KR102062447B1 - Target surfaces for MALDI mass spectrometry using Graphene films and Mass analysis method using the same - Google Patents

Abstract

The present invention relates to a method of applying graphene to the surface of a mass spectrometry plate to enable more reproducible MALDI mass spectrometry by increasing crystal uniformity of a MALDI sample spot consisting of a matrix-sample mixture.
In the method of the present invention, a target plate is formed of a graphene layer, and a droplet of the mattress-sample mixture is dropped on the graphene layer, and then dried and irradiated with a laser to measure mass. The graphene target plate of the present invention can induce spontaneous homogenous crystallization of the droplets by interacting with the matrix present in excess while the droplets (DROP) of the matrix-sample solution are drying. Therefore, when the target plate of the present invention is used, fine crystals are generated homogeneously on the target plate, thereby greatly reducing the sweet spot issue (the problem of poor reproducibility of mass measurement because the amount of the sample is varied depending on the laser irradiation position). Can be.
The present invention is simple in structure and manufacturing method in that the plate is replaced by graphene, which is easy to synthesize a large area, and can be applied to various samples in that a conventional organic matrix (CHCA, DHB, SA, etc.) can be used as it is. Do.

Description

Target surfaces for MALDI mass spectrometry using Graphene films and Mass analysis method using the same}

The present invention relates to a MALDI mass spectrometry plate using a graphene and a mass spectrometry method using the same. More specifically, when the graphene is applied to the surface of the mass spectrometry plate to determine the MALDI sample spot consisting of a matrix-sample mixture The present invention relates to a method for enabling more reproducible MALDI mass spectrometry by increasing uniformity.

Matrix-Assisted Laser Desorption / Ionization (MALDI) is a mass spectrometry that is widely used to analyze organic materials, biomaterials, and polymers. Way.

Specifically, crystals are prepared by mixing a sample and a matrix (for example, a-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapic acid (SA)), and then instantaneously. It is a method of inducing desorption ionization from the crystal by irradiating a pulsed UV laser to the crystal and releasing it as gaseous ions to measure the molecular weight or analyze the structure by mass spectrometry.

This method produces not only the ionization of high molecular biochemicals (proteins, peptides, DNA, etc.) that are difficult to turn into gas ions, but also the formation of gaseous ions without breakage for synthetic polymer materials with a large molecular weight. It also has the advantage of being able to analyze even samples (femtomol to picomolar levels). However, since the irradiated laser energy is transferred to the sample through the crystallized matrix and ionized, selection of an appropriate matrix and optimal crystallization have a very important effect on the experimental results according to the type of sample.

In addition, since the crystal mixed with the sample and the matrix is not homogeneous, an ion signal having a different intensity appears according to the analysis position, and thus, an effort to find a sweet spot is required to obtain an ion signal having an appropriate intensity for analysis. As a result, it is difficult to obtain a reproducible and reliable measurement result.

In addition, in the case of the analysis of the synthetic polymer sample in the low molecular weight region (1000 Da), there is a problem in that the accuracy of the mass measurement of the analytical sample is degraded due to interference by the peaks of the matrix used together.

To avoid this problem, various methods of matrix application are used. For example, a thin matrix (a-cyano-4-hydroxycinnamic acid (CHCA)) is prepared using organic solvents that rapidly dry the matrix-sample solution in vacuo, rapidly evaporate in the matrix solution, Deposition of the matrix layer using sublimation of 2,5-dihydroxybenzoic acid (DHB), CHCA), and spraying the matrix solution onto the target surface. These methods provide a fine and flat matrix crystal layer compared with the conventional MADLI method, but require a special additional process such as the process must be performed under vacuum conditions, and the problem is still applicable to a specific matrix.

Recently, instead of using a matrix to solve the MALDI problem, mass spectrometry using nanowires such as metals, metal oxides, and semiconductor materials has been proposed in Korean Patent Registration No. 10-0534204. The registered patent has an advantage that it is possible to easily match the center of the sample crystal to the laser irradiation point by using the nanowire, and also can be applied to a variety of samples because a nanowire having a specific band gap can be prepared. However, the Korean patent has a complicated process of manufacturing a nanowire having a specific size (long-ratio), and it is still difficult to manufacture each nanowire using a metal (having a band gap) suitable for a specific sample. .

The present invention provides a method by which the specimen can be formed into homogeneous and fine crystals on the target plate.

The present invention provides a mass measuring method that is simple in mass measuring method and easy to apply to various samples.

The present invention is to provide a method for improving the mass measurement reproducibility and quantitation of the analytical sample.

One aspect of the present invention

It relates to a MALDI mass spectrometry plate comprising a base plate and a graphene layer formed on the base plate.

The present invention relates to a method for producing a MALDI mass spectrometry plate comprising preparing a base plate and forming a graphene layer on the base plate.

The present invention provides a method for preparing analytical specimens by mixing a matrix and analytical sample, loading the analytical specimen on a graphene mass spectrometry plate, drying the loaded analytical sample at room temperature, and irradiating the analytical specimen with a laser sample. Desorption and ionization, and a method for mass spectrometry comprising analyzing the mass of the ionized sample.

In the method of the present invention, a target plate is formed of a graphene layer, and a droplet of the mattress-sample mixture is dropped on the graphene layer, and then dried and irradiated with a laser to measure mass. The graphene target plate of the present invention can induce spontaneous homogenous crystallization of the droplets by interacting with the matrix present in excess while the droplets (DROP) of the matrix-sample solution are drying. Therefore, when the target plate of the present invention is used, fine crystals are homogeneously generated on the target plate, thereby greatly reducing the sweet spot issue (the problem of poor reproducibility of mass measurement because the amount of the sample is varied depending on the laser irradiation position). Can be.

The present invention is simple in structure and manufacturing method in that the plate is replaced by graphene, which is easy to synthesize a large area, and can be applied to various samples in that a conventional organic matrix (CHCA, DHB, SA, etc.) can be used as it is. Do.

1 shows a cross section of a target plate of the invention.
2 is a schematic diagram of a MALDI mass spectrometry system of the present invention.
3A is an optical image of Comparative Example 1, and 3c is an optical image of Example 1. FIG.
4A and 4C are calibration lines of Comparative Example 1, and FIGS. 4B and 4D are calibration lines of Example 1. FIG.
5A is an optical image of Comparative Example 2, and 5c is an optical image of Example 1. FIG.
6A is a calibration line of Comparative Example 2, and FIGS. 6B and 6C are calibration lines of Example 2. FIG.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. However, the scope of the present invention is not limited to the description or the drawings for the following embodiments. In other words, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In addition, the terms "comprises" or "having" described herein are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, and one or the same. It is to be understood that the present invention does not exclude in advance the possibility of the presence or the addition of other features, numbers, steps, operations, components, parts, or a combination thereof.

1 is a cross-sectional view of the target plate of the present invention, Figure 2 is a schematic diagram of the MALDI mass spectrometry system of the present invention. Referring to FIG. 1, the matrix-assisted laser deposition / ionization (MALDI) mass spectrometry target plate of the present invention includes a base plate 11 and a graphene layer 12. A matrix (2) -analyte (1) mixture (A) is loaded onto the target plate (10).

In addition, the method of manufacturing a matrix-assisted laser deposition / ionization (MALDI) mass spectrometry target plate of the present invention includes preparing a base plate and forming a graphene layer on the base plate.

The base plate 10 may be a substrate on which the graphene layer may be supported or the graphene layer may be grown.

The base plate 10 may be a copper substrate, a nickel substrate, an SiO 2 substrate, stainless steel (SUS), or the like.

The graphene may be formed on the base plate by a known method. For example, the present invention may transfer (transfer) and attach graphene prepared by a chemical physical exfoliation method onto the base plate. In addition, the present invention can be used by pasting the graphene grown on the surface of copper or nickel foil with the foil to the base plate. In addition, the present invention can be formed on the base plate by a method such as chemical vapor deposition or epitaxial growth method.

The method for preparing (oxidized) graphene from graphite by a chemical method may be obtained by inducing peeling through the production of graphite oxide using a strong acid and an oxidizing agent, and then reducing it.

Chemical Vapor Deposition is a method of depositing a metal, which is easily adsorbed to carbon such as Ni, Cu, Pt, etc. at high temperature onto a SiO2 substrate as a catalyst layer, in a mixed gas atmosphere containing methane, hydrogen, etc. After carbon reacts with the catalyst layer, the carbon atoms that are dissolved in the catalyst layer crystallize on the surface to form graphene. Alternatively, large area graphene may be directly grown using chemical vapor deposition on foils such as Ni and Cu.

For example, the forming of the graphene layer may include forming a catalyst layer such as nickel on the base plate, vapor depositing graphene on the catalyst layer, and removing the catalyst layer.

In the epitaxial growth method, silicon is evaporated when a material containing carbon such as silicon carbide (SiC) is adsorbed and contained in the crystal structure at a high temperature of 1,500 ° C., and carbon in SiC forms graphene along the crystal surface. have.

The MALDI mass spectrometry method of the present invention comprises the steps of preparing an analytical specimen, loading the specimen onto the graphene plate, drying the loaded analyte at room temperature, irradiating the analytical specimen with a laser to desorb and ionize the sample. And analyzing the mass of the ionized sample.

The analysis sample (A) can be prepared by mixing the matrix (2) and the analysis sample (1) in a solvent. The present invention can use a known organic matrix without limitation. The matrix may be α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapic acid (SA), or 4-hydroxy-3-methoxycinnamic acid (4-hydroxy-3-). methoxycinnamic acid, picolinic acid, 3-hydroxy picolinic acid, 2,6-dihydroxyacetophenone, 1,5-diamino Naphthalene (1,5-diaminonapthalene), 2,4,6-trihydroxyacetophenone (2,4,6-trihydroxyacetophenone), 2- (4'-hydroxybenzeneazo) benzoic acid (2- (4'-hydroxybenzeneazo) benzoic acid), 2-mercaptobenzothiazole, chloro-cyanocinnamic acid or fluoro-cyanocinnamic acid, and mixtures thereof.

There are no particular limitations on the analytical samples that can be used in the present invention. As the solvent, water, acetonitrile, methanol, ethanol, or a mixture thereof can be used. In addition, additives for enhancing the ionization effect may include trifluoroacetic acid, acetic acid, NaCl, NaNO 3 and the like.

In the analytical specimen preparation step, the content of the matrix may be added in excess of the analytical sample. For example, the number of moles of the matrix and the sample can range from one thousand to one million.

The loading step is dropping the analyte specimen mixture onto the graphene plate in the form of droplets.

The drying step is a step of drying the loaded sample at room temperature. The solvent is removed through the drying step to form a matrix-sample crystal layer. In addition, the uniformity of the crystal can be more precisely controlled by adjusting the solvent removal and crystallization rate using a vacuum desiccator or the like.

Graphene plates are characterized by matrix-to-molecular attraction (including π-π interactions) between the surface of graphene molecules and the organic matrix molecules contained in excess while the droplets (DROP) of the matrix-sample mixed solution are dried. Induce uniform production of sample crystals.

3 is a mass image of the optical image (a, c) and the spatial distribution (b, d) after loading and drying the matrix-sample mixed droplets on the SUS target plate and the graphene target plate, respectively.

In more detail, FIG. 3A is an optical image of a dry assay specimen (loaded in spot form) loaded with 1 μl of analyte solution containing a CHCA matrix and a peptide on an SUS target plate, and 3c shows the same solution on a graphene film. This is an optical image of the analytical specimen, loaded on and dried. Referring to FIG. 3A, it can be seen that matrix-sample crystals are irregularly scattered in a non-uniform size adjacent to each other at a predetermined distance. In particular, FIG. 3A shows that matrix-sample crystals exist visually and clearly separated. In addition, 3a can clearly identify the rim of the matrix-sample crystals. On the other hand, in FIG. 3C, it can be seen that the matrix-sample crystals are densely located adjacent to each other and are evenly distributed. In Figure 3c it is not possible to see the border of the crystals. That is, even though the same amount of matrix-sample mixed droplets were dropped under the same conditions, in FIG. 3c, the crystal size was fine and uniformly formed so that the space between the matrix-sample crystals and the crystals was close to each other (ie, 3a). , Densely).

Figure 3b is a mass image showing the spatial distribution of the sample (peptide) in the assay specimen loaded and dried on the SUS target plate, 3d is a mass image showing the spatial distribution of peptide in the assay specimen loaded and dried on the graphene target plate to be.

Referring to FIG. 3B, the generation of peptide ions in the loaded sample spot on the SUS target plate is dependent on the position (position) (which reflects the scattering properties of the matrix-sample crystals). This is consistent with the optical image of 1a. However, 1d on graphene produces MALDI ion across the entire sample spot, which is much more uniform than 1b. This can greatly reduce the sweet spot.

Desorption ionization and mass spectrometry of the present invention can use a known mass spectrometer. For example, the present invention uses a MALDI mass spectrometer (Matrix-Assisted Laser Desorption / Ionization mass spectrometer, ASTA Model IDSys RT MALDI-TOF mass spectrometer) attached with a UV laser (emission wavelength 349 nm) to desorption ionization. And mass spectrometry.

There is no particular limitation on the molecular weight of the mass spectrometer.

In another aspect, it relates to a MALDI mass spectrometry system. Referring to FIG. 2, the mass spectrometry system of the present invention relates to a MALDI mass spectrometer system including a graphene target plate 10, a laser irradiator 20, a sensor 30, and a controller 40.

The laser irradiation unit 20, the sensor 30 and the control unit 40 can be replaced by a known mass spectrometer device. The sensor 30 measures the signal strength of ions.

The controller 40 may obtain a calibration curve through linear regression analysis from an ion signal intensity corresponding to a known molar concentration of a sample. In addition, the control unit may obtain the concentration of the sample from the calibration line and the measured ion signal intensity.

The system of the present invention shows an improved linear correlation between analyte sample concentration versus ionic strength compared to conventional SUS target plates.

Hereinafter, with reference to the accompanying embodiments and the drawings will be described in detail. However, the accompanying examples are only illustrative of the specific embodiments of the present invention, it is not intended to limit the scope of the invention to this.

Example  One

The large-area graphene film (10 cm x 10 cm; MCK Tech Co., Ltd.) produced by chemical vapor deposition on copper foil was purchased, cut into the size necessary for the experiment, and attached to a SUS plate for MALDI using double-sided tape. Was performed.

CHCA was dissolved in 50% acetonitrile and 0.1% trifluoroacetic acid (TFA) and the final concentration was adjusted to 5 mg / mL. A mixture of standard peptide solutions containing Substance P (monoisotopic mass = 1346.7 Da), renin (1759.9) and ACTH 18-39 (2464.2) was prepared, and each peptide was prepared to contain 20 pmol in 1 μl-drop. CHCA solution and standard peptide solution were mixed in equal amounts. A 1 μl-drop solution was dropped onto the graphene layer prepared above with a micropipette and dried at room temperature. Subsequently, mass spectrometry was performed with a MALDI mass spectrometer (Matrix-Assisted Laser Desorption / Ionization mass spectrometer, ASTA Model IDSys RT MALDI-TOF mass spectrometer).

Comparative example  One

The matrix and the sample were prepared in the same manner as in Example 1. The matrix-sample mixture was dropped on a SUS plate and dried at room temperature. Then mass spectrometry was performed.

Example  2

DHB (20 mg / mL) was dissolved in a solvent (acetonitrile: 20 mM NaCl = 8: 2) as a matrix. As analytical sample, glycan solution extracted from serum was used. DHB solution and glycan solution were mixed in equal amounts. A 1 μl-drop solution was dropped onto the graphene layer prepared above in Example 1 with a micro pipette and dried at room temperature. Mass was measured with a mass spectrometer.

Comparative example  2

The matrix and the sample were prepared in the same manner as in Example 2. The matrix-sample mixture was dropped on a SUS plate and dried at room temperature. Then mass spectrometry was performed.

Equipment Information and Experiment Conditions

ASTA Model IDSys RT MALDI-TOF Mass Spectrometer; Pulsed Nd: YLF laser (wavelength 349 nm), beam diameter 200 um; Laser pulse width: ˜5 nanoseconds; Laser intensity: 5 to 10 uJ; Final number of laser shots: 300-600 shots.

3A is an optical image of Comparative Example 1, and 3c is an optical image of Example 1. FIG. Referring to FIG. 3A, it can be seen that matrix-sample crystals are scattered adjacently at a predetermined distance. In particular, FIG. 3A shows that matrix-sample crystals exist visually and clearly separated. 3a can clearly identify the rim of the matrix-sample crystals. In contrast, in FIG. 3C matrix-sample crystals are dense (adjacent) so as to be indistinguishable visually. In 3c, the boundaries of the crystals are not visible.

Figure 3b is a mass image showing the spatial distribution of the peptide in the specimen of Comparative Example 1, 3d is a mass image showing the spatial distribution of the peptide in the specimen of Example 1.

4 is an assay line of the [glu ¹ ] -fibrinopeptide B (GluFib B) and angiotensin II (Angio II) peptides obtained from the graphene plate prepared in Example 1 and the SUS plate prepared in Comparative Example 1. FIG. Referring to Figure 4, the graphene plate shows a correlation (R ² ) of the black line better than the SUS plate.

5A is an optical image of Comparative Example 2, and 5c is an optical image of Example 2. FIG. FIG. 5A shows that matrix-sample crystals exist visually and clearly as compared to 5C. In FIG. 5C the edges of the matrix-sample crystals are dense (adjacent) so as to be virtually indistinguishable.

5b is an optical image showing the spatial distribution of glycan in the specimen of Comparative Example 2, 5d is an optical image showing the spatial distribution of glycan in the specimen of Example 2.

FIG. 6A is a calibration line for lysophosphatidylcholine 18: 0 obtained from SUS plate, and FIG. 6B is a calibration line for lysophosphatidylcholine 18: 0 obtained from graphene plate. Referring to Figure 6, it can be seen that the black line correlation (R ² ) of the graphene plate is superior to the SUS plate.

In the above, preferred embodiments of the present invention have been described in detail, but these are merely for the purpose of description and the scope of protection of the present invention is not limited thereto.

Claims (9)

Base plate; And
A MALDI mass spectrometry plate comprising a single film graphene layer formed on the base plate,
When the droplets (DROP) of the organic matrix-sample mixed solution are loaded onto the single layer graphene layer and dried, the single layer graphene layer interacts with the organic matrix to induce homogeneous crystallization of the droplets,
The organic matrix may be α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), or 4-hydroxy-3-methoxycinnamic acid (4-hydroxy-3). -methoxycinnamic acid, picolinic acid, 3-hydroxy picolinic acid, 2,6-dihydroxyacetophenone, 1,5-dia Minonaphthalene (1,5-diaminonapthalene), 2,4,6-trihydroxyacetophenone (2,4,6-trihydroxyacetophenone), 2- (4'-hydroxybenzeneazo) benzoic acid (2- (4'- hydroxybenzeneazo) benzoic acid), 2-mercaptobenzothiazole, chloro-cyanocinnamic acid, fluoro-cyanocinnamic acid or mixtures thereof MALDI mass spectrometry plate. The MALDI mass spectrometer plate of claim 1, wherein the base plate is a copper substrate, a nickel substrate, an SiO 2 substrate, or stainless steel (SUS). delete Preparing a base plate;
Forming a single layer graphene layer on the base plate;
The forming of the graphene layer may include forming a catalyst layer on the base plate, vapor depositing graphene on the catalyst layer, or removing the catalyst layer,
The forming of the graphene layer may include attaching a large-area graphene film grown on a copper or nickel foil surface to the base plate with a foil or transferring the same to form a graphene layer.
When the droplets (DROP) of the organic matrix-sample mixed solution are loaded onto the single layer graphene layer and dried, the single layer graphene layer interacts with the organic matrix to induce homogeneous crystallization of the droplets,
The organic matrix may be α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), or 4-hydroxy-3-methoxycinnamic acid (4-hydroxy-3). -methoxycinnamic acid, picolinic acid, 3-hydroxy picolinic acid, 2,6-dihydroxyacetophenone, 1,5-dia Minonaphthalene (1,5-diaminonapthalene), 2,4,6-trihydroxyacetophenone (2,4,6-trihydroxyacetophenone), 2- (4'-hydroxybenzeneazo) benzoic acid (2- (4'- hydroxybenzeneazo) benzoic acid), 2-mercaptobenzothiazole, chloro-cyanocinnamic acid, fluoro-cyanocinnamic acid or mixtures thereof MALDI mass spectrometry method. delete delete Preparing analytical specimens by mixing the matrix and analyte;
Loading the assay specimen into the MALDI mass spectrometry plate of claim 1;
Drying the loaded sample at room temperature;
Irradiating the sample with a laser to desorb and ionize the sample;
MALDI mass spectrometry method comprising the step of analyzing the mass of the ionized sample.
delete delete

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