KR101640084B1

KR101640084B1 - Matrix for ionizing n-glycan sample, method for manufacturing the same and method for mass spectrometry using the same

Info

Publication number: KR101640084B1
Application number: KR1020150053053A
Authority: KR
Inventors: 인용하; 박형순; 김양선; 조응준
Original assignee: 주식회사 아스타
Priority date: 2015-04-15
Filing date: 2015-04-15
Publication date: 2016-07-18

Abstract

A matrix for ionizing a sample including N-glycan may comprise: N,N-diisopropylethyl (DIEA), α-cyano-4-hydroxycinnamic acid (CHCA), ammonium dihydrogen phosphate (ADP), and sodium chloride. When a sample is analyzed by a matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) method by using the matrix, there are advantages in which signal homogeneity is high, linearity of intensity relative to concentration of an analysis sample is excellent, reproducibility of intensity between MALDI spots is high, and intensity of ionization of N-glycan increases and matrix noise decreases, compared to those of a conventional technology.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a matrix for ionizing an N-glycan sample, a method for producing the same, and a mass spectrometry method using the same. BACKGROUND ART MATRIX FOR IONIZING N-GLYCAN SAMPLE, METHOD FOR MANUFACTURING THE SAME AND METHOD FOR MASS SPECTROMETRY USING THE SAME,

More particularly, the present invention relates to a matrix for ionization of a sample, a method for producing the matrix, and a mass spectrometry method using the same. More particularly, the present invention relates to a matrix-assisted laser desorption / ionization The present invention relates to a matrix-related technique that can be used for mass spectrometry analysis of a Matrix Assisted Laser Desorption / Ionization Time of Flight (MALDI-TOF) method.

In recent years, research activities have been actively conducted to combine mass spectrometers into diagnostic applications. Matrix Assisted Laser Desorption / Ionization Time of Flight (MALDI-TOF) mass spectrometer is superior to other mass spectrometers in terms of analysis accuracy and data analysis. It is easy to approach, and it is easy to find a biomarker which is an index of a specific disease because it can profile various kinds of molecular substances at the same time with high sensitivity and resolution.

However, the MALDI-TOF mass spectrometer poses a problem of impairing analytical reproducibility when the matrix, which is the ionization energy transfer, is not crystallized uniformly in the course of the analysis and crystallization. This is because the signal is partially deflected in the MALDI spot and forms a hot spot. In this regard, it is known that the ionic liquid matrix maintains the liquid phase even at room temperature and vacuum, thereby not forming a hot spot in the MALDI spot, thereby improving the analytical reproducibility.

On the other hand, N-glycan is an indicator of abnormally glycosylation in the process of carcinogenesis and is known to function as a biomarker for various kinds of cancer. However, up to now, an ionic liquid matrix composition optimized for the purpose of analyzing N-glycan by MALDI method is not known.

Jeffrey A. Crank and Daniel W. Armstrong, "Towards a Second Generation of Ionic Liquid Matrices (ILMs) for MALDI-MS of Peptides, Proteins, and Carbohydrates", J. Am. Soc. Mass Spectrom. 2009, 20, 1790-1800

According to an aspect of the present invention, a matrix favorable for mass spectrometry of N-glycans, a method for producing the same, and a matrix sample processing condition are optimized to provide a matrix assisted laser desorption / A mass spectrometry method capable of quantitative analysis of ionization (Matrix Assisted Laser Desorption / Ionization) method can be provided.

According to one embodiment, the matrix for ionization of the sample comprises N, N-diisopropylethyl (DIEA), alpha -cyano-4- hydroxycinnamic acid (CHCA), ammonium dihydrogen phosphate (ADP), and sodium chloride.

In one embodiment, the ratio of DIEA and CHCA in the matrix is 1: 1. Also in one embodiment, the ratio of ADP and sodium chloride in the matrix is 5: 2.

In one embodiment, the matrix comprises 89-98 wt.% Of the combined DIEA and CHCA relative to the total weight, 1-10 wt.% Of the ADP, and 0.1-1 wt.% Of the sodium chloride.

According to one embodiment, a method of preparing a matrix for sample ionization comprises: producing a first solution comprising DIEA and CHCA; And mixing a third solution comprising the first solution, the second solution comprising ADP and sodium chloride.

In one embodiment, the step of producing the first solution comprises mixing the DIEA solution in which the CHCA is dissolved and the liquid phase such that the molar ratio of DIEA to CHCA is 1: 1. At this time, the step of producing the first solution may include: drying the solution produced by the mixing step; And dissolving the dried material in acetonitrile.

In one embodiment, the mixing of the first to third solutions includes mixing the first solution, the second solution and the third solution in a volume ratio of 2: 1: 1. At this time, the concentration of ADP in the second solution may be 5 mM to 10 mM. Also, the concentration of sodium chloride in the third solution may be 1 mM to 2 mM.

A mass spectrometry method according to one embodiment includes mixing a matrix comprising DIEA, CHCA, ADP, and sodium chloride with a sample solution; Drying the mixed solution; Ionizing the sample by irradiating the dried mixture with a laser; And deriving a mass spectrum of the sample based on the time until the ionized sample reaches the detector.

In one embodiment, the step of mixing the matrix with a sample solution comprises: drying a first fraction of a matrix solution comprising DIEA, CHCA, ADP and sodium chloride on a plate; Injecting a sample solution onto the dried first liquid and drying the dried liquid; And injecting a second fraction of the matrix solution onto the dried sample solution, followed by drying. At this time, the amounts of the first fraction, the sample solution, and the second fraction may be the same.

In one embodiment, the step of drying the mixed solution comprises drying the mixed solution in a gel state.

In one embodiment, the sample comprises N-glycans.

Mass analysis of N-glycan was performed by a matrix assisted laser desorption / ionization time of flight (MALDI-TOF) method using a matrix according to one aspect of the present invention. As a result, it was confirmed that the signal homogeneity was high in the MALDI spot, the linearity of sensitivity intensity versus analytical sample concentration was excellent, and the sensitivity intensity reproducibility between MALDI spots was also excellent. In addition, the addition of ammonium dihydrogen phosphate (ADP) to the matrix increased the ionization sensitivity intensity of N-glycans and decreased the matrix noise, and the 2,5-dihydroxy Compared to a 2,5-dihydroxybenzoic acid (DHB) matrix, the use of other matrices in one aspect of the invention has the advantage of greater sensitivity and less matrix noise.

1 is a flowchart showing a method of manufacturing a matrix according to an embodiment.
2 is a flowchart showing a mass spectrometry method using a matrix according to an embodiment.
FIG. 3 is a graph showing the detection sensitivity when a mass analysis of a sample containing N-glycan (N-glycan) is performed using a combination of various ionic liquids and a solid matrix.
FIG. 4 is a graph illustrating the signal distribution in a MALDI spot obtained as a result of performing Matrix Assisted Laser Desorption / Ionization (MALDI) mass spectrometry of N-glycans using a matrix according to one embodiment. Image.
FIGS. 5A to 5C are graphs showing sensitivity versus concentration of N-glycan when MALDI mass spectrometry of N-glycans is performed using the matrix according to the examples.
6A and 6B are graphs showing the results of mass spectrometric analysis of various types of N-glycans using the matrix according to the embodiments.
7 is a graph showing changes in the intensity of a detection signal according to the concentration of ammonium dihydrogen phosphate (ADP) in the matrix according to an embodiment.
8 is a graph showing a change in intensity of a detection signal when the conventional matrix and the matrix according to an embodiment are used, respectively.
Figure 9 shows a mass spectrum obtained using a conventional matrix and a matrix according to one embodiment.
10 is a graph showing a change in intensity of a detection signal according to the concentration of sodium chloride when a matrix according to an embodiment is used.
11 is a graph showing changes in the intensity of a detection signal depending on the concentrations of sodium chloride and ADP when the matrix according to one embodiment is used.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

1 is a flowchart showing a method of manufacturing a matrix according to an embodiment.

In the present specification, a matrix is a mass spectrometry technique involving ionization of a sample, such as a mass spectrometry of a matrix assisted laser desorption / ionization time of flight (MALDI-TOF) refers to a liquid or solid composition having a structure that is excited and used as a carrier for transferring ionization energy to a sample. Embodiments of the present invention provide an ionic liquid matrix wherein the ionic liquid matrix according to embodiments is selected from the group consisting of N, N-diisopropylethyl (DIEA) Cyano-4-hydroxycinnamic acid (CHCA), ammonium dihydrogen phosphate (ADP), and sodium chloride (NaCl).

The ionic liquid matrix is produced by mixing a solid matrix and an ionic liquid, and can be produced, for example, by mixing the molar ratios thereof equally. The inventors have tested the combination of various ionic liquids and solid matrices for carbohydrate ionization and found that the combination of DIEA and CHCA is the most sensitive to N-glycan analysis. The present inventors have also found that by adding ADP as an additive to a matrix, it is possible to increase the ionization sensitivity of N-glycans and reduce other noise while optimizing the concentration conditions by adding sodium chloride to the matrix and quantitatively analyzing N-glycans To an [M + Na] + ionizable structure (where M can be any atom), to facilitate the formation of the < RTI ID = 0.0 > That is, compared to the case of using only the DIEA / CHCA composition matrix, the addition of sodium chloride and ADP to the matrix of the DIEA / CHCA composition at an optimal composition ratio yields [M + K] + peak Can be removed. This will be described later in detail with reference to FIGS. 10 and 11. FIG.

Referring to FIG. 1, a method of manufacturing a matrix according to an embodiment of the present invention can dissolve powdered CHCA in a solvent for producing a matrix (S11). The solvent may be methanol (MeOH) at a concentration of 100%, but is not limited thereto. Next, the solution in which the CHCA is dissolved can be mixed with the liquid DIEA (S12). During the mixing, the molar ratio of CHCA solution to DIEA solution may be 1: 1, and the solution may turn yellow when the two substances react. Next, the mixed solution may be dried to evaporate methanol (S13). In one embodiment, the drying process may be in vacuum. If it is necessary to store the matrix for a long period of time, it can be stored at a low temperature (for example, minus 20 degrees Celsius) after drying.

If a matrix is to be used, the dried CHCA and DIEA mixture can be dissolved in the solvent (S14). The solvent may be the same amount as the methanol constituting the first solution before drying. The solvent may be, for example, acetonitrile, but is not limited thereto. Meanwhile, a solution in which ADP is dissolved and a solution in which sodium chloride is dissolved can be prepared (S15). For the sake of clarity, the CHCA / DIEA solution is also referred to herein as the first solution, the ADP solution as the second solution, and the sodium chloride solution as the third solution. The second and third solutions can be produced by dissolving ADP and sodium chloride in deionized water (DW), respectively. The second solution may be generated such that the concentration of ADP is 5 mM. Also, the third solution can be produced so that the concentration of sodium chloride is 2 mM. However, the above-mentioned ADP and sodium chloride concentrations are illustrative and may be determined differently in other embodiments. For example, in one embodiment, the ADP concentration of the second solution is between 5 mM and 10 mM. Also in one embodiment, the concentration of sodium chloride in the third solution is from 1 mM to 2 mM.

Finally, a matrix composition according to one embodiment is produced by mixing the first solution, the second solution and the third solution (S15). In one embodiment, the first solution, the second solution, and the third solution may be mixed in a volume ratio of 2: 1: 1, but are not limited thereto. In one embodiment, the ratio of CHCA and DIEA in the matrix composition is 1: 1. Also in one embodiment, the ratio of ADP and sodium chloride in the matrix composition is 5: 2. For example, in a matrix composition according to one embodiment, the molar ratio of DIEA / CHCA: ADP: NaCl is 26.5 mM: 1.25 mM: 0.5 mM, alternatively 53: 2.5: 1. In addition, in the matrix composition, the weight percent of DIEA and CHCA may be 89 to 98% together. Also, the weight percentage of ADP may be 1 to 10%. Further, the weight percentage of sodium chloride may be 0.1 to 1%. For example, in the matrix composition according to one embodiment, the weight% ratio of DIEA / CHCA: ADP: NaCl is 97.99: 1.67: 0.34.

2 is a flowchart showing a mass spectrometry method using a matrix according to an embodiment.

Referring to FIG. 2, the first fraction of the matrix produced as described above with reference to FIG. 1 may be placed on a sample plate and dried (S21). Here, the sample plate corresponds to a substrate for providing a reaction region for pretreatment and ionization of a sample. For example, the sample plate may be a well plate including a plurality of wells, 1 fraction can be injected and dried. The amount of the first fraction may vary depending on the kind of the sample, the shape of the plate, the laser used for subsequent ionization, etc. For example, the amount of the first fraction may be 0.8 μL, but is not limited thereto. In one embodiment, the drying process may be in vacuum.

Next, the sample solution may be placed on the dried first liquid and dried (S22). For example, 0.8 μL of the sample solution equal to the amount of the first fraction may be injected into each well of the well plate and dried. As with the first fraction, the drying of the sample solution may be done in vacuum.

The sample solution may include an analyte containing N-glycans. For example, the sample may be obtained from human or animal blood, and the steps such as serum separation for blood, protein denaturation, glycan separation by enzymes, protein precipitation and elimination, glycan elution, The resulting sample solution can be obtained. The method of pre-processing for extracting glycans from blood or the like is well known in the technical field to which the present invention belongs. Therefore, in order to clarify the gist of the present invention, detailed description is omitted here.

Next, the second fraction of the matrix may be placed on the dried first fraction and the sample solution, followed by drying (S23). The amount of the second fraction may be 0.8 μL, which is the same amount as the first fraction, but in other embodiments the amount of the second fraction may be more or less than the first fraction. As before, the drying of the second fraction may also be done in vacuum. At this time, drying causes the sample mixed with the matrix to maintain a gel state.

Next, the sample can be ionized by irradiating the sample mixed with the matrix with laser (S24). That is, the sample can be ionized by irradiating each well of the well plate with a laser. The laser may be a solid-state YAG laser, but is not limited thereto. As the matrix is ionized by absorbing energy from the laser, the sample can be indirectly ionized through the ion transfer process by the matrix material.

Next, the ionized sample may be moved under an electric field and detected by a detector (S25). The detector may be disposed at a position spaced apart from the plate into which the sample and matrix are injected, and the ions generated from the sample may be accelerated by the electric field and move toward the detector. At this time, the space between the plate on which the ions move and the detector can be kept in vacuum for accurate measurement. Depending on the solvent of the sample, ions having different polarities may be detected. Depending on the solvent, the electric field applied to the ions may be changed to a positive mode or a negative mode.

Next, the mass spectrum of the sample can be derived by measuring the detector arrival time of each ion (S25). The ions generated from the sample are accelerated by the electric field and are separated according to the mass to charge ratio during the movement. Thus, the time for each ion to reach the detector can be used to specify the mass to charge ratio of that ion. By performing the above procedure on all the ions constituting the sample, the mass-to-charge ratio and the intensity of the ions constituting the sample can be derived in a spectrum form.

Fig. 3 is a graph showing the detection sensitivity when a mass analysis of a sample containing N-glycans is performed using a combination of various ionic liquids and a solid matrix.

The present inventors have tested a combination of various ionic liquids and solid matrices to find out which kind of liquid matrix is suitable for analysis of N-glycans for carbohydrate analysis. The ionic liquids tested include DIEA and 1,1,3,3-tetramethylguanidinium hydrogen sulphate (TMG), and the solid matrix comprises CHCA, ferulate, and p-coumaric acid (CA). The two components were combined and tested for the following types: DIEA / CHCA, DIEA / ferulate, DIEA / CA, TMG / CHCA, TMG / ferulate, and TMG / CA. As a sample, a N-glycan standard substance having a mass to charge ratio (m / z) of 1647.6 and a concentration of 10 pmol / μL was used. As a result, as shown in FIG. 3, when the DIEA / CHCA was used as a matrix, the sensitivity intensity of the detected signal was the largest.

4 is an image showing signal distribution in a MALDI spot obtained as a result of MALDI mass spectrometry of N-glycans using a matrix according to one embodiment.

Figure 4 is an image of signal distribution in a MALDI spot using MALDI imaging analysis. MALDI imaging analysis is a technique for visualizing the distribution of signals by imaging the intensity of sensitivity for a specific peak by position. In FIG. 4, a relatively bright region represents a region where the signal is relatively heavily and a relatively dark region represents a region where the signal is relatively weak. As shown, it can be seen that the distributions of the signals obtained using the matrix according to one embodiment are uniform.

FIGS. 5A to 5C are graphs showing sensitivity versus concentration of N-glycan when MALDI mass spectrometry of N-glycans is performed using the matrix according to the examples.

The experimental results shown in FIGS. 5A to 5C are used to determine whether quantitative properties are obtained when a liquid matrix containing DIEA and CHCA is used. The N-glycane standard materials having mass-to-charge ratios of 1257.42, 1485.53, and 1647.59 The concentration was continuously diluted 1/2 times from 10 pmol / μL to prepare a sample such that the lowest concentration was 10 pmol / μL × (1/2) ⁸ . FIG. 5A shows the sensitivity of a detection signal when samples having various concentrations of N-glycans having a mass to charge ratio of 1257.42 are used. FIG. 5B shows a case where samples having various concentrations of N-glycans having a mass to charge ratio of 1485.53 were used 5c shows the sensitivity intensity of the detection signal when samples of various concentrations of N-glycans having a mass-to-charge ratio of 1647.59 are used. As shown in FIGS. 5A to 5C, as the concentration of the N-glycan sample increases, the sensitivity intensity also increases. When the linearity R is measured by analyzing the sample of each concentration, it is confirmed that all the R squares are 0.99 or more I could.

6A and 6B are graphs showing the results of mass spectrometric analysis of various types of N-glycans using the matrix according to the embodiments.

The present inventors have examined whether profile patterns are constant for each MALDI spot when various types of N-glycan samples separated from human serum are subjected to MALDI analysis using the matrix according to the embodiments. From the mass spectra obtained by repeatedly analyzing homogeneous samples in 8 MALDI spots, 13 of the peaks with a signal-to-noise ratio value of 100 or more were arbitrarily selected to see if the sensitivity intensity was constant. 6A and 6B, the abscissa represents the mass-to-charge ratio of N-glycans, and the ordinate axis of FIG. 6A represents the absolute sensitivity. In addition, the ordinate axis of FIG. 6B represents a normalized sensitivity intensity, that is, a value obtained by dividing the sensitivity intensity of each peak by the sensitivity intensity of all the detected peaks. The relative standard deviations of the absolute and normalized sensitivity intensities were 6.77% and 1.65%, respectively, as a result of calculating the relative standard deviation values of the rate of change of the sensitivity values shown.

7 is a graph illustrating a change in intensity of a detection signal according to the concentration of ADP in a matrix according to an embodiment.

The present inventors have confirmed that when ADP is added as an additive to a liquid matrix containing DIEA and CHCA, the sensitivity intensity is increased while the noise due to the matrix is reduced. FIG. 7 shows the sensitivity intensity of 861.05 m / z, which is one of the sensitivity and the noise peak of the base peak when the ADP concentration is 0, 1, 5 and 10 mM, respectively. As shown in the figure, when the ADP concentration is 5 mM, the sensitivity of the main peak is highest and the sensitivity of the noise peak is lower.

However, this is illustrative, and the concentration of ADP can be determined to an appropriate range that reduces the intensity of the noise without the absolute intensity of the main peak being low. For example, the mass-to-charge ratio (m / z) of the N-glycans to be analyzed is generally in the range of 800 to 3000, and the absolute intensity of the noise (861.05 m / z) contained in the analytical range is the lowest, As a less intense condition, the concentration of ADP in one embodiment may be between 5 mM and 10 mM.

8 is a graph showing a change in intensity of a detection signal when the conventional matrix and the matrix according to an embodiment are used, respectively. Figure 9 also shows a mass spectrum obtained using a conventional matrix and a matrix according to one embodiment.

In the past, a 2,5-dihydroxybenzoic acid (DHB) matrix was generally used to ionize an N-glycan sample. FIGS. 8 and 9 show the results of ionization using N-glycan samples separated from human serum using a conventional DHB matrix and ionization using a matrix according to one embodiment. FIG. 9 shows the sensitivity spectrum of the detection signal for the N-glycan of the large harbor, and Fig. 9 shows the mass spectrum of the whole sample. At this time, the matrix solution was prepared so that the concentration of DHB was 20 mg / mL in the DHB matrix, and 12 mM sodium chloride was added. In addition, the matrix according to one embodiment was prepared such that the ADP concentration was 5 mM and the sodium chloride concentration was 2 mM, and the volume ratio of DIEA / CHCA: ADP: NaCl was 2: 1: 1.

Referring to FIG. 8, when a liquid matrix containing DIEA and CHCA is used according to an embodiment of the present invention, the sensitivity is increased as compared with the prior art. Referring to FIG. 9, when a conventional DHB matrix is used, peaks corresponding to the matrix noise appear in the range of 500 to 800 m / z, whereas when the matrix according to an embodiment is used, the noise level is reduced .

10 is a graph showing a change in intensity of a detection signal according to the concentration of sodium chloride when a matrix according to an embodiment is used.

Specifically, Figure 10 shows that for 5 pmol / mu L of standard N-glycans with a mass to charge ratio (m / z) of 1485.5, [M + Na] + and [ M + K] + (where M is an arbitrary atom). In the case of complex samples, many peaks are detected at one time. When the [M + K] + peaks are mixed, the complexity of the peak increases, which is not easy to interpret. Particularly in quantitative analysis, As the peaks are dispersed and appear, it acts as a factor that hinders the quantification. To simplify the complexity of the MALDI spectrum in this example, the addition of sodium chloride to the matrix in which Na and K adducts can all be produced, thereby unifying the adducts with Na, To obtain a simple spectrum.

10, when the concentration of sodium chloride is 0 mM, that is, when only the DIEA / CHCA matrix is used, the ratio of [M + K] + peak is about 70% of the peak of [M + Na] + Value. &Lt; / RTI > However, as the concentration of sodium chloride increases, the ratio of [M + K] + peak decreases gradually, and when the concentration of sodium chloride is above 5 mM, the [M + K] + peak becomes almost invisible. However, as the concentration of sodium chloride increases, the size of the main peak [M + Na] + peak is also reduced, which may lead to problems in peak detection itself. Thus, in one embodiment, the concentration of sodium chloride added to the matrix is from 1 mM to 2 mM, with the absolute intensity of [M + K] + close to zero and the absolute intensity of the main peak not low.

In one embodiment, ADP may be added to the matrix together with sodium chloride to compensate for the decrease in peak intensity, which will be described in detail below with reference to FIG.

11 is a graph showing changes in the intensity of a detection signal depending on the concentrations of sodium chloride and ADP when the matrix according to one embodiment is used.

FIG. 11 shows the absolute intensity of the detection signal according to the concentration of sodium chloride and ADP when sodium chloride and ADP are added together as an additive in the DIEA / CHCA-based matrix for the N-glycan sample described above with reference to FIG. 10 . As described above, the addition of sodium chloride to the matrix can simplify the spectrum, while reducing the absolute intensity of the peak to be detected.

In this embodiment, the absolute intensity of the detection signal can be increased by adding ADP to the matrix together with sodium chloride. 11, when the concentration of sodium chloride / ADP was 2 mM / 0 mM, the absolute intensity was decreased. When the concentration of sodium chloride / ADP was 2 mM / 5 mM, the absolute intensity of the peak again increased . On the other hand, addition of ADP also suppresses peaks near 800 m / z, which may be referred to as noise, to increase the signal-to-noise ratio so as to more clearly detect the peaks of the region to be measured. As described above.

While the invention has been shown and described with reference to certain embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. However, it should be understood that such modifications are within the technical scope of the present invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims

N, N-diisopropylethyl (DIEA),? -Cyano-4-hydroxycinnamic acid (CHCA), ammonium dihydrogen phosphate ; ADP), and sodium chloride,
A first solution containing DIEA and CHCA, a second solution containing ADP at a concentration of 5 mM to 10 mM, and a third solution containing sodium chloride at a concentration of 1 mM to 2 mM were mixed at a volume ratio of 2: 1: 1 A matrix for ionization of the sample.

The method according to claim 1,
Wherein the ratio of DIEA and CHCA in the matrix is 1: 1.

The method according to claim 1,
Wherein the ratio of ADP and sodium chloride in the matrix is 5: 2.

The method according to claim 1,
Wherein the matrix comprises 89 to 98 wt.% Of the combined DIEA and CHCA relative to the total weight, 1 to 10 wt.% Of the ADP, and 0.1 to 1 wt.% Of the sodium chloride.

To produce a first solution comprising N, N-diisopropylethyl (DIEA) and? -Cyano-4-hydroxycinnamic acid (CHCA) step; And
Mixing a first solution, a second solution comprising ammonium dihydrogen phosphate (ADP), and a third solution comprising sodium chloride,
Mixing the first to third solutions comprises mixing the first solution, the second solution and the third solution in a volume ratio of 2: 1: 1,
The concentration of ADP in the second solution is 5 mM to 10 mM,
Wherein the concentration of sodium chloride in the third solution is 1 mM to 2 mM.

6. The method of claim 5,
Wherein the step of generating the first solution comprises:
Mixing the solution in which the CHCA is dissolved and the liquid DIEA in a molar ratio of DIEA to CHCA of 1: 1.

The method according to claim 6,
Wherein the step of generating the first solution comprises:
Drying the solution produced by said mixing step; And
&Lt; / RTI > further comprising the step of dissolving the dried material in acetonitrile.

delete

N, N-diisopropylethyl (DIEA),? -Cyano-4-hydroxycinnamic acid (CHCA), ammonium dihydrogen phosphate ; ADP), and sodium chloride, and a third solution comprising DIEA and CHCA, a second solution comprising 5 mM to 10 mM of ADP at a concentration of 1 mM to 2 mM, : Mixing the matrix prepared by mixing at a volume ratio of 1: 1 with the sample solution;
Drying the mixed solution; And
Ionizing the sample by irradiating the dried mixture with a laser; And
And deriving a mass spectrum of the sample based on the time until the ionized sample reaches the detector.

12. The method of claim 11,
The step of mixing the matrix with the sample solution comprises:
Drying a first fraction of a matrix solution comprising DIEA, CHCA, ADP and sodium chloride on a plate;
Injecting a sample solution onto the dried first liquid and drying the dried liquid; And
And injecting a second fraction of the matrix solution onto the dried sample solution, followed by drying.

13. The method of claim 12,
And the amounts of the first fraction, the sample solution and the second fraction are the same.

12. The method of claim 11,
Wherein the step of drying the mixed solution comprises drying the mixed solution in a gel state.

12. The method of claim 11,
Wherein said sample comprises N-glycans.