KR20050079525A - Tandem mass spectrometer and method using it - Google Patents

Abstract

The present invention relates to a two-stage mass spectrometry device and method, comprising: an ionization source for ionizing the polymer sample; A first time-of-flight analysis tube for classifying the same mass clusters based on the time at which the ionized polymer sample has flown, and generating daughter ions of the polymer sample; It includes; a second time-of-flight analysis tube for detecting the mass by changing the flight path of the daughter ion.

According to the present invention, there is an effect that the structural information of the mother ions can be collected by detecting and recording the daughter ions generated by decomposition from one mother ion.

Description

TANDEM MASS SPECTROMETER AND METHOD USING IT

The present invention relates to a two-stage mass spectrometry apparatus and method, and more particularly, by connecting two mass spectrometers in series to ionize a polymer sample such as a biopolymer, and generating a daughter ion by a method such as photolysis, and then mass Or a two-stage mass spectrometer for analyzing a sample for each element.

Mass spectrometry is called ionization of a sample, measurement of its mass to determine its molecular weight and elemental composition, and structural information from it. The mass spectrometer consists of an ion source that makes ions largely, an analyzer that separates ions generated in the ionization source in mass-to-charge ratio (m / z), and a detector and recorder that detects and records the separated ions. Can be distinguished.

In the ionization source, it is necessary to first make gaseous state to make ions. In case of high molecular weight sample, the vapor pressure is low, so if a lot of heat is applied for vaporization, the sample receives a lot of energy and is difficult to analyze.

However, with the recent development of various ionization methods, it is possible to ionize large-volume polymer samples without damage, such as biopolymers, and mass spectrometry, which has the advantage of being able to quickly analyze small amounts of samples, has been widely used in the study of biopolymers. Among the ionization methods for biopolymers, Matrix Assisted Laser Desorption Ionization (MALDI) and Electrospray Ionization (ESI) are the most used. MALDI is an ionization method used in the present invention by mixing an auxiliary material called matrix with a polymer sample to be analyzed and then making a sample in a solid state into a gaseous ion using a laser.

An object of the present invention is to provide a two-step mass spectrometer and a method capable of collecting structural information of a mother ion by detecting and recording daughter ions generated by decomposition from one mother ion.

On the other hand, an object of the present invention is to provide a two-stage mass spectrometer and a method capable of preventing the degradation of the spectrum resolution caused by the flight time of daughter ions by decomposing the mother ions into daughter ions by photolysis. .

On the other hand, it is an object of the present invention to provide an apparatus and method for a two-step mass spectrometry capable of giving sufficient time for the generation of daughter ions while improving the resolution of daughter ions by applying LPQ potential to reflectron. .

The present invention for solving the above problems is a two-step mass spectrometer, an ionization source for ionizing the polymer sample; A first time-of-flight analysis tube for classifying the same mass clusters based on the time at which the ionized polymer sample has flown, and generating daughter ions of the polymer sample; It includes; a second time-of-flight analysis tube for detecting the mass by changing the flight path of the daughter ion.

The ionization source is characterized in that a matrix desorption ionization device (Matrix Assisted Laser Desorption Ionization) using a matrix to mix the matrix with the polymer sample, and then to make the gaseous ions of the solid state sample using a laser.

The laser is characterized in that the nitrogen laser.

The first time-of-flight analysis tube is located between the ionization source and the second time-of-flight analysis tube, a portion to which a predetermined acceleration voltage is supplied to accelerate the ionized polymer sample, and an acceleration voltage is not supplied. It is characterized by being divided into parts.

The first time-of-flight analysis tube further includes an ion gate composed of two electrodes parallel to the traveling direction of the ionized polymer sample, and selectively supplies an electric potential to the ion gate to advance the path of the ionized polymer sample. By selectively changing only the ions to be detected are selectively passed.

The first time-of-flight analysis tube delivers energy to the ionized polymer sample by photodissociation using a laser to generate daughter ions of the polymer sample, and releases the daughter ions to the second time-of-flight analysis tube. It is characterized by.

The second time-of-flight analysis tube is connected to a state in which a predetermined angle is inclined with respect to the incident axis of the daughter ions reflector to reflect the daughter ions to change the flight path; And a detector for detecting daughter ions reflected from the reflectron.

The reflectron includes one or two or more electrodes, and an LPQ (Linear Plus Quadratic) potential is applied to the electrode so that an electric field in a direction opposite to the advancing direction of the daughter ion is formed inside the reflectron. And reflecting the daughter ions entering the reflectron in the inlet direction of the reflectron.

An input electrode is installed at the inlet of the reflectron, and a grid does not exist in the trajectory where the daughter ion enters from the input electrode, and in the trajectory where the daughter ion reflected by the reflecton is emitted from the input electrode. Characterized in that the grid for passing 95% of the daughter ions are installed.

A description with reference to the drawings is as follows.

1 is a structural diagram showing the configuration of a two-step mass spectrometer (hereinafter referred to as an "analyzer") according to an embodiment of the present invention. Referring to this, the analyzer 100 is an ionization source (MALDI Source, 102). , Nitrogen laser 104, first time of flight (TOF) 106, ion gate 108, photolysis laser 110, the first microchannel plate (MCP, 112), the second microchannel plate ( MCP 114, a second time-of-flight analyzer 116, and a preamplifier 118.

1. Mass Spectrometry

The ions exiting the ionization source 102 from the first time-of-flight analyzer 106 are accelerated (accelerated voltage V) to fly over a field free region. At this time, the accelerated energy is the same, so it has a different speed depending on the mass. Thus, the ions arrive at the detector at different times depending on their mass and record the time. Then, the energy (eV) and the flight distance (L) that accelerated the ion are known, so that the mass (m / z) can be obtained from the recorded time.

E = 1/2 m (L / t) ² = eV

2. Tandem Mass Spectrometry

In two-step mass spectrometry, two mass spectrometers (MS) are connected in series, and the ions (parent ions) to be observed with the first-stage MS are decomposed by a suitable method, and then the second stage To detect the fragment ion generated as a result of the decomposition reaction with MS.

If the internal energy when ions are generated is very small, they do not decompose until the ions reach the detector, which appears at the position of the mother ion. On the other hand, those with large internal energy are decomposed before exiting the ionization source, which appears at the position of the daughter ion on the mass spectrometry spectrum. Medium energetic mother ions decompose during flight and are called metastable ions or post source decay (PSD) ions, and in the case of MALDI, they are mainly called PSD ions. Unless a pure substance is analyzed, ions appearing in the normal mass spectrometry cannot be correlated with each other, so structural information cannot be obtained. However, only the daughter ions generated by decomposition in one mother ion are recorded. Get structural information.

The relationship between energy and reaction rate in monomolecular decomposition reactions is well known. As the internal energy increases, the rate constant of the reaction also increases. To obtain the same amount of product, the rate constant must be larger for the same time, or the time must be increased at the same rate constant. For example, suppose that the time that ions travel in a device is fixed at 1 ms. In this case, if the daughter ion signal is small, more energy must be given to increase the rate constant. Alternatively, even with the same energy, the yield can be increased by lengthening the distance that ions travel. However, it should not be overlooked that daughter ions obtained as a result of decomposition reactions can be decomposed again with sufficient energy. By simple theory, we found that the internal energy of the same rate constant is proportional to the molecular weight. Statistically, after the decomposition reaction, the internal energy will be proportional to each molecular weight, so if the energy is increased to increase the yield of the decomposition reaction, the probability of detecting only a small unit of ions by the continuous decomposition reaction increases.

When the first stage MS is a TOF device, it is common for the ion gate 108 to be used to select a desired ion among the various kinds of ions formed in the ionization stage. The ion gate 108 used in the present invention is composed of two plates lying parallel to the traveling direction of the ions. When unwanted ions pass between the plates, an electric field is applied to change the traveling path of the ions so as not to be detected. In addition, when the desired ions pass by, the electric field is eliminated so that the ions are detected without passing through.

The fractionation ability of this simple ion gate is about 100, and the fractionation capacity of the best reported ion gate does not exceed 1000. The separation of only mother ions and recording only daughter ions is called PSD spectrum.

However, PSD ions are generally small in type and amount, and there is not enough information available on the structure of the sample. In order to obtain sufficient information, additional energy must be transferred to induce more decomposition reactions. A widely used method is collision-induced dissociation (CID). This method is used in most commercial devices, where mother ions are decomposed by internal energy due to collision between fast ions and heavy molecules (collision gas).

In the collision-based CID method, the larger the molecular weight of the sample is, the less energy is delivered to the sample. Therefore, multiple collision conditions must be created to transfer the energy required for the decomposition reaction. It is possible to create multiple collision conditions by increasing the pressure of the collision gas, but this is a disadvantage of worsening the vacuum of the device. In addition, it is also a disadvantage that the ions traveling in the beam form under the multi-collision condition have different flight paths due to collisions, and thus the flight time is changed, resulting in reduced spectrum resolution.

Another method of delivering more energy to the sample is laser photodissociation (PD). The photolysis method delivers energy by crossing the laser beam with the ion beam at a desired time and can be tested without the disadvantages of using the collision-induced reaction described above.

A second MS is used for the analysis of the daughter ions, where the selected ions are energized and decomposed. In the present invention, a TOF with an Ion Mirror called Reflectron (116) is used.

In TOF devices, daughter ions have the same kinetic energy as mass ions and mass kinetic energy. The reflectron 116 has an electric field opposite to the direction of ions propagation so that the daughter ions entering the reflectron 116 proceed to the potential energy position corresponding to the respective kinetic energy and then reflect and exit the reflectron 116. At this time, since the energy is different depending on the mass, the flight distance is different, and thus the flight time is different, so that the daughter ions can be classified according to the mass.

The shape of the electric field in Reflectron 116 is selected and made, which determines the properties of Reflectron 116.

The MALDI source 102 uses the Delayed Extraction (DE) method. Nitrogen Laser 104 is used for ionization of the sample.

The ions generated in the source region are separated from each other in time according to mass while flying through the first flight time analyzer 106, which is the first analyzer. The ion gate 108 is located near the first time focusing position to serve to pass only the cluster of desired ions.

The selected ions meet the PD laser 110 at the first time-focused position. In this position, the separation between the different masses is good, and ions of the same mass are gathered together, which is a good place to cross the laser beam. As such, when the ions are concentrated in time at the position where they meet the PD laser 110 and the time intersecting with the PD laser 110 is well controlled, only one mass can be picked and decomposed.

In the conventional method using PD, the pulse width and intensity of the laser are used to maximize the energy transmitted through the laser, which has two disadvantages. Firstly, it is related to the generated ion of decomposition reaction according to energy. If the energy is too high, the decomposition ion is more likely to be decomposed again. As a result, only low mass peptides of amino acids or 2-3 amino acids are obtained. Therefore, the spectra obtained under these conditions do not give enough information about the structure of the selected ion. The design to overcome this is described in the following description of Reflectron 116.

The second disadvantage is that the pulse width of the PD laser 110 is large. In a TOF device in which ions are separated in time with respect to adjacent masses, several mass ions interact with the laser to select only a single isotope during the laser's time duration. Cannot be decomposed into

Fig. 2 is a graph showing the amount of mother ions reduced by the PD laser, recorded by the mass spectrometer.

The dotted line is when it does not meet the PD laser 110, the solid line is when it is met, it can be seen that the mother peptide is reduced by the PD laser (110).

It can also be seen that only one mass can be decomposed by adjusting the time of the PD laser 110. In the case of ions composed of the most abundant isotope, the resolution in the fragment spectrum is improved because the isotope pattern is not seen even when decomposed.

In the present invention, a removable detector is installed at the first time concentration position. Since the time that the PD laser 110 meets ions is 5 ns (5 × 10 ^-9 seconds) or less and the time that ions fly is several tens of microseconds (10 ^-6 seconds), it is necessary to cross the PD laser 110 with the ion beam. Very hard.

 However, knowing the time at that point using a removable detector, it is very easy to cross the PD laser 110 and the ion beam. We can also see that the ions are actually focused at the PD position.

As a second MS, a second time-of-flight analyzer with Reflectron 116 was used.

3 is a structural diagram showing a trajectory of daughter ions reflected by Reflectron.

Many physical properties will vary depending on the shape of the potential placed inside Reflectron 116. The most commonly used is the constant field (linear potential) Reflectron (116).

In general, the ions are installed at a slight inclination with respect to the axis of incidence, and have two time concentration positions with respect to the mother ions, with the ion gate in the first position and the detector 114 in the second position. The Reflectron 116 has a sharply different time-intensive condition depending on the energy, and the resolution becomes worse as the mass gets smaller.

In general, a method of joining only a high resolution region by changing the voltage of the reflectron 116 to obtain a daughter ion spectrum is used.

There is a quadratic potential using Reflectron with completely different characteristics. It was originally designed by Rockwood and called a parabolic reflectron. The equation of motion of ions in reflectron is the same as that of harmonic oscillator, and time concentration is possible regardless of energy. Unlike linear potential reflectrons, the spectrum of daughter ions can be obtained with good resolution at one time, but due to the nature of Reflectron, there is only one time-focused position so that the ion gate, PD laser, detector, etc. must be concentrated in one place. It has the disadvantage that it cannot give enough time to decompose afterwards.

Meanwhile, a linear plus quadratic potential (LPQ) Reflectron was developed that combines these two potentials. This LPQ was first introduced by Yoshida, which did not mention the possibilities in Tandem MS, and also many mathematical functions have limitations for general instrument design, considering the linear potential term to be very small.

Cotter has also reported Reflectron with similar functionality, but its characteristics are very demanding and it's generally not enough to design a device. Using this LPQ Reflectron (116) in mind is not a way to increase the rate constant to increase the daughter ion, but to give more time for the decomposition reaction, it is possible to obtain a definite resolution daughter ion spectrum from various mathematics The equipment obtained was produced.

2-1. LPQ reflectron

d1 and d2 represent the respective distances from the reflectron 116 inlet to the first focusing position 112 and the detector 114. dr is the distance from the entrance of Reflectron 116 to the last electrode. The potential V is at the end of the electrodes, the ions exiting the source, let's assume that when m ⁺ ions with a kinetic energy K in a field free to go into Reflectron (116). At this time, the following equation is obtained according to the energy conservation.

Where t is the position in Reflectron 116 where x = 0 at the entrance of Reflectron 116, the time of flight at x . This equation is mathematically integral and physically meaningful when V is a function of x that is linear (linear reflectron), quadratic (parabolic reflectron), or linear-plus-quadratic (LPQ reflectron). For example, functions such as the number sin ² x can be integrated but are not physically significant. When the LPQ potential is given by Equation 2,

V _r can be thought of as being divided into two parts, V ₁ and V ₂ .

At this time, V ₁ and V ₂ are as follows.

Integrating Equation 1 gives Equation 6.

The total flight time, t _r , in Reflectron 116 is double the time taken to return to the turning point at the entrance to Reflectron 116. Considering the condition that the velocity is 0 (v = 0) at the turning point position and the law of energy conservation, Equation 6 is

Considering the time in the field free area and the time spent in Reflectron 116 before entering Reflectron 116, the time can be written as

This result is basically the same as that obtained by Yoshida. Yoshida used Taylor expansion to find time-concentration conditions for mother ions, but they are generally not applicable due to errors in expansion itself and terms that are ignored during the simplification of the equation. However, the time-intensive condition is that the TOF does not change even with a slight kinetic energy difference that ions can have.

In order to satisfy the condition,

Using equations 4 and 5

For linear reflectrons, V ₂ = 0, so ( d ₁ + d ₂ ) / d _r Is 4 K / eV ₁ and 4 when eV ₁ is K , which is well known as the time focusing condition of the linear reflectron. For parabolic reflectrons ( V ₁ = 0), d ₁ + d ₂ is zero. Equation 11 shows that for ions produced in the source and accelerated to the same potential, the second time concentration position is independent of mass. LPQ Reflectron can be used to improve resolution for normal MALDI spectra.

Now consider the m ₂ ⁺ ions produced by the decomposition of the mother ion, m ₁ ⁺ .

m ₁ ⁺ → m ₂ ⁺ + m ₃

When kinetic energy of m ₂ ⁺ is K ₂ , the kinetic energy of m ₁ ⁺ , K ₁ has the following relationship.

Looking at Equation 11, it is clear that the focusing condition is set for m ₁ ⁺ , but may not be set for m ₂ ⁺ . The condition for m ₂ ⁺ is obtained as follows.

In the case of Parabolic Reflectron ( V ₁ = 0), dT / dK ₂ becomes 0, allowing focusing on all daughter ions.

From the standpoint of daughters, daughters created before entering Reflectron (116) will be observed as signals unless they are further broken in Reflectron (116). On the other hand, ions generated in Refelctron 116 become chemical noise. The ions generated when exiting the reflectron 116 and going to the detector 114 arrive at the detector 114 at the same speed as the mother ion and thus do not affect the daughter ion signal. If the time before Reflectron (116) is t ₁ , the time in Reflectron (116) is t _r , and the time out of Reflectron (116) to detector (114) is t ₂ , t ₁ is equal to t _r + t _2. Keep it as long as possible. This is equivalent to lengthening d ₁ compared to d _r + d ₂ . This increases the daughter's signal and reduces chemical noise.

Since d ₁ + d ₂ is constant at the same Reflectron potential, it is advantageous to attach d ₂ very small, ie the second time concentration position very close to the entrance of Reflectron 116, in order to increase the signal of the PD. It is not possible to increase the ratio of d ₁ / d _r very largely to increase the signal, because the resolution of daughter ions is poor, as shown in Eq.

Equation 14 shows that reducing the linear portion can increase the resolution of daughter ions, which is close to the parabolic situation where the value of ( d ₁ + d ₂ ) / d _r decreases. In other words, good signal-to-noise (S / N) ratio and good resolution of daughter ions cannot be satisfied at the same time.

2-2. Increasing S / N

Most of the noise is chemical noise that disintegrates inside Reflectron 116 and appears at an unspecified location. To reduce this, we made Reflectron (116) slightly smaller and increased d ₁ to make the signal larger. However, reducing the length of Reflectron 116 and increasing d ₁ were both in the range of maintaining the proper resolution because the resolution of daughter ions decreased.

4 is a graph showing the spectrum of PSD and PD ions by the mass spectrometer, and FIG. 5 is a graph showing the spectrum of PSD and PD ions according to the prior art.

Comparing the spectrum obtained by adjusting the power of the laser with the spectrum published by Reilly in 2003, we can see the difference between increasing the speed constant with a lot of energy and raising the signal with a lot of time.

FIG. 6 is a graph showing the respective spectra when the Parabolic potential and the LPQ potential are applied to the Reflectron 116.

7 is a structural diagram showing the structure before and after the improvement of the reflectron inlet electrode, which will be described with reference to this.

Since the ions and heavy constituents colliding with the grids 202 and 204 of the reflectron 116 are another source of noise, a reflectron base is introduced that covers the unnecessary part of the ion without the grid.

Here, the grid 204 does not exist in the trajectory of the ions incident on the reflectron 116, whereas the grid 204 exists in the trajectory of the ions reflected and reflected back from the reflectron 116.

Although a two-step mass spectrometer and method according to an embodiment of the present invention have been described above, the scope of the present invention is not limited to these embodiments, and the scope of the invention can be easily invented by those skilled in the art.

According to the present invention, there is an effect that the structural information of the mother ions can be collected by detecting and recording the daughter ions generated by decomposition from one mother ion.

In addition, according to the present invention, by decomposing the mother ions into daughter ions by the photolysis method, there is an effect that the resolution degradation of the spectrum caused by the flight time of the daughter ions can be prevented.

In addition, according to the present invention, the LPQ potential is applied to the reflectron to improve the resolution of the daughter ions while providing sufficient time for the daughter ions to be generated.

1 is a structural diagram showing the configuration of a two-step mass spectrometer according to an embodiment of the present invention.

Figure 2 is a graph showing the amount of mother ions reduced by photolysis, recorded with a mass spectrometer.

3 is a structural diagram showing a trajectory of daughter ions reflected by reflectron;

4 is a graph showing the spectra of PSD and PD ions by a mass spectrometer;

5 is a graph showing the spectrum of PSD and PD ions according to the prior art;

Figure 6 is a graph showing the spectrum of each when applying the parabolic potential and LPQ potential to the reflectron.

Fig. 7 is a structural diagram showing the structure before and after the improvement of the reflectron inlet electrode;

Claims (9)

In the mass spectrometer for measuring the mass and element composition of the polymer sample, An ionization source for ionizing the polymer sample; A first time-of-flight analysis tube for classifying the same mass clusters based on the time at which the ionized polymer sample has flown, and generating daughter ions of the polymer sample; And a second time-of-flight analysis tube for changing the flight paths of the daughter ions and detecting the masses by mass. The method of claim 1, The ionization source is Mixing a matrix with the polymer sample, and then using a laser laser desorption ionization device (Matrix Assisted Laser Desorption Ionization) using a matrix to make a gaseous state of the solid state sample using a laser. The method of claim 2, The laser A two-step mass spectrometer, which is a nitrogen laser. The method of claim 1, The first flight time analyzer Located between the ionization source and the second time-of-flight analysis tube, Two-step mass spectrometer, characterized in that divided into a portion that is supplied with a predetermined acceleration voltage for accelerating the ionized polymer sample, and a portion that is not supplied with an acceleration voltage. The method of claim 4, wherein The first flight time analyzer Further comprising an ion gate consisting of two electrodes parallel to the traveling direction of the ionized polymer sample, And selectively supplying a potential to the ion gate to change an advancing path of the ionized polymer sample to selectively pass only ions to be detected. The method of claim 5, The first flight time analyzer Two-step mass, characterized in that by generating energy to the ionized polymer sample by the photodissociation method using a laser to generate daughter ions of the polymer sample, the daughter ions are released to the second time-of-flight analysis tube Analysis device. The method of claim 6, The second flight time analyzer A reflectron connected in a state in which a predetermined angle is inclined with respect to the incident axis of the daughter ions to change the flight path by reflecting the daughter ions; And a detector for detecting daughter ions reflected from the reflectron. The method of claim 7, wherein The reflectron is One or more electrodes, An LPQ (Linear Plus Quadratic) potential is applied to the electrode such that an electric field in a direction opposite to the advancing direction of the daughter ions is formed inside the reflectron. A two-step mass spectrometer, characterized by reflecting in the inlet direction of the reflectron. The method of claim 8, An input electrode is installed at the inlet of the reflectron, The grid does not exist in the trajectory where the daughter ion enters from the input electrode, and a grid that passes 95% of the daughter ion is installed in the trajectory where the daughter ion reflected by the reflecton is emitted from the input electrode. Two-step mass spectrometer, characterized in that.