究所 | An in-situ thermal analysis-mass spectrometry measurement method for a film material |
| CN105445308B (en) * | 2014-08-28 | 2019-10-15 | 中国科学院上海硅酸盐研究所 | A kind of heat analysis in situ-mass-spectrometer measurement method of thin-film material |
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| US10181395B2 (en) | 2015-01-21 | 2019-01-15 | Central China Normal University | Mass calibration kit and calibration method for low-mass region of high-resolution mass spectrometer in negative ion mode |
| CN111386463A (en) * | 2018-08-30 | 2020-07-07 | 株式会社Lg化学 | Method for relative quantitative analysis of polymers using MALDI mass spectrometry |
| CN111386463B (en) * | 2018-08-30 | 2022-11-01 | 株式会社Lg化学 | Method for relative quantitative analysis of polymers using MALDI mass spectrometry |
| CN111527391A (en) * | 2018-09-11 | 2020-08-11 | 株式会社Lg化学 | Method for preparing test piece for MALDI mass spectrometry using water-insoluble substance and method for quantitatively analyzing water-insoluble substance using MALDI mass spectrometry |
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