JP2010117245A - Mass spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide mass spectrometry capable of performing high-sensitive mass spectrometry of substances of high molecular weight. <P>SOLUTION: The mass spectrometry is performed using a transparent substrate as a substrate 1, preparing a matrix mixture sample with a matrix agent M mixed, as a sample S, supplying the sample S to the front surface 1a of the substrate 1, generating evanescent light Le on the front surface 1a of the substrate 1 by making exciting light Lo incident on the substrate 1 from the back surface 1b of the substrate 1 in such a manner that the exciting light Lo is totally reflected by the front surface 1a of the substrate 1, separating an analysis target substance A from the substrate 1 by irradiation with the evanescent light Le, ionizing the analysis target substance A, and capturing the ionized analysis target substance A. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a mass spectrometry method in which a sample in contact with a substrate surface is desorbed from the substrate surface and ionized, and an analyte in the sample is captured and mass analyzed.

  In analysis methods used for material identification, etc., a mass spectrometry method is known in which a substance to be analyzed attached to the surface of a substrate is desorbed from the substrate surface, ionized, and the substance is identified by the ratio of the mass and charge of the substance. It has been. For example, Time of Flight Mass Spectroscopy (TOF-MS) is a method in which an ionized analyte is made to fly a predetermined distance between high-voltage electrodes and the mass of the substance is analyzed based on the time of flight. is there.

  As one of the desorption and ionization of the analyte in such mass spectrometry, there is a laser desorption ionization method using laser irradiation. In the ionization method using laser light, analysis with a very small amount of sample is possible, but high-power laser light is required. When a high-power laser beam is used, the analyte may be damaged (fragmentation or denaturation occurs), and in particular, desorption and ionization of high molecular weight substances are difficult. As means for solving this problem, matrix-assisted laser desorption / ionization (MALDI) has been proposed and widely known.

  The MALDI method uses a matrix mixed sample in which a sample containing an analyte is mixed with sinapinic acid or glycerin, which is called a matrix agent, and vaporizes the analyte together with the matrix agent using light energy absorbed by the matrix agent. This is a method of ionizing an analyte. The MALDI method is widely used for mass spectrometry of non-volatile substances, biomolecules, and high molecular weight substances such as synthetic polymers as a soft ionization method with little chemical influence on the analyte, such as fragmentation and denaturation. (Patent Document 1, etc.).

  On the other hand, in the MALDI method, since the matrix and the analyte in the sample form a mixed crystal state, the existence ratio of the analyte in the sample changes depending on the location, the sample surface is rough, and the light penetration length is It is difficult to quantify the analyte because it cannot be defined.

In order to discuss the quantitativeness, a method of desorbing and ionizing a sample that does not use a matrix agent is caused by making a laser beam incident from a prism arranged on the back surface of the substrate and totally reflecting it inside the substrate surface. A method and apparatus using evanescent light is proposed in Patent Document 2.
JP 9-320515 A JP 2007-225395 A

  As described above, when the sample is directly irradiated with irradiation light such as a laser beam to desorb and ionize the sample, there is a problem that the analyte is fragmented and denatured.

  FIG. 5 is a diagram schematically showing desorption and ionization of the sample from the substrate 51 by the MALDI method. As shown in FIG. 5, according to the MALDI method, the substance A to be analyzed is mixed with the matrix agent M, and the influence of the direct irradiation of the laser beam L can be partially mitigated. Since not only M but also the analyte A is irradiated at the same time, it cannot be said that the damage suppression by direct irradiation of the analyte A with the laser beam is still sufficient, and a part of the analyte A is fragmented, Desorption and ionization are performed in a state in which denaturation or the like has occurred (indicated by symbol a in the figure).

  FIG. 6 is a diagram schematically showing a form of desorption and ionization of a sample using evanescent light, which is disclosed in Patent Document 2. As shown in FIG. 6, the laser beam L is incident on the sample attached to one surface (sample surface) 61a of the prism 61 so as to be totally reflected by the sample surface 61a from the other surface 61b of the prism 61. Evanescent light Le is generated on the surface 61a, and the analyte A is desorbed and ionized by irradiation with the evanescent light Le. According to this method, it is considered that fragmentation and denaturation of the analyte A can be suppressed as compared with the case of direct irradiation with laser light. However, in the analysis method that presupposes quantitative analysis as in Patent Document 2 and does not use a matrix agent, the evanescent light Le is directly irradiated to the analyte A, and therefore, one of the analytes A is also used. The part is desorbed and ionized in a state in which fragmentation, denaturation, etc. have occurred (indicated by a in the figure), and the problem of damage to the analyte due to direct irradiation of light still remains.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a mass spectrometry method capable of suppressing the damage to the analyte by irradiation light more than before and capable of mass spectrometry of a high molecular weight substance. It is what.

In the mass spectrometry method of the present invention, the analyte in the sample is desorbed from the substrate by being irradiated with light, which is supplied to the surface of the substrate for mass spectrometry and attached to the substrate, and is ionized. In a mass spectrometry method for capturing the ionized analyte and performing mass spectrometry,
As the substrate, a transparent substrate is used,
As the sample, a matrix mixed sample mixed with a matrix agent is prepared,
Supplying the sample to the surface of the substrate;
By causing excitation light to enter the substrate so as to be totally reflected from the back surface side of the substrate, the evanescent light is generated on the surface of the substrate,
The analyte is desorbed from the substrate by irradiation with the evanescent light.

  As the substrate, it is desirable to use a substrate having a light absorbing material, an electric field enhancing material and / or a desorption promoter on the surface of the substrate. In particular, it is desirable to use a nitro compound as the elimination accelerator. Examples of the nitro compound include nitrocellulose, trinitrotoluene (TNT), dinitrotoluene (DNT), nitronaphthalene, nitrate ester, alkylnitratoethylnitramine, nitroguanidine, hexogen, 3-nitro-1,2,4- Examples include triazol-5-one and hexanitrohexazayl soul titanium.

  The mass spectrometric method of the present invention uses a matrix agent mixed sample as a sample, and desorbs and ionizes an analyte by evanescent light generated by total reflection of excitation light. Such a configuration can suppress damage (damage of the analyte, fragmentation, etc.) due to light irradiation to the sample as compared with the case of directly irradiating the matrix agent mixed sample with laser light. Compared with the case where the analyte is irradiated with evanescent light without using it, damage due to light can be suppressed more effectively. According to the mass spectrometric method of the present invention, damage to the sample due to light can be suppressed more effectively than before, so that measurement with higher accuracy than before can be performed and damage due to light is large. Measurement of a substance to be measured having a large molecular weight becomes possible.

  Hereinafter, a mass spectrometry method according to an embodiment of the present invention will be described with reference to the drawings.

  First, an embodiment of a mass spectrometer for carrying out a mass spectrometry method will be described with reference to FIG. The mass spectrometer of this embodiment is a time-of-flight mass spectrometer (TOF-MS). FIG. 1 is a schematic diagram showing a configuration of a mass spectrometer 10 of the present embodiment.

  As shown in the figure, the mass spectrometer 10 is applied to the substrate 1 so that it is totally reflected by the surface 1a of the substrate 1 from the back surface 1b side of the substrate 1 for mass analysis in the box 11 kept in a vacuum. By making the excitation light Lo incident, the excitation light irradiation means 20 including the prism 21 and the light source 22 for generating the evanescent light Le on the surface 1a of the substrate 1, and the substrate 1 is detached from the substrate 1 by the irradiation of the evanescent light Le. An analysis means for detecting the analyte A and analyzing the mass of the analyte A, and disposed between the mass analysis substrate 1 and the analysis means 14 at a position facing the surface 1a of the substrate 1; The lead grid 15 and the end plate 16 disposed to face the surface of the lead grid 15 opposite to the surface on the mass analysis substrate 1 side are provided.

  The substrate 1 for mass spectrometry is composed of a translucent member, and the sample S to be measured is supplied to the surface 1a, and is placed on the one surface 21a of the prism 21 with the sample S attached to the surface 1a. Is.

  In the excitation light irradiation means 20, the prism 21 is a light guide member for causing the excitation light Lo to enter the substrate 1 so as to totally reflect the excitation light Lo at the interface 1 a between the substrate 1 and the sample S, and a light source 22. Is arranged and configured so that the laser light as the excitation light Lo is totally reflected from the other surface 21b of the prism 21 at the surface 1a of the substrate 1 (interface between the substrate and the sample). As the light source 22, for example, a pulse laser light source having a wavelength of 337 nm and a pulse width of about 50 ps to 50 ns is used. A light guide system such as a mirror or a lens for guiding the excitation light Lo emitted from the light source 22 may be provided between the light source 22 and the prism 21 as necessary. Refractive index matching oil is applied on the prism 21, and the prism 21 and the substrate 1 are in contact with each other through the refractive index matching oil. The prism 21 and the substrate 1 may be configured to be inserted into and removed from the mass spectrometer as a sensor chip formed integrally.

  The analysis means 14 detects the analyte A that has been desorbed from the surface 1a of the mass analysis substrate 1 by irradiation with the evanescent light Le, and has flown through the central holes of the extraction grid 15 and the end plate 16. And a data processor 19 for processing an output signal from the amplifier 18. The amplifier 17 amplifies the output of the detector 17.

  A mass spectrometry method according to an embodiment of the present invention using the mass spectrometer 10 having the above configuration will be described below.

  First, as the sample S, a mixed sample of the sample containing the analyte A and the matrix agent M is prepared. As the matrix agent M, a known matrix agent used in the conventional MALDI method can be used. Specifically, nicotinic acid, picolinic acid, 3-hydroxypicolinic acid, 3-aminopicolinic acid, 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, sinapinic acid, 2- (4-hydroxy Phenylazo) benzoic acid, 2-mercaptobenzothiazole, 5-chloro-2-mercaptobenzothiazole, 2,6-dihydroxyacetophenone, 2,4,6-trihydroxyacetophenone, disranol, benzo [a] pyrene, 9-nitro Anthracene, 2-[(2E) -3- (4-tret-butylphenyl) -2-methylprop-2-enylidene] malononitryl and the like can be used as the matrix agent M.

  The substrate 1 on which the sample S is supplied to the front surface 1 a is placed on the one surface 21 a of the prism 21 of the apparatus 10. Thereafter, a voltage Vs is applied to the mass analysis substrate 1, and excitation light Lo that is laser light of a specific wavelength is output from the light source 22 by a predetermined start signal, and from the back surface 1 b of the mass analysis substrate 1 through the prism 21. Incident. The excitation light Lo is incident on the surface 1a of the substrate 1 (interface between the substrate and the sample S) at an angle greater than or equal to total reflection, and is totally reflected on the surface 1a. As a result, evanescent light Le is generated on the surface 1a of the substrate 1, and the sample S is irradiated with the evanescent light Le. The matrix agent M in the sample S absorbs the light energy of the evanescent light Le and converts it into thermal energy. By this thermal energy, the analyte A is vaporized together with the matrix agent M, desorbed from the substrate surface 1a, and at the same time. Ionized.

  The desorbed and ionized analyte A is extracted and accelerated in the direction of the extraction grid 15 by the potential difference Vs between the mass spectrometry substrate 1 and the extraction grid 15, and passes through the central hole in the direction of the end plate 16. The air travels almost straight and then passes through the hole of the end plate 16 and reaches the detector 17 to be detected.

  The output signal from the detector 17 is amplified to a predetermined level by the amplifier 18 and then input to the data processing unit 19. In the data processing unit 19, a synchronization signal synchronized with the start signal is input, and the flight time of the analyte A can be obtained based on the synchronization signal and the output signal from the amplifier 18. A mass spectrum can be obtained by deriving mass from time.

  In the present embodiment, the configuration in which everything is provided in the box 11 has been described. However, at least if the drawer grid 15, the end plate 16, and the detector 17 are arranged in the box 11, the others are outside the box. It may be arranged.

  In the present embodiment, the case where the mass spectrometer 10 is TOF-MS has been described as an example. However, the apparatus for performing mass analysis of ionized sample ions is not limited to the TOF type, but IT (Ion Trap; Ion trap type), FT (ICR) (Fourier-Transform Ion Cyclotron Resonance; Fourier transform type), and QqTOF (Quadrupole-TOF; Quadrupole-TOF type), TOF- A mass spectrometer such as TOF (TOF connection type) can be used.

  Moreover, as mass spectrometry of this invention, it may replace with the substrate for mass spectrometry used in the said embodiment, and may use the substrates for mass spectrometry 2-4 shown in FIGS. 2 to 4 are schematic diagrams showing a method for desorbing an analyte when another mass analysis substrate is used, which is applicable to the mass spectrometry method of the present invention.

  The mass spectrometric substrate 2 shown in FIG. 2 includes a metal thin film 32 that is an electric field enhancing material on the surface of a transparent substrate 31. The material of the metal thin film 32 may be any metal having free electrons, and examples thereof include Au, Ag, Cu, Pt, Ni, and Ti. Au, Ag, and the like having a high electric field enhancing effect are particularly preferable. A sample S to be measured is supplied onto the metal thin film 32. When the mass spectrometry substrate 2 shown in FIG. 2 is used, the excitation light Lo is incident as p-polarized light at a specific angle at which surface plasmons are generated on the metal thin film 32. If such a substrate for mass spectrometry 2 is used, surface plasmon SP is generated by the irradiation of the excitation light Lo, and the electric field on the surface of the substrate 2 is enhanced accordingly, so that the light energy is enhanced and absorbed. Since the action of the matrix agent that converts heat is also enhanced, the analyte A can be effectively desorbed and ionized.

  Note that the electric field enhancing material may include a metal nanostructure that generates localized plasmons when irradiated with light. As such a metal nanostructure, a substrate for mass spectrometry in which a metal mesh, metal nanoparticles, metal nanorods and the like are fixed on the transparent substrate 31 is also preferable because an electric field enhancing effect on the substrate surface can be obtained. The arrangement of the meshes, particles, and rods may be random or regular, but the arrangement pitch is preferably smaller than the wavelength of the excitation light Lo. These metal materials may be any metal having free electrons, and examples thereof include Au, Ag, Cu, Pt, Ni, and Ti. Au, Ag, and the like that have a high electric field enhancing effect are particularly preferable.

  The mass spectrometric substrate 3 shown in FIG. 3 is obtained by applying a desorption accelerator 34 to the surface of a transparent substrate 33. The desorption accelerator 34 may be any agent that assists (promotes) the desorption of the sample S adhering to the explosion caused by the irradiation with the evanescent light Le, and specifically, nitrocellulose. , Trinitrotoluene (TNT), dinitrotoluene (DNT), nitronaphthalene, nitrate ester, alkylnitratoethylnitramine, nitroguanidine, hexogen, 3-nitro-1,2,4-triazol-5-one and hexanitrohexa Nitro compounds such as Azai Soul Titanium. When the matrix agent M in the sample S absorbs the light energy of the evanescent light Le and converts it into thermal energy, the analyte A is vaporized together with the matrix agent M and desorbed from the substrate surface 1a by this thermal energy. At the same time, due to the generation of vanescent light, the desorption promoter 34 explodes, and the desorption of the analyte can be promoted.

The mass spectrometric substrate 4 shown in FIG. 4 is obtained by fixing gold nanoparticles 36 having light absorption and electric field enhancing properties to the surface of a transparent substrate 35 and further applying a desorption accelerator 37. When such a mass analysis substrate 4 is used, localized plasmons are generated by the evanescent light, and the electric field on the substrate surface is enhanced with the generation of localized plasmons, so that the optical energy is enhanced and absorbed. Since the action of the matrix agent that converts heat is also enhanced, the analyte A can be desorbed and ionized effectively, and the desorption promoter 37 is exploded by irradiation with the evanescent light. It is possible to further accelerate the detachment of the sample adhering thereto. In addition, if it is a light absorption material, the effect of a desorption promoter can be further strengthened with the heat | fever accompanying photothermal conversion.
In addition to gold nanoparticles, light absorbing materials include pigments (or dyes, pigments), specifically carbon black, azo dyes, metal complex azo dyes, pyrazolone azo dyes, anthraquinone dyes, phthalocyanine dyes, carbonium. Examples thereof include dyes, quinoneimine dyes, polymethine dyes, cyanine dyes, and other metal nanoparticles, which may be used together with other electric field enhancing materials or by being applied or fixed to the substrate surface alone.

  According to the mass spectrometric method of the present invention, the analyte is desorbed and ionized using absorption of the light energy of the evanescent light by the matrix agent, so that the analyte is damaged (fragmentation, denaturation, etc.). Can be effectively suppressed, and even if the analyte is a hardly volatile substance or a high molecular weight substance, fragmentation or denaturation of the analyte is suppressed compared to the conventional method, and mass spectrometry is performed with high sensitivity. Can do.

Mass spectrometry was performed by the methods of Examples 1 and 2 and Comparative Example 1 shown below using angiotensin, which is a type of polypeptide, as an analyte, and the respective mass spectra were measured. The respective mass spectra are shown in FIG. 7A, FIG. 8A and FIG. FIGS. 7B and 8B are enlarged views of parts (parts surrounded by a dotted line in the figure) of FIGS. 7A and 8A, respectively. In each figure, the horizontal axis is the m / z value (where m is the dimensionless quantity obtained by dividing the mass of the ion by the unit of unified atomic mass, z is the valence of the charge of the ionized analyte) The vertical axis represents the signal value (au) indicating the amount of ions. From this mass spectrum, the intensity (signal intensity at m / z≈1294.5) of angiotensin ion (non-fragmented ionized analyte) signal was determined, and the intensity and ratio were compared.
In each of Examples 1 and 2 and Comparative Example 1, a mixed sample in which an analyte is mixed with a matrix agent is prepared, and a mass spectrometer (autoflex (TM) III MALDI-TOF-MS manufactured by BRUKER) is prepared. A mass spectrum was obtained using. Α-Cyano-4-hydroxycinnamic acid was used as the matrix agent. It mixed with the matrix agent so that the density | concentration of the to-be-analyzed substance Angiotensin I which is a measurement sample might be set to 1 micromol, and it was set as the mixed sample.

(Embodiment 1) As shown in FIG. 1, a matrix mixed sample is supplied onto a transparent substrate, and excitation light is incident from the back side under total reflection conditions to generate evanescent light, and evanescent light is applied to the mixed sample. Irradiated.
Example 2 As shown in FIG. 3, a matrix mixed sample was supplied onto a transparent substrate coated with a desorption accelerator, and the mixed sample was irradiated with evanescent light in the same manner as in the example.
(Comparative example) As shown in FIG. 5, the matrix mixed sample was supplied on the board | substrate, and the laser beam was directly irradiated to the mixed sample.

In the case of the comparative example, as shown in FIG. 9, the amount of angiotensin ions was extremely small as compared with Example 1 and Example 2. This is due to the fact that the sample was much more fragmented compared to Example 1 and Example 2.
A region F on the horizontal axis in FIGS. 7A and 8A is a signal due to fragmented components. In Example 1, the angiotensin ion signal is doubled as compared with the comparative example, and fragmentation is considered to be suppressed as compared with the conventional example. Example 2 also had a high angiotensin ion signal value compared to Example 1, and fragmentation was very effectively suppressed. This indicates that fragmentation can be suppressed by adding a desorption accelerator. In the first and second embodiments, unlike the comparative example in which the laser beam is directly irradiated, since evanescent light is used as the irradiation light, fragmentation is suppressed as compared with the conventional example. By adding the agent, the power of the evanescent light can be reduced, so that it is considered that a further fragmentation suppressing effect could be obtained.

Schematic configuration diagram of a mass spectrometer for carrying out a mass spectrometry method according to an embodiment of the present invention Schematic diagram showing the method for desorbing the analyte when using the second mass spectrometry substrate Schematic diagram showing a method for desorbing an analyte when a third mass spectrometry substrate is used Schematic diagram showing a method for desorbing an analyte when a fourth mass spectrometry substrate is used Schematic diagram showing a conventional matrix-assisted laser desorption method Schematic diagram showing a conventional desorption method using evanescent light Mass spectrum of Example 1 Partial enlarged view of the mass spectrum of Example 1 shown in FIG. 7A Mass spectrum of Example 2 Partial enlarged view of the mass spectrum of Example 2 shown in FIG. 8A Mass spectrum of comparative example

Explanation of symbols

1, 2, 3, 4 Mass analysis substrate 10 Mass spectrometer 11 Case 14 Analysis means 17 Detection means 31, 33, 35 Transparent substrate 32 Metal thin film 34, 37 Desorption accelerator 36 Gold nanoparticle A Analyte a Fragmented (or denatured) analyte M matrix agent S sample L laser light Lo excitation light Le evanescent light

Claims (3)

By irradiating the sample supplied to the surface of the substrate for mass spectrometry with light, the analyte in the sample is desorbed from the substrate, ionized, and the ionized analyte is In a mass spectrometry method for capturing and performing mass spectrometry,
As the substrate, a transparent substrate is used,
As the sample, a matrix mixed sample mixed with a matrix agent is prepared,
Supplying the sample to the surface of the substrate;
By causing excitation light to enter the substrate so as to be totally reflected from the back surface side of the substrate, the evanescent light is generated on the surface of the substrate,
A mass spectrometry method, wherein the analyte is desorbed from the substrate by irradiation with the evanescent light.   The mass spectrometric method according to claim 1, wherein a substrate having a light absorbing material, an electric field enhancing material and / or a desorption promoter is used on the surface of the substrate.   The mass spectrometry method according to claim 2, wherein a nitro compound is used as the desorption accelerator.

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