Patent Grant
7034287
This application claims priority under 35 USC §119 to Japanese patent application P-2003-045345 filed Feb. 24, 2003, the entire disclosure of which is hereby incorporated herein by reference.
The present invention relates to a mass spectrometric approach, and in particular to a mass spectrometer combining an ion trap and a Time-Of-Flight Mass Spectrometer (TOFMS) together and a mass spectrometric method.
Against the background of an advance in genome-sequence research, attention has been shifted to proteome analysis, in which proteins expressed in living bodies are exhaustively analyzed. Mass spectrometry is a high-sensitivity and high-throughput protein identification method and considered to be one of major approaches for proteome analysis.
Proteome is analyzed by following the procedure described below. First, the molecular weights of peptide fragments resultant from enzyme-catalyzed digestion of protein are measured. Then, the resulting peptide fragments are further dissociated in a mass spectrometer to measure the molecular weights of individual fragments. The molecular weights of original peptide fragments and of their fragments are searched in a database to identify the target protein.
A method for dissociating sample molecules in the mass spectrometer and analyzing the masses of the fragments thereof is called a MS/MS analysis, an essential approach for proteome analysis.
As one of mass spectrometers capable of MS/MS analysis, an ion trap mass spectrometer is well known. (See, for example, Patent document 1, U.S. Pat. No. 2,939,952.) In this ion trap mass spectrometer, RF voltage is applied between a ring electrode and a pair of end cap electrodes composing the ion trap, forming a quadrupole field in the ion trap to trap and store ions. At that time, the introduction of a neutral gas, for example helium gas, causes kinetic energy of ions to be lost because the ions coming into the ion trap collide against the introduced gas, improving efficiency of ion trapping. After being stored, the ions are ejected from the ion trap starting from one having the smallest m/z ratio by scanning the amplitude of RF voltage and detected, forming a mass spectrum (MS spectrum).
MS/MS analysis is performed using an ion trap mass spectrometer by following the procedure described below. First, ions are stored in the ion trap and by following the procedure described above, a mass spectrum is formed. The ion to be dissociated (a precursor ion or parent ion) is selected among those on the resulting mass spectrum. Then, after being stored again in the ion trap, all the ions excluding the parent ion are ejected from there. This step is commonly called isolation.
As one of the parent ion isolation methods, such a method that auxiliary AC voltages are applied to two endcap electrodes. When the amplitude of auxiliary AC voltage exceeds a certain level, the orbits of the ions go into the unstable state, and the ions are ejected from the inner space of the ion trap.
Next, the parent ion remaining in the ion trap is dissociated. Ion dissociation is commonly performed with Collision Induced Dissociation (CID) With CID, auxiliary AC voltage is applied to two end cap electrodes to increase the kinetic energy of the parent ion, causing it to collide and to dissociate it against a neutral gas, for example, helium gas, which is introduced in the ion trap as a target gas. The target gas also serves as a buffer gas for improving ion trapping efficiency.
Since all of or part of the fragment ions resultant from CID remain trapped and stored in the ion trap, finally the mass spectrum of fragment ions (MS/MS spectrum) can be obtained by scanning the RF voltage to eject the fragment ions stored in the ion trap from there starting from one having the smallest m/z ratio and detect them.
With the ion trap mass spectrometer, the MSn (n>2) analysis can be performed, in which the parent ion is further selected among the fragment ions and dissociated into smaller fragments to analyze the masses of them. The MSn analysis provides such an advantage that more detailed information on the structure of the original ion can be obtained. The MSn analysis is performed by following the procedure described below. First, the MS(n−1) analysis is performed and the parent ion is selected among those on the resulting mass spectrum (MS(n−1) spectrum) Next, the steps up to the immediately before the step for obtaining an additional MS(n−1) spectrum are repeated. After ion isolation and dissociation, the mass spectrum (MSn spectrum) of the resultant fragments is obtained.
Such a structure that a quadrupole filter is disposed at the front of the ion trap is known (see, for example, Patent document 2, U.S. Pat. No. 5,572,022). In this structure, ions can be isolated inside the quadrupole filter, enabling ion storage and isolation to be performed simultaneously, which improves the duty ratio for ion trapping and resultantly the detection sensitivity in MS/MS analysis.
Such a mass spectrometer is known that the ion trap and a TOFMS are combined in the direction orthogonal to the direction of ion traveling (see, for example, Patent document 3, JP-A No. 297730/2001). With this type of mass spectrometer, ion storage, ion isolation, and CID are performed at the ion trap and the masses of the ions are analyzed in the TOFMS. Mass analysis is performed by following the procedure described below. After the ions are stored in the ion trap, the application of RF voltage is stopped and an electrostatic field is formed to eject the stored ions. The ejected ions go into the inside of the TOFMS, where is being pumped to a high vacuum. Then the ions are accelerated by an electric field orthogonally to the direction of ion travel and the time-of-flight of the ions are measured.
As mentioned above, in the case of an ion trap mass spectrometer, a neutral gas must have been introduced in the ion trap for two purposes, one being the improvement of ion trapping efficiency and the other being the achievement of CID. The pressure of this neutral gas may affect not only ion trapping efficiency and CID efficiency but also mass resolution of the mass spectrum and isolation resolution.
The duty ratio of the ion trap of a prior art (Patent document 1) is calculated as follows, considering a typical assumption that 100 ms is required for ion storage, 20 ms for isolation, 30 ms for CID, and 200 ms for mass analysis, respectively;
(100 ms)/(100 ms+20 ms+30 ms+200 ms)=0.285.
According to a prior art (Patent document 2), because ion storage and isolation can be simultaneously performed, the duty ratio is calculated as follows:
(100 ms)/(100 ms+30 ms+200 ms)=0.303.
In this case, the duty ratio is slightly improved from 0.285 to 0.303. Moreover, since only parent ion is introduced into the ion trap, the injected ions/unit period of time can be reduced and therefore the period of time for storing ions until the ion trap is filled with ions can be increased. As the result, the duty ratio and the detection sensitivity can be improved.
For example, if the period of time for storing ions until the ion trap is filled with ions can be prolonged to 500 ms, the duty ratio will be improved to the value obtained from the formula below:
(500 ms)/(500 ms+30 ms+200 ms)=0.684.
For this reason, it is expected that the sensitivity can be improved by a factor obtained from the formula below;
0.684/0.285=2.4.
From the descriptions above, it can be known that the main cause for deterioration in duty ratio in the ion trap mass spectrometer is a relatively long dead-time, about 200 ms, during mass analysis.
According to a prior art (Patent document 2), however, the dependency of mass resolution, CID efficiency, and ion trapping efficiency on the gas pressure are identical to those for the ion trap mass spectrometer disclosed in Patent document 1 and no gas pressure cannot be attained for providing all of acceptable performances. For this reason, the gas pressure is set within the same region as that of the ion trap mass spectrometer according to the prior art (Patent document 1).
In the system according to the prior art (Patent document 3), the subject of improving the duty ratio described in Patent document 2 has been spontaneously solved without a quadrupole filter disposed at the front of the ion trap, thanks for high speed of TOF mass spectrometry. This means that even if 100 ms is required for ion storage, 20 ms for ion isolation, and 30 ms for CID, respectively, only less than 1 ms is needed for mass analysis in the TOF system. For this reason, since the duty ratio has already reached to the level obtained from the formula below: (100 ms)/(100 ms+20 ms+30 ms+1 ms)=0.662, without a quadrupole filter disposed. Even if the duty ratio is increased toward a value 1 by omitting the isolation time or prolonging the ion storage period of time as the result of disposing a quadrupole filter at the front of the ion trap, the effect of the sensitivity improvement is small against the new problems of more complicated instrument and increased cost, which occur by the disposition of the quadrupole filter. Consequently, in the system according to the prior art (Patent document 3), no quadruple filter needs to be disposed at the front of the ion trap TOF analyzer only from the knowledge of the improvement of the duty ratio of the system according to the prior art (Patent document 2).
On the other hand, the mass resolution of the TOFMS becomes higher as the initial ion state, namely ion dispersion in the space and energy distribution at the moment of voltage being applied to form a electric field for ion acceleration, are smaller in the direction of accelerating ions. The ion dispersion in the space and the energy distribution are smaller as the gas pressure becomes higher inside the ion trap. That is because since the ion dispersion in the space and the energy distribution are smaller as the gas pressure inside the gas trap becomes higher, the ion dispersion in the space and the energy distribution can be easily controlled in the direction orthogonal to the ion ejected from the ion trap. Thus, the mass spectrometer according to the prior art (Patent document 3) has a feature that higher mass resolution is attained as the gas pressure inside the ion trap becomes higher, contrary to the ion trap mass spectrometer.
However, like the ion trap mass spectrometer, the isolation resolution 104 deteriorates as the gas pressure becomes higher. For this reason, the system according to the prior art disclosed in Patent document 3 has a problem that no gas pressure can be attained for providing all of optimal isolation resolution 104, ion trapping efficiency 102, mass resolution 103, and CID efficiency 101.
As described above, with the ion trap mass spectrometer, a neutral gas, for example helium gas, must have been introduced into the ion trap serving as both of a target gas for CID and a buffer gas for improving ion trapping efficiency. Either of CID efficiency and ion trapping efficiency depends on the gas pressure and has optimal values.
On the other hand, mass resolution and isolation resolution deteriorate as the gas pressure becomes higher. For this reason, no gas pressure can be attained for providing all of approximately maximum ion trapping efficiency, mass resolution, isolation resolution, and CID efficiency simultaneously.
In the system according to the prior art (Patent document 2), isolation resolution does not depend on the gas pressure inside the ion trap because a quadrupole filter is disposed at the front of the ion trap for isolating ions there. No gas pressure, however, can be attained for providing all of approximately maximum ion trapping efficiency, mass resolution, and CID efficiency simultaneously.
The mass spectrometer according to the prior art combining the ion trap and the TOFMS together (Patent document 3) has a feature contrary to the instruments according to the prior arts (Patent document 1) and (Patent document 2) in that mass resolution is more improved as the gas pressure becomes higher. Nevertheless, no gas pressure can be attained for providing all of maximum ion trapping efficiency, mass resolution, and isolation resolution simultaneously.
The object of the present invention is to provide a mass spectrometer combining an ion trap and a TOFMS together, which can provide approximately maximum CID efficiency, mass resolution, and CID efficiency simultaneously, and a mass spectrometric method using it.
To attain the object described above, according to the present invention, in a mass spectrometer, which has the ion trap and the TOFMS combined together non-coaxially, for example in the direction orthogonal to the direction of ion ejection from the ion trap, a mass filter (for example, a quadrupole filter) is disposed at the front of the ion trap for isolating ions there. The gas pressure inside the mass filter and the gas pressure inside the ion trap are controlled independently, the gas pressure inside the mass filter being optimized for maximizing isolation resolution and the gas pressure inside the ion trap being optimized for approximately maximizing all of ion trapping efficiency, mass resolution., and CID efficiency simultaneously.
According to the present invention, the mass spectrometer is structured so that it has a 3D quadrupole ion trap for ejecting the ions, after ions generated at an ion source are stored in the ion trap for a certain period of time, and a TOFMS for accelerating the ions ejected from the ion trap orthogonal to the direction of the ejection and measuring the time-of-flight of the accelerated ions, wherein a mass filter is disposed between the ion source and the ion trap, and the gas pressure inside the ion trap and the gas pressure inside the mass filter are controlled independently.
The gas pressure inside the trap is set to a higher level than that inside the mass filter, the ions being passed through the mass filter and stored in the ion trap are dissociated therein, and the masses of the fragment ions resultant from ion dissociation are analyzed using the TOFMS. The mass filter may be comprised of three-stage of quadrupoles, the gas pressure in the second-stage of quadrupole is controlled to the lower level than those of the first-stage and third-stage quadrupoles. Among the peaks on the mass spectrum which has the intervals between neighboring peaks exceeding the value pre-determined based on the isolation resolution of the mass filter on the mass spectrum, is selected and the ion corresponding to the selected peak is isolated at the mass filter. The selected peak is displayed on the monitor screen.
The mass spectrometric method of the present invention includes a step for generating sample ions at an ion source, a step for ejecting the ions, after storing ions generated at the ion source in the 3D quadrupole ion trap for a certain period of time, a step for analyzing the masses of the ions and/or fragment irons resultant from ion dissociation using the TOFMS, which accelerates the ions ejected from the ion trap in the direction orthogonal to the direction of the ejection, and a step for controlling the gas pressure inside the mass filter disposed between the ion source and the ion trap and the gas pressure inside the ion trap independently.
In the controlling step, the gas pressure inside the ion trap is set to a higher level than that inside the mass filter. The method of the present invention includes a step for dissociating the ions stored in the ion trap through the mass filter therein to produce fragment ions resultant from ion dissociation. Moreover, it may have a mass filter comprised of three-stages quadrupoles and further include a step for controlling so that the gas pressure inside the second-stage quadrupole to lower level than that inside of the first-stage and third-stage quadrupole. Further, it may include a step for selecting mass spectral peaks, which has the intervals between neighboring peaks exceeding the value pre-determined based on the isolation resolution of the mass filter, among the peaks on the mass spectrum and a step for isolating the ion associated with the selected peak at the mass filter, wherein the selected peak is displayed on the monitor screen.
Samples are ionized at an atmospheric-pressure ion source 1. The ions generated at the ion source 1 go into a first vacuum chamber 3 through a sampling orifice 2 and then into a second vacuum chamber 4. The ions go through a mass filter (for example, a quadrupole filter) 8 disposed inside the second vacuum chamber 4 and a gate electrode 19. Next, the ions go into a third vacuum chamber 5 and then into a 3D quadrupole ion trap 9 disposed inside of it. At that time, voltage has been applied to the gate electrode 19 for providing the ions to go through there.
Inside the ion trap, a neutral gas (for example, helium, nitrogen, or argon) has been introduced through a tubing 17 and the ions passing into the ion trap are trapped around its center while losing their kinetic energy though repeated collisions against the neutral gas. The gas pressure inside the ion trap can be controlled by adjusting a flow rate of gas using a valve 15.
The quadrupole filter 8 is disposed inside a housing 20, inside which the neutral gas (for example, helium, nitrogen, or argon) is introduced through a gas tube 16. Since the quadrupole filter 8 may improve the rate of ion introduction into the ion trap 9 by focusing ion beams, a certain level of gas pressure is required. The gas pressure inside the quadrupole filter 8 can be controlled by adjusting the gas flow rate of a gas tube 16 using a valve 14.
After the ions being introduced into the ion trap 9 and stored for a certain period of time, a DC power source 51 is changed to a DC power source 50 using a switch 52 to set the voltage applied to the gate electrode 19 to a level, at which the ions cannot pass through there, stopping ion introduction into the ion trap 9.
The ion trap is comprised of a pair of end cap electrodes 23 and 25, and a ring electrode 24. During ion storage RF voltage is applied to the ring electrode and the potentials at the end cap electrodes are at 0 V level. When the masses of the ions stored in the ion trap are analyzed, a switch 48 is used to change from a AC power source 42 to a DC power source 41, from a RF power source to a DC power source 43, and the AC power source to the DC power source 41, respectively, stopping the application of RF voltage to the ring electrode 24 and at the time, appropriate DC voltages are applied to two end cap electrode 23 and 25, and the ring electrode 24, respectively to form a electrostatic field for ejecting the ions.
The ions are ejected from the ion trap and come into a fourth vacuum chamber 7. The ions coming into the fourth vacuum chamber fly in the inner space of an orthogonal accelerating element 18 disposed therein. During ion passing through the inner space of the orthogonal accelerating element 18, a switch 49 is used to change a DC power source 47 to a DC power source 46 to apply about 1 kV to 10 kV of pulse voltage to an accelerating electrode 21, which accelerates the ions in the electric field in the direction orthogonal to the direction of ion traveling. The accelerated ions are further accelerated between electrodes 22 and 11, flying in a field-free space defined by the electrode 11, and come into a reflectron 12.
Note that the first vacuum chamber 3, the second vacuum chamber 4, the third vacuum chamber 5, and the fourth vacuum chamber 7, is exhausted of the air independently.
The ions are reversed in the reflectron 12 and fly through the field-free space into a detector 13. Measured is the time-of-flight of the ions from the application of voltage to the orthogonal accelerating element 18 to the arrival of the ions to the detector 3. Using such a characteristic that the time of flight depends on the ion's m/z value, a mass spectrum can be obtained.
A controlling element 70 controls the timings for switching switches 48, 49, and 52, respectively. In addition, the controlling element 70 changes operating modes of the quadrupole filter 8 by controlling a power source 60.
Depending on the method for applying voltage to the electrodes composing the quadrupole filter 8, the quadrupole filter can be operated as either an ion guide or a mass filter.
When MS analysis is performed, the quadrupole filter 8 is operated as an ion guide to introduce the ions in the whole m/z range into the ion trap 9. In MS/MS analysis, during ion storage in the ion trap 9, a quadrupole filter is operated as a band pass filter to introduce only the parent ion into the ion trap 9. Then, the ions stored in the ion trap 9 are dissociated by CID and the masses of the fragrant ions, which are stored in the ion trap, are analyzed in the same procedure as that for MS analysis.
During a period from the application of DC voltages for ejecting the ions to the ion trap 9 to the application of pulse voltage to the orthogonal accelerating element 18, the next ion storage process is initiated. This interval is usually about 10 to 50 μs, while the time for the ion storage is about 10 ms to 1 s, at which any loss of the sample ions is negligible.
By adjusting the pumping speeds, the diameter of ions' passes between adjacent vacuum chambers, and the diameter of a sampling orifice 2 (ions' pass between the atmospheric-pressure space and the first vacuum chamber 3), and further adjusting the gas flow rate using the valve 15, the gas pressure inside the ion trap 9 can be set to Pmax (about 10−1–10−2 Torr) so that ion trapping efficiency, mass resolution, and CID efficiency may be approximately maximized and by adjusting the gas flow rate using he valve 14, the gas pressure inside the quadrupole filter can be set to the level lower than Pmax.
The degree of vacuum in the fourth vacuum chamber 7, where the TOFMS is disposed, is kept at a level, at which the TOFMS can demonstrate sufficiently its performances, by increasing the pumping speed for the third vacuum chamber 5 or that for the fourth vacuum chamber 7, because the ion trap is operated at a region of gas pressure higher than that for the mass spectrometer according to the prior art.
According to an embodiment of the present invention, the ion trap 9 is operated in the gas-pressure region, where all of, one of, or two of ion-trapping efficiency 102, mass resolution 103, and CID efficiency 101 are maximized or in the vicinity of the gas-pressure region described above. The gas-pressure region for operating the quadrupole filter is set and controlled independently from the gas-pressure region for operating the ion trap 9 and optimized for isolation resolution. The gas-pressure region 105′ for operating the quadrupole filter 8 is set and controlled to the lower level than that of the gas-pressure region 105 for operating the ion trap 9.
Like the control element shown in a schematic view shown in
The first-stage quadrupole 8-1 is disposed in a housing 20, into which the neutral gas (for example, helium, nitrogen, or argon) is introduced through a gas tube 123. The gas pressure inside the quadrupole 8-1 is controlled by adjusting the gas flow rate of gas tube 123 using a valve 124. The third-stage quadrupole 8-3 is disposed in the housing 20, into which the neutral gas (for example, helium, nitrogen, or argon) is introduced through a gas tube 16. The gas pressure inside the quadrupole 8-3 is controlled by adjusting the gas flow rate of the gas tube 16, using the valve 14.
Similarly, in the schematic view shown in
The gas-pressure region for operating the quadrupole 8-1, 8-2, and 8-3 is set and controlled independently from the gas-pressure region 105 for operating the ion trap 9, and optimized for isolation resolution. The degree of vacuum in the fourth vacuum chamber 7, where the TOFMS is disposed, can be kept at a level, at which the TOFMS demonstrates sufficiently its performances by increasing the pumping speed for pumping air from the third vacuum chamber 5 or for pumping air from the fourth vacuum chamber 7 in the schematic view shown in
In the example of the schematic view shown in
It is possible to set the gas pressure inside the second stage quadrupole 8-2 to the level as lower as possible (about 10−3–10−4 Torr) and use the quadrupole 8-2 for isolation and set the gas pressure P inside the first-stage quadrupole 8-1 and the second-stage quadrupole 8-3 to the level required for focusing the ion beam (about 10−1–10−2 Torr).
The gas pressure inside the quadrupole 8-2 can be controlled to the level lower than those inside the quadrupoles 8-1 and 8-3 by adjusting the valves 124 and 14.
Although the ion beam is focused in the first-stage quadrupole 8-1, they may defocus at the interface between the first-stage quadrupole 8-1 and the second-stage quadrupole 8-2. The third-stage quadrupole 8-3 has a function for focusing the defocused beam again.
When MS/MS analysis is performed, first, MS analysis is made to obtain a mass spectrum. A parent-ion peak is selected among the peaks on the mass spectrum. Next, during ion storage into the ion trap, the quadrupole is operated as a band pass filter, through which only the selected parent ion may pass.
(1) The ions are stored in the ion trap for a certain period of time and the masses of the stored ions are analyzed (MS in
(2) Only ions having an m/z ratio of M1 are stored in the ion trap for certain period of time while isolation is being performed using the quadrupole. Next, the stored ions are dissociated by CID to analyze the masses of the fragments resultant from ion dissociation (MS2 (1st) in
(3) In the same manner as those described in (2), the ions having the m/z ratio of M2 are analyzed (MS2 (2nd) in
In this way, MS/MS analysis is performed on the ions having up to the m/z ratio of Mn. M1 to Mn are selected among those on the mass spectrum obtained in (1), for example in the order of the intensity of peak being larger. The user (the measurer) is responsible for setting the value for n. Note that generally, to improve the S/N ratio, the individual sequences are repeated and the mass spectra are integrated several times.
In MS/MS analysis, isolation resolution can be improved without ion trapping efficiency, mass resolution, and CID efficiency being deteriorated because isolation can be performed at the low gas-pressure quadrupole element. In addition, the duty ratio is improved because ion storage and isolation are simultaneously performed and, the effect of improving detection sensitivity can be also attained.
(1) MS(n−1) is performed.
(2) While isolation is being performed at the quadrupole element, only the ions having an m/z ratio of M1 are stored in the ion trap for a certain period of time and the stored ions are dissociated by CID.
(3) Only the ions having an m/z ratio of, for example M2, among the fragment ions are isolated in the ion trap and the isolated ions are dissociated by CID.
(4) The step (3) is repeated (n−1) times.
(5) The masses of the fragment ions are analyzed.
When MSn (n>2) is performed, the first isolation is performed at the quadrupole element (
Generally, in the first isolation, higher isolation resolution is desired because random noise ions, any other peptide fragments, and solvent-derived ions must be removed, though lower isolation resolution may be acceptable in the second or succeeding isolations compared with that in the first isolation. For this reason, the gas pressure inside the ion trap can be set to the level, at which ion trapping efficiency, mass resolution, and CID efficiency may be maximized.
The parent ion can be selected in either the manual or auto-select mode.
In the auto-select mode, generally, a specified number of ions are selected by software in the order of the intensity of peak being higher. Any adjacent peaks, which cannot be completely removed by isolation, may exist in the vicinity of the selected peak. In this case, the mass spectrum of fragments may be misunderstood, leading to an error in identifying original ions. To solve this problem, such a preventive means may be considered that the presence of peaks in the vicinity to the target peak, which cannot be removed, is determined based on the isolation resolution of the instrument and if any, the peak is not selected. Note that it goes without saying that the criterion for determination depends on the place, where the isolation is performed, the quadrupole filter or the ion trap, because isolation resolution is different.
In
In the mass spectrometer combining the ion trap and the TOFMS, the quadrupole element is disposed at the front of the ion trap, at which isolation is performed. This structure enables the gas pressure inside the ion trap to be set in the region, where ion trapping efficiency, mass resolution, and CID efficiency are simultaneously maximized. On the other hand, the gas pressure inside the quadrupole element can be set to a relatively low level appropriate for isolation.
Thus, detection sensitivity, mass resolution, and CID efficiency can be improved without isolation resolution deteriorated. Using the mass spectrometer with enhanced performances, analysis efficiency can be improved in especially, analyzing proteome.
According to the present invention, the mass spectrometer combining the ion trap and the TOFMS non-coaxially, wherein ion trapping efficiency, mass resolution, and CID efficiency can be simultaneously improved and the mass spectrometric method using it may be implemented.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-045345 | Feb 2003 | JP | national |
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| 2939952 | Paul et al. | Jun 1960 | A |
| 5572022 | Schwartz et al. | Nov 1996 | A |
| 5652427 | Whitehouse et al. | Jul 1997 | A |
| 5696376 | Doroshenko et al. | Dec 1997 | A |
| 6627883 | Wang et al. | Sep 2003 | B1 |
| 6707033 | Okumura et al. | Mar 2004 | B1 |
| Number | Date | Country |
|---|---|---|
| 2001-297730 | Apr 2000 | JP |
| Number | Date | Country | |
|---|---|---|---|
| 20040164240 A1 | Aug 2004 | US |
