JP2008108739A - Mass spectroscope and measurement system provided with the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a practical mass spectroscope for proteome analysis. <P>SOLUTION: In a quadrature acceleration type ion trap-coupling time-of-flight mass spectrometer, a range of a mass-load ratio which can be analyzed at one time is enlarged by providing a means to decrease a speed distribution of ion emitted from an ion trap. An efficiency of a protein identification in a proteome analysis is improved. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a time-of-flight mass spectrometer coupled with an ion trap, and more particularly to a mass spectrometer for proteomic analysis.

In the field of proteome analysis, a protein mixture extracted from cells is digested with digestive enzymes, and the obtained peptide fragments are separated by liquid chromatography, and then one type of peptide is selected in a mass spectrometer to induce collision. A so-called shotgun method is widely used in which decomposition is performed by dissociation (CID), the molecular weight of each fragment is determined from the mass spectrum of the decomposition product, and the original protein is identified by collating with a genome database. A technique of selecting and decomposing one kind of ion inside the mass spectrometer and performing mass analysis on the decomposition product is generally called MS / MS analysis. Depending on the type of mass spectrometer, it is possible to select one of the decomposition products generated by MS / MS analysis and further decompose it for mass analysis. Such a sequence can be repeated n times and is generally called MSn analysis.

A quadrupole ion trap mass spectrometer (ITMS) is capable of MSn analysis of n = 3 or more, and has a feature of high sensitivity and efficiency because CID is performed after ions are accumulated in the ion trap. However, in proteomic analysis, a mass-to-charge ratio range of up to about 3000 and a mass resolution of about 5000 or more are desired, whereas an ion trap mass spectrometer generally has a mass-to-charge ratio range and mass resolution of about 2000. Furthermore, since the mass accuracy is also low, the application range is limited, and the protein identification efficiency is low.
Known Example 1 (BMChien, SMMichael and DMLubman, Rapid Commun. Mass Spectrom. 7 (1993
837.) discloses a mass spectrometer in which a quadrupole ion trap and a time-of-flight mass spectrometer (TOFMS) are coaxially coupled. MSn analysis of n = 3 or more using this device
It is possible to use TOFMS with high mass accuracy and high mass resolution in a high mass-to-charge ratio range.

However, in this apparatus, the ion trap and the TOFMS are coaxially coupled, and the ion trap also serves as an acceleration part of the TOFMS. Therefore, collisions between ions and neutral gas for CID frequently occur during acceleration. As a result, ions are scattered, and as a result, it is difficult to obtain high resolution. If the acceleration voltage is increased, ions can be ejected in a short time, so that the scattering is reduced and the resolution is improved. However, since the collision energy is increased, there is a problem that the ions are easily decomposed.
Ions decomposed in the middle of acceleration become chemical noise, causing a lower detection limit.
In the mass spectrometer disclosed in the known example 2 (US Patent 6011259), CID is performed in a multipolar ion guide, ions are discharged from the ion guide, and analysis is performed by orthogonal acceleration type TOFMS. Since the orthogonal acceleration part can be arranged in a high vacuum part, collision with neutral gas during acceleration can be almost ignored. In general, the CID efficiency of a multipolar ion guide is lower than that of an ion trap, but the CID efficiency can be improved to some extent by making the ion guide function as a two-dimensional ion trap (or called a linear trap).
However, since the spatial distribution and energy distribution of ions in the axial direction of the ion guide are large, the accelerated ions diverge, resulting in a low detection sensitivity. Also, unlike a quadrupole ion trap, MSn with n = 3 or more is impossible with a linear trap.

Known Example 3 (C. Marinach, A. Brunot, C. Beaugrand, G. Bolbach, J. -C. Tabet, Proc
eedings of the 49 ^th ASMS Conference on Mass Spectrometry and Allied Topics, Chic
ago, Illinois, May 27-31, 2001) discloses a mass spectrometer in which a quadrupole ion trap and a TOFMS are coupled non-coaxially. In this apparatus, ions are once ejected from the ion trap, and then the ions are accelerated in a direction perpendicular to the axis of the ion trap and subjected to TOFMS analysis.
In this device, while the ions spatially focused on the center of the ion trap are transported from the ion trap to the orthogonal acceleration part as spatially as possible in the axial direction, a substantially continuous ion flow is formed. , Accelerating voltage pulses are continuously applied at a constant period and many times
FMS analysis. Since ions focused spatially and energetically in the ion trap are converted into a continuous ion flow, there is a problem similar to the apparatus of the known example 2 as a result.

B.M.Chien, S.M.Michael and D.M.Lubman, Rapid Commun. Mass Spectrom. 7 (1993) 837.)

As described above, the conventional mass spectrometer has a problem that it is difficult to achieve both a wide mass-to-charge ratio range, high mass resolution, and sufficient detection sensitivity.

In the present invention, the problem is solved by configuring a mass spectrometer by combining an ion trap and an orthogonal acceleration type TOFMS. In the mass spectrometer according to the present invention, ions ejected from the ion trap are transported to the orthogonal acceleration type part, and an acceleration voltage is applied in a direction transverse to the traveling direction of the ions. In the present invention, the mass-to-charge ratio range is controlled by setting the time from when ions are ejected from the ion trap to when the acceleration voltage pulse is applied to a predetermined value.

As a means for ejecting ions from the ion trap, an acceleration electric field may be formed inside the ion trap after the application of the RF voltage for ion accumulation is stopped. When an accelerating electric field is formed with an RF voltage applied, ions in the acceleration region are caused by the spatial distribution of ions inside the ion trap, the kinetic energy distribution of ions inside the ion trap, and collisional scattering with neutral gas. However, if this method is used, such an increase effect is not produced.
Since ions have a certain spatial distribution inside the ion trap, even if the ion ejection means described above is provided, the initial potential when ions are ejected differs depending on the difference in the initial positions of the ions. Ions far from the exit are ejected later than ions near the exit, but the velocity is greater than ions near the exit, so it overtakes it at some position. This position is called a spatial convergence position. By forming an electric field for accelerating ions in the direction of ion travel from the ion trap outlet to the orthogonal acceleration part, the spatial convergence position can be adjusted by the well-known multistage acceleration principle. By optimizing the spatial convergence position based on this principle, it is possible to improve the detection efficiency of ions existing at the end of the acceleration region.

In addition, a means for reducing the ion velocity distribution may be provided while the ions are transported from the ion trap to the orthogonal acceleration unit. The means for reducing the ion velocity distribution may be provided inside or outside the ion trap.
The ions ejected from the ion trap reach the orthogonal acceleration unit with a time difference corresponding to the mass number-to-charge ratio (m / z), but are accelerated by the orthogonal acceleration unit, that is, ions sent to the detector. Are only ions present in the acceleration region at the time when the acceleration voltage is applied. In other words, the mass-to-charge ratio range of ions accumulated in the ion trap is limited by the length of the orthogonal acceleration unit, the length of the detector, etc. There are limitations. Although the mass-to-charge ratio range can be expanded by lengthening the orthogonal acceleration part, the spread of the ion beam in the acceleration region becomes large, and it becomes difficult to realize high resolution over the entire region. In addition, it is necessary to enlarge the detector according to the length of the acceleration region,
Detectors are expensive and have a large size dependency on the price.

Therefore, by providing a means for reducing the velocity distribution width, the mass-to-charge ratio range that can be analyzed by one-time ion accumulation accumulated in the ion trap can be expanded. Such expansion of the mass-to-charge ratio range is particularly useful in proteomic analysis.
Specific examples of means for reducing the velocity distribution of ions include (1) increasing the acceleration electric field inside the ion trap before ions are ejected from the ion trap, or (2) ejecting ions from the ion trap. There is a means for reducing the velocity distribution of ions in the axial direction by changing the electric field in the region between the ion trap outlet and the orthogonal acceleration unit or a part of the region after the ion trap exit.

In addition, as a means for expanding the mass-to-charge ratio range other than reducing the ion velocity distribution, (3) dividing the mass-to-charge ratio range to be analyzed into a plurality of areas, and sequentially analyzing each area and connecting the data (4) Among the ions accumulated in the ion trap, a low mass-to-charge ratio range is analyzed using ion trap mass spectrometry, and the remaining ions are analyzed by orthogonal acceleration type TOFMS. The mass-to-charge ratio range can be further expanded by combining with an ion trap and an orthogonal acceleration type TOFMS.

In an orthogonal acceleration ion trap coupled time-of-flight mass spectrometer as a high-resolution, high-sensitivity MSn device, by expanding the mass-to-charge ratio range that can be analyzed by one-time ion accumulation, practicality in proteomic analysis As a result, the efficiency of protein identification was improved.

(Example 1)
FIG. 1 shows the configuration of a mass spectrometer of the present invention and a measurement system using the same. The apparatus and measurement system will be described by taking proteome analysis as an example. This analysis example is a proteome analysis example for a biological species whose genome has been decoded, and is called a so-called shotgun method. In the shotgun method, the molecular weight of a partial fragment of a protein is determined by mass spectrometry, and the original protein is identified by collating it with an amino acid sequence database translated from a genomic base sequence. First, a protein mixture extracted from cells is decomposed with a digestive enzyme or the like to produce a peptide mixture. A sample solution containing the generated peptide mixture is loaded into an injector of a liquid chromatograph (LC) 60 and injected into the LC flow path. The peptide mixture in the sample is separated according to the molecular weight while passing through the separation column, and reaches the electrospray (ESI) ion source 1 sequentially connected to the end of the LC channel for several minutes to several hours after the sample injection. To do. Note that the ion source is not limited to ESI. The ion source is always in an operating state and is ionized sequentially from the peptide fragment that has reached the ion source.

The generated ions are introduced into the mass spectrometer through the pore 2, pass through the gate electrode 4,
The light enters the ion trap 5 provided in the first vacuum part. Reference numerals 51 and 52 denote power sources connected to the gate electrode. The ion trap consists of a ring electrode 15 and two end cap electrodes 1
6 and 17. A DC power source 43 and a high frequency power source are connected to the ring electrode 15, and DC power sources 41, 42, 44, 45 are connected to the end cap electrodes 16, 17 via a switch 48. It is controlled by the control device 14. Although the gas supply pipe 6 is shown in FIG. 1, it is not necessary in principle.
When accumulating ions, a high-frequency voltage is applied to the ring electrode, and the two end cap electrodes are set to the ground potential. Thereby, a quadrupole electric field is formed inside the ion trap, and ions having a mass number to charge ratio (m / z) or more corresponding to the amplitude of the high-frequency voltage can be captured among the incident ions. After ions are accumulated for about 1 to 100 ms in this way, the voltage of the gate electrode is changed to stop the incidence of ions. In this state, the trapped ions are stabilized for about 0 to 10 ms.

Next, the application of the high-frequency voltage to the ring electrode is stopped, and immediately thereafter, a DC voltage of about 0 to 100 V (rising about 10 to 100 ns) is applied to the ring electrode and the two end cap electrodes to accelerate the electric field inside the ion trap. Form. The accelerated ions are discharged from the ion trap and pass through the pinhole 7 which is the ground potential. The kinetic energy in the axial direction of the ion trap after passing through the pinhole is determined by the potential Vtrap at the center of the ion trap,
It does not depend on the mass number of ions.
Ions that have passed through the pinhole fly at a velocity v determined by M / z · v ² = ² eVtrap and pass through the orthogonal acceleration unit 18. Here, M is the mass of the ion, z is the valence of the ion, and e is the elementary charge.
Therefore, ions with a smaller m / z reach the acceleration part earlier. The orthogonal acceleration unit 18 is composed of two parallel plate electrodes 9 and 10 and is provided in the second vacuum unit 8. While the orthogonal acceleration unit 18 is filled with ions, the two electrodes are at the ground potential, and when the ions are filled, a high voltage pulse (rising 10 to 100 ns) is applied to the acceleration electrode 9. The electrode 10 has a mesh shape for allowing ions to pass therethrough, but the outer peripheral portion has a plate shape, and the entire outer shape is substantially equal to that of the electrode 9. Therefore, ions that enter the orthogonal acceleration portion after the acceleration voltage is applied to the acceleration electrode 9 are immediately accelerated and collide with the outer peripheral portion of the electrode 10 and do not reach the detector. Ions that have passed through the mesh portion of the electrode 10 fly in the drift space 11 of an electric field and enter the reflectron 12,
It reverses inside the reflectron, flies again in the drift space, and enters the MCP detector 13. The use of the reflectron has the advantage that the time spread caused by the spatial spread of ions in the orthogonal acceleration section (related to the acceleration direction) is converged and the resolution is improved, and the apparatus is downsized. By dividing the orthogonal acceleration section into two-stage acceleration electric fields and adjusting the spatial convergence position using the principle of the so-called two-stage acceleration method, the convergence effect by the reflectron can be optimized.

The flight direction of ions incident on the drift space has an angle α with respect to the direction of the acceleration electric field. The flight angle α of ions is determined by the ratio of Vtrap to the initial potential Vacc in the orthogonal acceleration unit m / z
It does not depend on. Therefore, in order to detect all accelerated ions, a detector having a length equal to or greater than the length of the acceleration region is used. The magnitudes of Vtrap and Vacc are, for example, 20 V and 7.5 kV, respectively, where α = about 3 degrees.
At this time, if the ion trajectory is converged using the electrostatic lens 32, the detector can be miniaturized. At the same time, by placing the electrostatic lens 30 between the ion trap outlet and the pinhole,
The detection sensitivity can be improved by increasing the amount of ions passing through the pinhole, and at the same time, since the spread of the ion beam can be suppressed, the resolution is improved. The control unit 14 includes switches 48 and 4
By switching between 9 and 52, a gate electrode, a ring electrode, an end cap electrode,
Controls the magnitude and timing of the voltage applied to the orthogonal acceleration unit.

The time from when ions are ejected from the ion trap to when the pulse voltage is applied to the orthogonal acceleration unit is controlled by a delay circuit provided in the control unit 14. The relationship between the delay time and the m / z range of ions to be detected is determined by the electrode arrangement from the ion trap to the orthogonal acceleration part and the respective electrode voltages when ions are transported from the ion trap to the orthogonal acceleration part. Therefore, the delay time is determined in advance according to the m / z range of the ions to be detected. The control unit 62 is a higher-order control device of the control unit 14, and links the measurement start timing of the detection unit, the operation control of the orthogonal acceleration unit by the control unit 14, and the like.

FIG. 2 shows a voltage sequence applied to each electrode in the case of performing normal MS analysis. After ions are ejected from the ion trap, the voltage of each electrode constituting the ion trap is switched from a DC voltage for forming an accelerating electric field to a voltage for forming a quadrupole electric field. Immediately thereafter (after about 1 μs), the gate voltage is changed to restart the ion implantation into the ion trap. Thereafter, an acceleration voltage pulse is applied to the orthogonal acceleration unit. The pulse width of the acceleration voltage pulse is set slightly longer than the time required for all ions existing in the acceleration region to enter the drift space. This time depends on the mass-to-charge ratio range of ions present in the acceleration region. This mass-to-charge ratio range (hereinafter referred to as mass window) depends on the time (Tacc in the figure) from immediately after the acceleration electric field is formed inside the ion trap to when the acceleration voltage pulse is applied. The mass window is determined by the measurer and entered from the computer keyboard. Maximum value of the mass window Mmax
The ratio Mmax / Mmin between the minimum value Mmin and the minimum value Mmin is constant regardless of Vtrap. Therefore, the measurer is Mmin
(Or Mmax) need only be entered. Alternatively, a method in which a plurality of appropriate mass windows are prepared in advance on the screen of a personal computer and the measurer selects may be used. The acceleration pulse application timing and acceleration pulse width are automatically calculated by software.

Usually, mass spectrum analysis is repeated about 10 to 1000 times to obtain an integrated spectrum. Next, the strongest peak is selected from the MS spectra thus obtained, and MS / MS analysis is performed. This selection is automatically performed by software. MS / MS
In the analysis, first, ions are accumulated in the ion trap in the same manner as in the MS analysis. Next, ions other than the ions corresponding to the selected peak (referred to as parent ions) are excluded from the ion trap, and the parent ions are decomposed by CID. All or part of fragment ions (called daughter ions) generated by decomposition of parent ions are trapped and accumulated in the ion trap. Next, daughter ions are ejected from the ion trap using the same sequence as in FIG.
MS analysis. The above sequence is usually repeated about 10 to 100 times, and the obtained MS /
MS spectrum data is stored in a recording medium. After the analysis of the sample solution is complete, the resulting M
The S / MS spectrum is integrated and the molecular weight of the daughter ions is calculated. In the ESI method, multivalent ions are particularly likely to be generated, so it is necessary to first determine the valence of the ions. Since proteins contain many carbons, the valence of fragment ions can be determined from the interval between isotope peaks due to carbon stable isotopes. Next, the average molecular weight of the daughter ions is determined from the intensity ratio and valence of the isotope peak. The obtained protein is checked against the database to identify the original protein.

Next, the second strongest peak is selected from the MS spectrum, and the MS is similarly selected.
/ MS analysis. Hereinafter, the MS / MS analysis of the nth strongest peak is performed. n is usually about 1 to 5 and is set in advance by the measurer. Until the analysis of the sample solution is completed, the mass spectrometer repeats the above series of measurements. Usually, one MS spectrum measurement and one MS / MS spectrum measurement each take 0.1 to several seconds, and a series of measurements requires a total of several seconds to several tens of seconds. In contrast, each peptide fragment eluted from the LC is introduced into the mass spectrometer for several tens of seconds to several minutes. Therefore, a series of measurements from several times to several tens of times are repeated for one kind of peptide fragment.

FIG. 3 shows a configuration of a quadrupole ion trap suitable for the mass spectrometer of the present invention. This ion trap is composed of four parallel plate electrodes 21 to 24, and two on both ends are end cap electrodes 2.
1 and 24, the middle two are ring electrodes 22 and 23.
In the case of storing ions, a high frequency voltage having the same amplitude, frequency and phase is applied to the two ring electrodes 22 and 23, and the two end cap electrodes are grounded. When ions are ejected, an appropriate DC voltage is applied to the four electrodes to form an accelerating electric field. If a flat quadrupole ion trap is used, a uniform accelerating electric field can be formed. (1) The ion beam spread is small. (2) The spatial focusing position can be easily controlled by two-stage acceleration. (3) There is an advantage that the spatial convergence effect is also good. By setting the spatial convergence position by the two-stage acceleration at or near the detection position, the spread of ions within the detection surface can be reduced, and the decrease in detection sensitivity at the end of the mass-to-charge ratio range can be reduced.

Resonance emission is used as a means for eliminating unnecessary ions other than the parent ion from the ion trap. In resonance emission, an AC voltage having a frequency f is applied between a pair of end cap electrodes. At this time, the trajectory of ions having m / z corresponding to the frequency f is rapidly expanded and excluded from the ion trap. By scanning this frequency f in a predetermined frequency region excluding the vicinity of the frequency f0 corresponding to m / z of the parent ion, ions other than the parent ion are excluded from the ion trap. This resonance emission can be performed simultaneously with trapping and accumulation of ions in the ion trap. In this case, since ion accumulation and elimination of unnecessary ions are performed at the same time, the repetition cycle of analysis is shortened, resulting in improved sensitivity. Instead of scanning the frequency f, the frequency f0
Also, unnecessary ions can be eliminated by superimposing desired frequency components excluding the vicinity thereof and applying them simultaneously. If this method is used, frequency scanning is unnecessary, and thus there is an advantage that the time required for eliminating unnecessary ions can be shortened. As a method for eliminating unnecessary ions, other methods such as superimposing a DC voltage and a high frequency voltage on the ring electrode can be used. However, the voltage operation is complicated, and the method using resonance emission is more practical.

FIG. 4 shows an example of an ion trap control method capable of reducing the ion velocity distribution.
After accumulating ions in the ion trap, the application of the high-frequency voltage is stopped, and then a DC voltage is applied to the two end cap electrodes and the ring electrode to form an accelerating electric field inside the ion trap. At this time, the gradient of the acceleration electric field is increased by gradually changing each electrode voltage from the ground potential. The gradual change of the electrode voltage is performed by a voltage scanning circuit provided in the DC power supply. The voltage scanning circuit realizes arbitrary voltage scanning by setting a maximum voltage value (absolute value) and a time required to reach the maximum voltage value.
When ions are ejected with a constant acceleration electric field, the kinetic energy of the ions ejected from the ion trap is constant. The velocity v of the ejected ions is determined by v = √ (2 · z / M · eV). Here, M is the mass of the ion, and V is the potential at the center of the ion trap. That is, when the acceleration electric field is increased, the kinetic energy of the ejected ions increases as m / z increases. That is, V in the velocity equation is larger as m / z is larger. By appropriately setting the increasing amount and increasing speed of the accelerating electric field, it is possible to expand the mass-to-charge ratio range that can be analyzed at one time, and at the same time to downsize the detector.

FIG. 5 shows schematic diagrams of ion trajectories when (a) the acceleration electric field is not increased and (b) the acceleration electric field is appropriately increased. A similar effect can be realized by increasing the acceleration electric field stepwise.

FIG. 6 shows an ion trap control method in which the acceleration electric field is changed in steps. The method of increasing the accelerating electric field in a stepwise manner has the advantage that the spatial expansion of ions due to the turnaround time can be suppressed.

FIG. 7 shows an example of an apparatus configuration and a control method that can reduce the velocity distribution of ions. An electrode 65 is disposed between the ion trap 5 and the orthogonal acceleration unit 18. The electrode 65 is normally set to a potential such that a decelerating electric field is formed between the electrode 65 and the ion trap outlet side. The application of the RF voltage to the ring electrode is stopped, an acceleration electric field is formed inside the ion trap, and ions accumulated in the ion trap are ejected. While the ejected ions pass through the deceleration electric field, the potential of the electrode 65 is changed, and as shown in the figure, (a) the inclination of the deceleration electric field is decreased, (b) the deceleration electric field is extinguished, or (c) Create an accelerating electric field. By optimizing the amount of change in the deceleration electric field and the timing of the change, the same effect as that shown in FIG. 5 can be realized. Optimal conditions are formulated and written into the measurement software, and the measurer only needs to specify the minimum mass (or maximum mass).

FIG. 8 shows an example of the calculation result regarding the mass-to-charge ratio range expansion effect by the present method. The electrode configuration and voltage control method are as shown in FIG. A flat plate type is used as the ion trap, and a multistage acceleration method for optimizing the spatial convergence position is used. An electrode is arranged after the exit of the multistage acceleration section, and a deceleration electric field is formed between the multistage acceleration section exit (ground potential) and the electrode,
At some timing during the passage of ions, the electrode was changed to the ground potential to extinguish the deceleration electric field. In the figure, (b) shows the calculation result when this method is used, and (c) shows the calculation result when this method is not used, that is, when the electrode is always at the ground potential. The first vertical axis in each figure indicates the position of ions at the timing of applying an acceleration pulse to the orthogonal acceleration section. Here, the position 0 mm is the entrance of the acceleration part,
The position 50 mm corresponds to the exit of the acceleration part. From the figure, it can be seen that when this method is used, ions of m / z 500 to 3100 are present in the acceleration region at the time of application of the acceleration pulse. The ratio of maximum mass to minimum mass (Mmax / Mmin) is 6.2. On the other hand, when this method is not used, ions existing in the acceleration region are m / z 600 to 1600, and Mmax / Mmin = 2.7. That is, the mass window is enlarged by about 2.3 times. The second vertical axis in each figure represents the kinetic energy of ions in the orthogonal acceleration section. The ion trajectory in the TOF portion when the position and kinetic energy obtained by this calculation are used as initial conditions is represented by the ion trajectory analysis software “S
As a result of calculation using "IMION", it was found that when this method is used, the spatial distribution of ions on the detection surface of the detector is within 13 mm. As described above, the spatial distribution on the detection surface when this method is not used is equal to the length of the acceleration region and is 50 mm.
The detector can be downsized to about 1/3.

As an alternative to this method, the same effect can be obtained by using a method in which the potential of the outlet end cap electrode is changed while ions pass between the ion trap outlet end cap and the electrode. . Or you may change both the electric potential of an exit side end cap and an electrode. In short, it is only necessary to change the electric field between the two electrodes so that the ratio of the kinetic energy of the preceding ions and the following ions among the ions flying between the two electrodes becomes small. However, in order to reduce the spread of the ion beam, a method of decelerating preceding ions is preferable to accelerating subsequent ions.

This method is also effective in an orthogonal acceleration type TOFMS using a linear trap (two-dimensional ion trap). As a means for reducing the velocity distribution of ions, a magnetic field can be used in addition to the electric field.
As a means for ejecting ions from the ion trap, a method of forming an accelerating electric field inside the ion trap after the application of the RF voltage for ion accumulation is stopped is used. When an accelerating electric field is formed with an RF voltage applied, ions in the acceleration region are caused by the spatial distribution of ions inside the ion trap, the kinetic energy distribution of ions inside the ion trap, and collisional scattering with neutral gas. However, if this method is used, such an increase effect is not produced.

Since ions have a certain spatial distribution inside the ion trap, even if the ion ejection means described above is provided, the initial potential when ions are ejected differs depending on the difference in the initial positions of the ions. Ions far from the exit are ejected later than ions near the exit, but the velocity is greater than ions near the exit, so it overtakes it at some position.
This position is called a spatial convergence position. By forming an electric field for accelerating ions in the direction of ion travel from the ion trap outlet to the orthogonal acceleration part, the spatial convergence position can be adjusted by the well-known multistage acceleration principle. By optimizing the spatial convergence position based on this principle, it is possible to improve the detection efficiency of ions existing at the end of the acceleration region.

(Example 2)
FIG. 9 shows an example of an analysis sequence according to the segment system of the present invention. In the segment method, the mass-to-charge ratio range to be analyzed is divided into several degrees. Here, the case of analyzing m / z 300 to 3000 using an apparatus of Mmax / Mmin = 2 is illustrated. In this case, the total mass-to-charge ratio range is divided into 300 to 600 (mass window 1), 550 to 1100 (mass window 2), 1000 to 2000 (mass window 3), and 1600 to 3200 (mass window 4). Considering the sensitivity reduction at the end of the mass window, the end of each mass window is overlapped by an appropriate amount. When the mass spectra are connected, the one with the higher intensity among the spectra of each window is adopted as the overlapping mass region.
First, ions are accumulated in the ion trap, ions are then ejected from the ion trap, and an acceleration pulse is applied to analyze the mass window 1. Subsequently, the mass window 3 is analyzed by applying a second acceleration pulse. Next, ions are accumulated again, and mass windows 2 and 4 are similarly analyzed. Even when the number of mass windows further increases, it is possible to analyze the entire region with two ion accumulations by increasing the number of acceleration pulses applied with one ion accumulation. The measurer only needs to set the mass-to-charge ratio range to be analyzed, and the setting of the mass window and the timing of the acceleration pulse are automatically calculated by software.
Since the probability that the daughter ion peak overlaps with the parent ion peak is low, there is little need to analyze the region in the vicinity of the parent ion peak in the MS / MS analysis. In the ion trap, 1 / of the parent ion.
Daughter ions having m / z of 3 or less and 3 or more times are not accumulated in the ion trap. Therefore, if an apparatus with Mmax / Mmin = 3 is used, two regions before and after the vicinity of the parent ion peak may be analyzed by one ion accumulation.

(Example 3)
FIG. 10 shows a configuration of a hybrid apparatus of an ion trap mass spectrometer and an orthogonal acceleration ion trap coupled time-of-flight mass spectrometer according to the present invention.
The apparatus includes a deflection electrode 66 and 67 and a detector 68 for detecting ions deflected by the deflection electrode during the configuration of the orthogonal acceleration ion trap coupled time-of-flight mass spectrometer.
It is comprised by arranging. In the ion trap mass spectrometry, the mass spectrum is acquired by scanning the amplitude of the high-frequency voltage and discharging the ions from the ion trap in order from the smallest m / z. In this hybrid device, a potential difference is applied between the two electrodes of the deflection electrode, and then a high frequency voltage is scanned, and the ejected ions are deflected and guided to the detector. Of the two deflection electrodes, the electrode through which ions pass has a mesh shape. Instead of the mesh electrode, a potential difference can be provided between the incident surface of the detector and the ions can be deflected. This detector may be arranged after the orthogonal acceleration unit. In this case, since the deflection electrode is unnecessary, the apparatus configuration is simple. However, since a pinhole exists in the middle, sensitivity is sacrificed.
Next, the amplitude of the high-frequency voltage is fixed to an appropriate value, the ions remaining in the ion trap for about 0 to 10 ms are stabilized, and the function of the deflection electrode is stopped during that time, and then the TOFMS analysis is performed. In this method, even with an apparatus having Mmax / Mmin = 2, for example, m / z 100-1500 is analyzed by ion trap mass spectrometry, and m / z 1500-3000 is analyzed by TOFMS once. It is possible to analyze a wide region from 100 to 3000 by the ion accumulation. The method can be used in combination with a method of enlarging the mass window by reducing the ion velocity distribution, whereby a wider mass-to-charge ratio range can be measured with high resolution.
In proteomic analysis using the shotgun method, the higher the mass resolution, the more advantageous when determining the valence of daughter ions. However, when selecting a parent ion, resolution as high as that of a daughter ion is not necessary, but rather detection sensitivity is more important. In general, MS rather than MS measurement
/ MS measurement is more sensitive. This is because, in MS / MS measurement, ion accumulation conditions can be set only for the target parent ion, other ions and chemical noise can be greatly reduced during the isolation process, and the parent ion decomposes to lower the molecular weight. This reduces the number of isotope peaks and increases the peak intensity per line. When comparing ITMS and orthogonal acceleration type IT-TOFMS, ITMS may be more sensitive depending on measurement conditions and apparatus configuration. If this hybrid apparatus is used, it is possible to use ITMS for MS spectrum measurement and TOFMS for MS / MS spectrum measurement. As a result, the parent ion selection efficiency is improved, and as a result, the protein identification efficiency is improved.

Example 4
FIG. 11 shows still another configuration example of the mass spectrometer of the present invention. Ions generated by the ion source are introduced into a quadrupole ion trap disposed in the first vacuum unit 3 inside the vacuum apparatus. Ions are trapped in the ion trap and accumulated for a certain time, and then ejected from the ion trap. The ejected ions pass through the pinhole and enter the second vacuum part where the time-of-flight measuring part is arranged. An orthogonal acceleration unit is disposed in the second vacuum unit 8, and an electric field for accelerating ions passing through the pinhole in a direction orthogonal to the axial direction of the ion trap (the direction in which ions are ejected) is provided. Can be formed. An electric field is not initially formed in the orthogonal acceleration portion, and an acceleration electric field is formed by applying a pulse voltage while ions to be detected pass through the orthogonal acceleration portion.

The ion m / z is determined from the time of flight until the accelerated ions reach the detector. A neutral gas (such as helium or argon) is introduced into the ion trap for the purpose of increasing ion trapping efficiency. Therefore, the degree of vacuum inside the ion trap is about 1 mTorr, and the ion trap in the first vacuum part. The external vacuum is about 10 μTorr.
The second vacuum part and the first vacuum part are separated by a partition wall having only a pinhole having a diameter of about 1 to 2 mm, and is a high vacuum of about 0.1 μTorr. Since the acceleration part is located in a high vacuum part of about 0.1μTorr, there is almost no collision with the neutral gas during acceleration or until it reaches the detector after acceleration, thus realizing high mass resolution. Is done.
Since the ions ejected from the ion trap reach the orthogonal acceleration unit in ascending order of m / z of ions, only the ions passing through the acceleration unit are detected when a pulse voltage is applied to the orthogonal acceleration unit. However, in this device, quadrupole ion traps can be used to focus ions in a very narrow region (considered to be about 1 mm or less in diameter) at the center of the ion trap. There is a feature that there is little spatial expansion in the axial direction at, and therefore the detection sensitivity of ions to be detected is high.

(Example 5)
In Example 1, the ion velocity distribution was narrowed by switching the polarity of the voltage applied to the ring electrode and end cap electrode provided in the ion trap from AC to DC.
For the same effect, a means for reducing the ion velocity distribution may be provided outside the ion trap. By providing a parallel electrode connected to a pair of DC power sources outside the ion trap and applying a DC voltage to the ions ejected from the ion trap, an effect of reducing the velocity distribution of ions can be obtained.

The structure of the mass spectrometer of this invention. The voltage sequence in the mass spectrometer of this invention. Configuration of a flat plate quadrupole ion trap suitable for the present invention A first ion trap control method capable of reducing the velocity distribution of ions. The schematic diagram which shows the mass-to-charge ratio range expansion effect by speed distribution reduction of ion. A second ion trap control method capable of reducing the ion velocity distribution. Electrode configuration and control method capable of reducing ion velocity distribution. The calculation result which shows the mass to charge ratio range expansion effect. Explanatory drawing of the segment system of this invention. The structure of the hybrid apparatus of this invention. The structure of another mass spectrometer of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Ion source, 2 ... Fine hole, 3 ... 1st vacuum part, 4 ... Gate electrode, 5 ... Quadrupole ion trap, 6 ... Gas pipe, 7 ... Pinhole, 8 ... 2nd vacuum part, 9 ... acceleration electrode, 10 ... electrode, 11
... Drift space 12 ... Reflectron, 13 ... Detector, 14 ... Control part, 15 ring electrode,
16, 17 ... End cap electrode, 18 ... Orthogonal acceleration part, 19 ... Partition, 21, 24 ... End cap electrode, 22, 23 ... Ring electrode, 30, 32 ... Electrostatic lens, 41, 43, 44
... DC power supply, 42, 45, 47 ... AC power supply, 50, 51, 53 ... Power supply, 48, 49, 52
... switch, 60 ... liquid chromatograph, 61 ... database, 62 ... control unit, 65 ... electrode, 66, 67 ... deflection electrode, 68 ... detector.

Claims (8)

An ion source;
An ion trap for accumulating and ejecting ions generated by the ion source;
Accelerating means for accelerating ions ejected from the ion trap in a direction transverse to the ejected direction;
A first detector for detecting ions accelerated by the acceleration means;
A mass scanning means for discharging a part of the ions accumulated in the ion trap in ascending order of mass-to-charge ratio by sweeping the amplitude of the RF voltage applied to the ion trap;
And a second detector for detecting ions ejected from the ion trap by the mass scanning means.   2. The mass spectrometer according to claim 1, further comprising orbital deflecting means for introducing ions discharged from the ion trap into the second detector. An ion source;
An ion trap for accumulating and ejecting ions generated by the ion source;
Accelerating means for accelerating ions ejected from the ion trap in a direction transverse to the ejected direction;
A detector for detecting ions accelerated by the acceleration means;
Timing control means for controlling the timing of ion ejection from the ion trap;
Linkage control means for linking the acceleration means and the timing control means;
The cooperative control means determines the time between the timing of starting the ejection of ions and the timing of starting the acceleration operation by the acceleration means according to the range of the mass-to-charge ratio of the ions to be measured, Mass spectrometer.   4. The mass spectrometer according to claim 3, wherein the cooperative control means includes a timing for starting ion ejection and a timing for starting an acceleration operation by the acceleration means in order to analyze a plurality of mass-to-charge ratio regions. A mass spectrometer characterized by sequentially changing the time between them.   4. The mass spectrometer according to claim 3, wherein a plurality of mass-to-charge ratio regions are analyzed by repeating the acceleration operation a plurality of times after ion ejection is started, wherein the ion ejection and analysis are performed. The mass spectrometer is characterized in that the timing of the plurality of acceleration operations is different every time one or a limited number of ions are ejected.   4. The mass spectrometer according to claim 3, wherein the cooperation control unit detects a time between a timing at which ion ejection is started and a timing at which the acceleration operation is started every time the acceleration operation is performed. The mass spectrometer is characterized in that the mass-to-charge ratio regions are partially overlapped with each other.   A measurement system comprising the mass spectrometer according to any one of claims 1 to 6 and a liquid chromatograph.   A proteome analysis system comprising the mass spectrometer according to any one of claims 1 to 6 and a database.

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