CN112557497A - Use of hydralazine as a matrix in matrix-assisted laser desorption ionization mass spectrometry - Google Patents
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Abstract
The invention discloses application of hydralazine as a matrix in matrix-assisted laser desorption ionization mass spectrometry, which is used for analyzing organic small molecular compounds, small molecular peptides, body fluid, tissue homogenate or tissue slices and mass spectrometry imaging. When the hydralazine is used as a matrix-assisted laser desorption ionization mass spectrum matrix, the ionization efficiency is high, the mass spectrum background is very simple in the range of m/z50-2000, and the analysis of small molecular compounds and complex biological samples is not interfered; when the hydralazine is used as a matrix of matrix-assisted laser desorption ionization mass spectrometry, simultaneous detection of positive and negative ions can be realized, and the hydralazine is suitable for high-coverage detection in different molecular weight ranges, so that the analysis requirements of various complex biological samples can be met; the hydralazine is a known compound existing in the prior art, and is low in obtaining cost.
Description
Technical Field
The invention belongs to the field of analysis, relates to a matrix of matrix-assisted laser desorption ionization mass spectrometry, and particularly relates to application of hydralazine as a matrix in matrix-assisted laser desorption ionization mass spectrometry.
Background
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a soft ionization mass spectrometry technology which is started at the end of the 80 th 20 th century, and has the advantages of no marking property, high sensitivity, high permeability, molecular specificity and the capability of simultaneously detecting and positioning various biomolecules directly from a tissue sample. The selection of the matrix is critical to the MALDI MSI analysis technique. The choice of the substrate for analyzing the sample to be tested in the experiment depends mainly on the kind and properties of the sample to be analyzed. 2,5-dihydroxy benzoic acid (DHB) is often used as a substrate to analyze proteins and polypeptides on the surface of a tissue, and can be used in a positive and negative ion mode; the 9-Aminoacridine (9-Aminoacridine,9-AA) generally has a better detection effect in a negative ion mode. Sinapinic Acid (SA) is commonly used as a substrate for detecting biological macromolecules such as proteins; alpha-4-Hydroxy-cinnamic acid, (alpha-4-Hydroxy-cinamic acid, CHCA) is commonly used to detect small polypeptide molecules, primarily in the positive ion mode. However, these matrices are prone to fragmentation and association between molecules during analysis, often generate serious matrix background interference in the range of charge-to-mass ratio (m/z) below 500, and have the disadvantages of poor salt tolerance, etc., which seriously hinder the analysis of small molecule compounds (with the charge-to-mass ratio below 500) and complex biological samples. In addition, the conventional matrix is difficult to realize simultaneous detection of positive and negative ions, and has certain limitation on the coverage of a molecular weight range.
In order to solve the above problems, matrix materials such as nanomaterials (silica, carbon nanomaterials, etc.), organic salts (naphthylethylenediamine hydrochloride, naphthylhydrazine hydrochloride, 1, 5-naphthylenediamine hydrochloride), and proton sponges have been developed in succession in recent years. The matrixes have small background interference and good salt tolerance, and have certain advantages in the aspects of small molecular compounds and complex biological sample analysis. However, although the background interference of the nano material is small, the preparation process is complex, the cost is high, and the salt resistance is poor. Proton sponges as a matrix can only be analyzed for organic acid compounds. Although the salt resistance of the organic salt matrix is good, many isotope peaks appear in the process of analyzing small molecular compounds, and the determination of other small molecules is interfered. In addition, most of these matrices still do not allow high coverage detection of positive and negative ions and different molecular weight ranges.
Therefore, the matrix which is simple in operation method, low in cost, high in ionization efficiency and wide in detection range is provided, and is an important and powerful supplement for the mass spectrometry based on MALDI at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the application of hydralazine as a matrix in matrix-assisted laser desorption ionization mass spectrometry.
The above purpose of the invention is realized by the following technical scheme:
use of hydralazine as a matrix in matrix-assisted laser desorption ionization mass spectrometry.
Use of hydralazine as a matrix for the analysis of small organic molecular compounds, small molecular peptides, body fluids, tissue homogenates or tissue sections in matrix-assisted laser desorption ionization mass spectrometry.
Further, the method is used as a matrix for analyzing organic small molecule compounds or small molecule peptides, and the analysis method comprises the following steps: (1) preparing a matrix solution containing hydralazine; (2) preparing a sample solution containing an organic small molecule compound or a small molecule peptide; (3) the mixture of matrix solution and sample solution was placed on the target plate of a mass spectrometer for mass spectrometry.
Further, for use as a matrix for analyzing a body fluid or tissue homogenate, the analysis method comprises the steps of: (1) preparing a matrix solution containing hydralazine; (2) precipitating the body fluid or tissue homogenate by an organic solvent to prepare a sample solution; (3) the mixture of matrix solution and sample solution was placed on the target plate of a mass spectrometer for mass spectrometry.
Further, the tissue section is analyzed as a matrix, and the analysis method includes: (1) preparing a matrix solution containing hydralazine; (2) coating the matrix solution on the tissue slice; (3) mass spectrometry was performed on the tissue sections coated with the matrix solution.
Still further, the solvent of the matrix solution is selected from one or more of water, methanol, ethanol and acetonitrile.
Further, 0.1% to 0.5% trifluoroacetic acid was added to the matrix solution for positive ion mode analysis.
Further, 0.1% to 0.5% ammonia was added to the matrix solution for negative ion mode analysis.
Still further, the solvent of the sample solution is selected from one or more of water, methanol, ethanol, and acetonitrile.
Use of hydralazine as a matrix for mass spectrometry imaging in matrix-assisted laser desorption ionization mass spectrometry.
Has the advantages that:
1. the hydralazine can be used as a matrix of matrix-assisted laser desorption ionization mass spectrometry, has high ionization efficiency, has a very simple mass spectrometry background in the range of m/z50-2000, and has no interference on the analysis of small molecular compounds and complex biological samples, so the defect that the small molecules (m/z <500) cannot be effectively analyzed due to the fact that the serious matrix background interference phenomenon is easily generated in a low molecular weight region by a common organic small molecular matrix can be overcome;
2. when the hydralazine is used as a matrix of matrix-assisted laser desorption ionization mass spectrometry, simultaneous detection of positive and negative ions can be realized, and the hydralazine is suitable for high-coverage detection in different molecular weight ranges, so that the analysis requirements of various complex biological samples can be met;
3. the hydralazine is a known compound existing in the prior art, and is low in obtaining cost.
Drawings
FIG. 1 is a structural formula of hydralazine hydrochloride.
FIG. 2 is a background mass spectrum of hydralazine with trifluoroacetic acid as a matrix in the molecular weight range of m/z50-2000 in the positive ion mode of example 1 (panel (a) m/z 50-650, panel (b) m/z 650-2000).
FIG. 3 is the background mass spectrum of the anion mode of example 1 with hydralazine with ammonia water as matrix in the range of m/z50-2000 (graph (a) m/z 50-650, graph (b) m/z 650-2000).
FIG. 4 is a mass spectrum of brain homogenate measured in the m/z50-2000 molecular weight range in the positive ion mode of example 2 using hydralazine plus trifluoroacetic acid as a matrix (panel (a) m/z 50-650, panel (b) m/z 650-1000, and panel (c) m/z 650-2000).
FIG. 5 is a mass spectrum of brain homogenate measured in the molecular weight range of m/z50-2000 in the negative ion mode of example 2 using hydralazine with ammonia water as a matrix (graph (a) m/z 50-650, graph (b) m/z 650-1000, and graph (c) m/z 650-2000).
FIG. 6 is an image of mass spectrum of kidney tissue section (one kidney tissue image under each characteristic peak) measured in m/z50-2000 molecular weight range by detecting kidney tissue section with hydralazine and trifluoroacetic acid as substrates in positive ion mode of example 3.
FIG. 7 is an image of mass spectrum of kidney tissue section (one kidney tissue image under each characteristic peak) measured in m/z50-2000 molecular weight range by detecting kidney tissue section with hydralazine and ammonia water as matrix in positive ion mode of example 3.
Detailed Description
The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The hydralazine used in the following examples of the present invention was prepared from hydralazine hydrochloride, which was obtained from sigma, usa and has the structural formula shown in fig. 1.
The hydralazine is prepared as follows: taking 100mg of hydralazine hydrochloride, adding 1-2mL of deionized water, ultrasonically mixing uniformly, adding 1mL of concentrated ammonia water, carrying out vortex oscillation for 3-5min, adding 3mL of chloroform for extraction, continuously extracting for 3 times, combining chloroform layers, and removing a solvent under reduced pressure to obtain hydralazine yellow powder for later use.
The matrix solution was prepared as follows: dissolving hydralazine in an ethanol solvent at room temperature to obtain a matrix solution of 10mg/mL, and adding 0.1% trifluoroacetic acid into the matrix solution when the method is applied to a positive ion detection mode; when applied to the negative ion detection mode, 0.1% ammonia was added to the matrix solution.
The matrix assisted laser desorption ionization time-of-flight mass spectrometer model used below was Bruker Ultraflexreeme TOF/MS.
The mass spectrum background of the matrix is very simple, and has no interference to a test sample including a complex system, and mass spectrograms of the hydralazine in a positive ion mode and a negative ion mode by adding trifluoroacetic acid and ammonia water are shown in figures 2 and 3 (figure (a) m/z 50-650, figure (b) m/z 650-2000).
Example 1: background interference with hydralazine as a substrate
1 mu L of hydralazine is added with trifluoroacetic acid matrix solution and spotted on a MALDI target plate, and mass spectrum detection is carried out by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Bruker Ultraflexreeme TOF/MS), wherein the laser wavelength is 355nm, the pulse frequency is 1000Hz, the intensity is 75 percent, and the positive ion mode is adopted.
1 mu L of hydralazine is added with ammonia water matrix solution and spotted on a MALDI target plate, and mass spectrum detection is carried out by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Bruker UltraflexreeTem TOF/MS), wherein the laser wavelength is 355nm, the pulse frequency is 1000Hz, the intensity is 75 percent, and the negative ion mode is adopted.
As can be seen from fig. 2 and 3, the background of mass spectrometry using the hydralazine compound as the matrix is usually simple and clean, and has low background interference on the test sample.
Example 2: detection of hydralazine on mouse brain tissue homogenate extract
Taking fresh mouse brain tissue, rinsing in normal saline, removing blood, putting into a 2mL homogenate tube, adding about 0.2mL cold normal saline, grinding for 2min by using a tissue grinder, sucking the tissue homogenate into a 1.5mL centrifuge tube, precipitating with acetonitrile, centrifuging, taking supernatant, drying by using nitrogen, and dissolving by using methanol to obtain the solution to be detected.
And mixing the solution to be detected and the matrix solution in equal volume, adding 1 mu L of the mixed solution onto a MALDI target plate, and performing mass spectrum detection by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Bruker UltraflexreeTem TOF/MS), wherein the laser wavelength is 355nm, the pulse frequency is 1000Hz, the intensity is 75 percent, and the positive and negative ion modes are adopted.
As can be seen from FIGS. 4 and 5, the hydralazine can satisfy the detection of different lipids in biological samples (m/z is 700-900 in a positive and negative ion mode), and in addition, the matrix has excellent detection performance in a negative ion mode, especially a small molecular weight region (m/z is less than 500), and can satisfy the analysis of fatty acid compounds.
Example 3: mass spectrometry imaging of mouse kidney tissue sections
Fresh kidney tissues of mice taken out by operation are put into a cavity of a freezing microtome at the temperature of minus 80 ℃ after being frozen quickly by dry ice, are taken out every other day, are put into the cavity of a freezing microtome at the temperature of minus 20 ℃ for balancing for 30min, are frozen and sliced, the thickness of the sample slice is 12 mu m, and are transferred to an ITO slide to be sprayed with a matrix solution (a positive ion mode: a trifluoroacetic acid solution is added to hydralazine; a negative ion mode: an ammonia water solution is added to hydralazine).
The matrix solution is sprayed by adopting an automatic electrospray device constructed in a laboratory, 100 mu L of the matrix solution is absorbed and is input into the device through an injection pump, the flow rate is 1mL/h, the temperature of a heating connection module is set to be 90 ℃, nitrogen is used for assisting atomization and drying under the action of a heating device, the high pressure is set to be 5.90kv, the distance between a capillary nozzle and a sample slice is 13cm, and a uniform matrix layer is formed on the surface of the slice under the auxiliary action of an electric field. Bruker UltraFlextrememe TOF/MS; 150 μm resolution (10 μm limit resolution), laser wavelength 355nm, pulse frequency 1000Hz, 75% intensity, m/z 60-2000, positive and negative ion mode.
Fig. 6 is a mass spectrogram and an imaging image of a characteristic peak of a kidney tissue slice in a positive ion mode obtained by adding trifluoroacetic acid matrix to hydralazine, and fig. 7 is a mass spectrogram and an imaging image of a characteristic peak of a kidney tissue slice in a negative ion mode obtained by adding ammonia water to hydralazine. The tissue slices were not further processed except during the spraying of the matrix. As can be seen from the figure, the hydralazine compound is taken as a matrix, and the space specificity distribution of different small molecular compounds in mouse kidney tissues can be obtained, so that the hydralazine compound is suitable to be taken as the matrix to realize tissue section mass spectrum imaging.
From the above experimental results, it can be seen that:
1. the hydralazine can be used as a matrix of matrix-assisted laser desorption ionization mass spectrometry, has high ionization efficiency, has a very simple mass spectrometry background in the range of m/z50-2000, and has no interference on the analysis of small molecular compounds and complex biological samples, so the defect that the small molecules (m/z <500) cannot be effectively analyzed due to the fact that the serious matrix background interference phenomenon is easily generated in a low molecular weight region by a common organic small molecular matrix can be overcome;
2. when the hydralazine is used as a matrix of matrix-assisted laser desorption ionization mass spectrometry, simultaneous detection of positive and negative ions can be realized, and the hydralazine is suitable for high-coverage detection in different molecular weight ranges, so that the analysis requirements of various complex biological samples can be met;
3. the hydralazine is a known compound existing in the prior art, and is low in obtaining cost.
The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.
Claims (10)
1. Use of hydralazine as a matrix in matrix-assisted laser desorption ionization mass spectrometry.
2. Use of hydralazine as a matrix for the analysis of small organic molecular compounds, small molecular peptides, body fluids, tissue homogenates or tissue sections in matrix-assisted laser desorption ionization mass spectrometry.
3. Use according to claim 2 as a matrix for the analysis of small organic molecule compounds or small peptides, the analysis method comprising the steps of: (1) preparing a matrix solution containing hydralazine; (2) preparing a sample solution containing an organic small molecule compound or a small molecule peptide; (3) the mixture of matrix solution and sample solution was placed on the target plate of a mass spectrometer for mass spectrometry.
4. Use according to claim 2 as a matrix for analyzing a body fluid or tissue homogenate, the analysis method comprising the steps of: (1) preparing a matrix solution containing hydralazine; (2) precipitating the body fluid or tissue homogenate by an organic solvent to prepare a sample solution; (3) the mixture of matrix solution and sample solution was placed on the target plate of a mass spectrometer for mass spectrometry.
5. Use according to claim 2, as a matrix for analyzing tissue sections, the analysis method comprising the steps of: (1) preparing a matrix solution containing hydralazine; (2) coating the matrix solution on the tissue slice; (3) mass spectrometry was performed on the tissue sections coated with the matrix solution.
6. Use according to any one of claims 3 to 5, characterized in that: the solvent of the matrix solution is selected from one or more of water, methanol, ethanol and acetonitrile.
7. Use according to claim 6, characterized in that: 0.1% -0.5% trifluoroacetic acid was added to the matrix solution for positive ion mode analysis.
8. Use according to claim 6, characterized in that: 0.1% -0.5% ammonia was added to the matrix solution for negative ion mode analysis.
9. Use according to any one of claims 3 to 5, characterized in that: the solvent of the sample solution is selected from one or more of water, methanol, ethanol and acetonitrile.
10. Use of hydralazine as a matrix for mass spectrometry imaging in matrix-assisted laser desorption ionization mass spectrometry.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200401896A (en) * | 2002-01-25 | 2004-02-01 | Ciphergen Biosystems Inc | Monomers and polymers having energy absorbing moieties of use in desorption/ionization of analytes |
| US20040038258A1 (en) * | 2002-04-29 | 2004-02-26 | Harley John B. | Methods for detecting DNA polymorphisms |
| CA2528370A1 (en) * | 2003-06-06 | 2005-05-12 | President And Fellows Of Harvard College | Capture and release based isotope tagged peptides and methods for using the same |
| CN101222942A (en) * | 2005-07-18 | 2008-07-16 | 诺沃-诺迪斯克有限公司 | Peptides for use in the treatment of obesity |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200401896A (en) * | 2002-01-25 | 2004-02-01 | Ciphergen Biosystems Inc | Monomers and polymers having energy absorbing moieties of use in desorption/ionization of analytes |
| US20040038258A1 (en) * | 2002-04-29 | 2004-02-26 | Harley John B. | Methods for detecting DNA polymorphisms |
| CA2528370A1 (en) * | 2003-06-06 | 2005-05-12 | President And Fellows Of Harvard College | Capture and release based isotope tagged peptides and methods for using the same |
| CN101222942A (en) * | 2005-07-18 | 2008-07-16 | 诺沃-诺迪斯克有限公司 | Peptides for use in the treatment of obesity |
Non-Patent Citations (2)
| Title |
|---|
| WEIWEI TANG ET AL: "Hydralazine as a Versatile and Universal Matrix for High-Molecular Coverage and Dual-Polarity Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging", 《ANAL. CHEM》 * |
| 张红敏等: "基质辅助激光解吸电离飞行时间质谱技术在分枝杆菌检测中的应用进展", 《新发传染病电子杂志》 * |
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