Patent Application
20090278043
The present application claims priority from Japanese Patent Application No. 2005-341365 filed on Nov. 28, 2005, the content of which is hereby incorporated by reference into this application.
The present invention relates to a method and device for analysis of sequence structures of biological macromolecules with the use of mass spectrometry.
In the mass spectrometry, sample molecules are ionized and introduced into vacuum (or ionized in vacuum), and mass to charge ratios of target molecular ions are measured by measuring movements of the ions in an electromagnetic field. Since the obtained information represents macroscopic quantities of the mass to charge ratios, it is difficult to obtain information on internal structure by a single mass analysis. Accordingly, a method called tandem mass spectrometry is used. That is, sample ions are isolated or selected in the first mass analysis. These ions are referred to as precursor ions. Subsequently, the precursor ions are dissociated by a certain technique. The dissociated ions are referred to as fragment ions. The dissociated ions are further mass-analyzed, thereby obtaining information on the generation patterns of the fragment ions. Since there is a rule for dissociation patterns depending on each dissociation technique, it is possible to infer the sequence structure of the precursor ions. Particularly, in the fields of analysis of biomolecules composed of protein, adiabatic reactions, such as charged particle reaction, using Collision Induced Dissociation (CID), Infra Red Multi Photon Dissociation (IRMPD), and Electron Capture Dissociation (ECD), Electron Transfer Dissociation (ETD), Proton Transfer charge Reduction (PTR), and Fast Atomic Bombardment (FAB), are used for the dissociation technique.
CID is currently widely used in the fields of the protein analysis. A kinetic energy is provided to the precursor ions to allow them to collide with gas. Molecular vibration is excited by the collision and the molecular chain is dissociated at sites susceptible to cleavage. Further, a method that has recently come to be used is IRMPD. The precursor ions are irradiated by infra red laser to allow them to absorb multiple photons. The molecular vibrations are excited and a molecular chain is dissociated at a site susceptible to cleavage. The sites susceptible to cleavage by CID or IRMPD are sites designated as a-x and b-y in the backbone consisting of an amino acid sequence. It is known that a complete structural analysis cannot be carried out only by CID or IRMPD, because even when sites correspond to a-x and b-y, those are sometimes hard to be cleaved depending on the kind of the amino acid sequence pattern. Therefore, a pretreatment using an enzyme or the like is necessary, which hampers high speed analysis. Further, when CID or IRMPD is used for post-translationally modified biomolecules, side chains involved in the post-translational modification tend to be easily cleaved. Due to facile cleavage of the side chains, it is possible to determine, based on lost mass, molecular species involved in the modification and whether or not it is modified. However, important information on modification sites concerning which amino acids are modified is lost.
On the other hand, ECD, ETD, and the like, which are the adiabatic dissociating methods using an electron, as other dissociation means, are less dependent on an amino acid sequence (as an exception, proline residue with a cyclic structure is not cleaved) and cleave only one c-z site on the backbone of the amino acid sequence. Therefore, a complete analysis of the backbone chain sequence of a protein molecule can be performed only by the mass spectrometric approach. In addition, ECD, ETD, and the like are suitable for research and analysis of post-translational modification owing to its property of hardly cleaving side chains. Therefore, the dissociation techniques of ECD and ETD attract particular attention in recent years. CID and IRMPD, ECD and ETD, and the like can be utilized mutually complementarily because they provide different sequence information, respectively.
In the mass spectrometers of an ion trap type, a quadrupole type, or the like, a radio frequency voltage is applied to a three-dimensional ion trap or to multipole electrodes, thereby focusing the ions to their trajectories.
Non-Patent Document 3 describes a principle that ions are focused to their trajectories in the radial direction by an application of a radio frequency voltage with the use of an idea of pseudopotential. The pseudopotential is the one obtained by expressing a potential in the radial direction formed by a radio frequency voltage with such a potential that is formed by a DC voltage. The ion trap using the radio frequency voltage features that ions can be focused and captured regardless of a positive ion or a negative ion.
Non-Patent Document 1 describes an ETD technique inside a radio frequency ion trap. In a structure of three-series of quadrupole ion traps (LTQ mass spectrometer) provided with wall electrodes, positive ions are introduced from one of two input/output ports for ions placed on both sides and then are kept captured, subsequently negative ions are introduced from the opposite port, and the both positive and negative ions enter into a potential generated by an application of a DC voltage. Then, a secondary radio frequency voltage is applied to the quadrupole and the wall electrodes to react the positive ions and negative ions to each other, thereby causing an ETD reaction.
An ECD technique inside the three-dimensional and radio frequency linear ion traps is described in Patent Document 1 and Patent Document 2. Here, there is proposed the ECD technique, in which a magnetic field is applied onto ion trajectories of the three-dimensional ion trap and linear ion trap, and this magnetic field restricts the electron trajectories so as to avoid the heating of electrons. In the configuration using a three-dimensional ion trap, there is proposed a method, in which a magnet is placed inside a ring electrode or outside an end cap, and electrons are introduced from the outside of the ion trap. Moreover, in the configuration using a linear ion trap, there is proposed a method, in which a magnetic field is applied onto the central axis of the linear ion trap, and electrons are introduced from within the magnetic field onto the ion trajectories.
An ECD technique inside a radio-frequency linear ion trap is described in Non-Patent Document 2. Here, there is described the ECD method, in which a magnetic field is applied to the ion trajectories of a linear quadrupole electrode ion trap to restrict the electron trajectories, thereby avoiding the heating of electrons.
Patent Document 3 discloses a method, in which ions are transported using a DC voltage in a fragment device comprising a serially arranged plurality of electrodes. Namely, a potential hill and well are formed with a DC voltage, and ions are pushed out at this potential hill and are captured at this potential well, whereby the ions are transported by transferring the potential hill and potential well. Moreover, by changing a applying method of the DC voltage, the speed of the potential hill and well can be adjusted, and consequently the transporting speed of ions can be adjusted. This approach can adjust the transit time of ions.
Patent Document 1: U.S. Pat. No. 6,800,851 B1
Patent Document 2: U.S. Patent Application Publication No. U.S. 2004/0155180 A1
Patent Document 3: U.S. Pat. No. 6,884,995 B2
Non-Patent Document 1: John E. P. Syka et al., PNAS, vol. 101, No. 26, pp. 9528-9533
Non-Patent Document 2: Takashi Baba et al., Analytical Chemistry, 2004, vol. 76, pp. 4263-4266
Non-Patent document 3: H. G. Dehmelt et al., Adv. At. MolPhys 353 (1967), pp. 53-72
A triple-quadrupole mass spectrometer and a quadrupole-TOF mass spectrometer are widely used in protein analysis because the triple-quadrupole mass spectrometer allows for high throughput analysis and quantification, such as a precursor scan or a neutral loss scan, while the quadrupole-TOF mass spectrometer also allows for high throughput analysis. In these devices, although CID is implemented as an ion dissociation technique, not only CID but other new ion dissociation techniques, such as ECD and ETD, are reasonably expected to be implemented for the purpose of improving the efficiency of protein analysis in the future. However, currently, in implementing ETD or ECD in an ion dissociation chamber using a triple-quadrupole mass spectrometer or a quadrupole-TOF mass spectrometer, there are the following problems.
The both triple-quadrupole mass spectrometer and quadrupole-TOF mass spectrometer comprise a quadrupole mass filter at the preceding stage of the ion dissociation chamber. The quadrupole mass filter plays a role to allow only ions with a specific mass to charge ratio to pass therethrough and removes the other ions. Moreover, the quadrupole mass filter scans the mass to charge ratio to be passed therethrough. The mass scan is performed at the scanning speed of no less than 1000 amu/sec (amu: atomic mass unit). For example, at the scanning speed of 1000 amu/sec, ions having a different mass by 1 amu are sequentially ejected at every one millisecond. In this case, in order to respond to ions coming one after another at every one millisecond, the dissociation such as CID is implemented in a short time of no more than 1 msec in the ion dissociation chamber. In the same way, if ECD, ETD, or the like is performed in the ion dissociation chamber, ions are requested to be dissociated in a short time of no more than 1 msec.
However, in order to perform ETD, ECD, or the like by means of the triple-quadrupole mass spectrometer or the quadrupole-TOF mass spectrometer, there are currently two problems. A first problem is that according to the previous reports the reaction time of ECD, ETD or the like takes no less than 10 msec and thus a longer reaction time by about one digit as compared with CID is required. For example, for the quadrupole mass filter with the scanning speed of 1000 amu/sec, the reaction time needs to be set to no more than 1 msec in order to maintain the mass resolution of 1 amu. However, if the reaction time of ETD, ECD, or the like is set to a short time of no more than 1 msec, the quantity of fragment ions will be reduced and it is thus difficult to obtain a spectrum with reasonably good S/N. It is therefore necessary to secure the reaction time of no less than 10 msec in the current ETD, ECD, or the like. This leads to a reduction in the throughput. A second problem is that an ion can pass through a quadrupole ion guide in about several hundreds of microseconds. Since the energy of a sample ion is around several tens of eV, the sample ion passes through the ion trap of about 10 cm in length in about several hundreds of microseconds. Under such conditions allowing the ion to pass straight therethrough, the reaction time of 10 msec cannot be secured. Conventionally, in order to secure the reaction time of 10 msec by reducing the throughput or mass information, a method for capturing ions with a DC voltage has been employed.
Namely, as the configuration, as described in non-Patent Document 2, a quadrupole linear ion trap and wall electrodes at the both ends thereof are placed and then a DC voltage is applied to the wall electrodes to form a wall of potential at the both ends of the linear ion trap. Thereby, a focusing effect in the radial direction is provided by a pseudopotential due to a radio frequency voltage and thus the ions are focused onto the central axis of the quadrupole, and at the same time, with a DC voltage potential at the wall electrodes, the ions are focused in the axial direction (direction parallel to the quadrupole electrode) and captured. This is an approach of non-Patent Document 2, which makes it possible to capture ions for no less than 10 msec and secure the reaction time. However, ETD, ECD, or the like needs to be implemented while the wall of potential is formed with the wall electrodes and the electrons are confined, and thereafter the potential of the wall electrodes needs to be reduced to eject the ions. Therefore, there are processes of the accumulation, reaction, and ejection of ions. For this reason, ions cannot be introduced into the ion trap during the reaction or ejection of ions. For this reason, the conventional method must sacrifice either the mass resolution or the throughput, as in the following two examples.
The first one is a method, in which a preliminary ion trap used for ion accumulation is placed at the subsequent stage of the quadrupole mass filter and before the ion dissociation chamber, whereby the ions ejected from the quadrupole mass filter are accumulated for 10 msec by means of the preliminary ion trap and thereafter the ions are allowed to enter into the ion dissociation chamber. In this way, the ions can be introduced into the ion dissociation chamber without loss of the ions. However, in the case of the mass filter with 1 amu/msec, since the ions are accumulated for 10 msec, the ions of the mass by the amount of 10 amu are mixed to each other inside the preliminary ion trap, which leads to a degradation of the mass resolution, causing a problem that the mass information on the ions is lost.
The second one is the fact that the scanning speed of the quadrupole mass filter is reduced to 100 amu/sec. However, in scanning the same mass range, the sample analyzing time takes 10 times longer, causing a problem of a degradation of the throughput.
On the other hand, when a method of Patent Document 3 is employed, difficulty is expected in implementing ETD reaction. For example, consider to inject negative ions in order to cause the ETD reaction while positive ions are captured in a potential well and being transported. However, in this case, negative ions cannot enter into a potential in which the positive ions are being captured, the potential consisting of a DC voltage. This is because only either of the positive ions and negative ions can be captured by the potential generated by the DC voltage (i.e., the both cannot exist at the same place at the same time) since the positive ion and the negative ion have different electric charge signs to each other. As an alternative method, a high energy may be applied to negative ions so that the negative ions are caused to pass through the potential portion, in which positive ions are present, thereby causing the reaction. However, it is expected that with negative ions having such high energy the reaction efficiency of ETD is low and the reaction will hardly occur.
The present invention solves these conventional problems of the charged particle reaction, and discloses a fast charged particle reaction device, a mass analyzer (i.e., mass spectrometer) including the same, and a method of operating the same.
An ion guide, an ion reactor, and a mass spectrometer of the present invention comprises: a plurality of electrodes each having a circular hole opened therein, the plurality of electrodes being serially arranged in the axial direction; and two or more power supplies that periodically change a radio frequency voltage amplitude of a voltage applied to the electrodes, wherein phases of the periodically changed radio frequency voltages differ to each other, wherein ions are captured and transferred by a radio frequency electric field formed on a central axis of the plurality of electrodes each having a circular hole opened therein, the plurality of electrodes being serially arranged in the axial direction.
In such configuration, a radio frequency voltage obtained by modulating the radio frequency voltage amplitude is applied, and with this modulation of the radio frequency electric field the transfer speed of an ion is adjusted. The amplitude of the radio frequency voltage is controlled so as to periodically change, and the resulting radio frequency voltage is applied so that the phases at the adjacent electrodes differ from each other by a certain value. Generating a pseudopotential in the radial direction by the radio frequency voltage so as to focus ions is the same as the conventional ion guide or ion trap. However, by modulating the radio frequency voltage amplitude, the ups and downs of the pseudopotential are also generated in the axial direction. Furthermore, a field where the bottom of the ups and downs of the pseudopotential moves at a certain speed is formed, whereby ions are captured by an ion packet of the bottom of the ups and downs of this pseudopotential and the ions are transported along with the movement of the ion packet. The ion packet caused by this pseudopotential features the capability of capturing the positive ions and negative ions at the same time. Moreover, the frequency for modulating the amplitude determines the transfer speed of the ion packet, so that the transit time of the ions inside the charged particle reaction cell can be adjusted.
Moreover, in the device of the present invention, the particle reaction is caused by providing a particle source that generates a medium particle, such as an ion or an electron, capable of changing the electric charge of a sample ion.
As in Patent Document 3, also in the present invention, ions are focused and captured by means of a radio frequency voltage. However, the methods for adjusting the transfer speed of ions differ from each other. In the method of Patent Document 3, with the use of a DC voltage for ions, the DC voltage is sequentially applied to serially arranged electrodes to push out the ions. At this instance, if the electric charge sign of the ion differs, the sign of the DC voltage to be applied becomes opposite and therefore the DC voltage corresponding to each of the positive ion and the negative ion needs to be applied. For this reason, the positive ion and negative ion cannot be transferred as the same ion packet at the same time.
According to the present invention, since the incoming and outgoing of ions are not controlled with the wall electrodes or the like as in the ordinary ion trap, ions can be incident to the charged particle reaction cell at intervals from several milliseconds to several hundreds of microsecond and also the residence time of ions can be extended by about 10 msec or more. Furthermore, by putting the positive ions and negative ions into the same ion packet, 10 msec or more required for the reaction time of the charged particles in ETD can be secured. Moreover, since this is a method, in which the incident positive and negative ions pass through the interior of the ion trap and are sequentially ejected with their incident order being kept, it is possible to efficiently cause the reaction during the transportation.
In this way, in the charged particle reaction device using a radio frequency ion trap, the speed of the charged particle reaction can be accelerated. The lowered throughput and mass resolution, which are the problems in implementing the charged particle reaction, can be solved, and the speed of the structure analysis of a measurement sample can be accelerated.
Other objects, features, and advantages of the present invention will be apparent from the following description of the embodiments of the present invention, with reference to the accompanying drawings.
For a sample of analyte, a sample separated by liquid chromatograph or the like is ionized in an ion source 8. The ionized sample is incident upon a quadrupole ion guide part 24 to 25 inside a vacuum device, and passes therethrough and is introduced into a linear ion trap part 26 to 28. He gas, Ar gas, or the like is introduced into the ion trap part, where the sample ion is cooled by collision with the gas. In the linear ion trap part, the accumulation, separation, and ejection of ions are performed and the ejected ions are incident to an electron transfer dissociation cell. The electron transfer dissociation cell for implementing electron transfer dissociation comprises a plurality of electrodes 1 each having a circular hole opened therein. In order to perform the electron transfer dissociation reaction, a negative ion source 9 for generating negative ions is placed, and as shown in the diagram the negative ions are introduced onto the central axis of the plurality of electrodes 1 each having a circular hole opened therein. Immediately after positive ions are incident to the electron transfer dissociation cell, negative ions are introduced to cause the electron transfer dissociation reaction. The ions ejected from the electron transfer dissociation cell are incident to a collision attenuator (Collisinal-damping chamber) 29 to 30, in which He gas, Ar gas, or the like is introduced, and these ions are focused onto trajectories, whereby the mass to charge ratio is measured in a time-of-flight mass spectrometer part 32 to 34.
The present disclosure is with regard to a method for implementing electron transfer dissociation reaction in the electron transfer dissociation reaction cell comprising the plurality of electrodes 1 each having a circular hole opened therein of
V
rf
=V
0·cos(2πωt) Equation 1
V0 represents the amplitude of a radio frequency voltage, and ω represents the frequency of the radio frequency voltage.
In the present invention, there are proposed two or more power supplies 35 that sinusoidally temporally modulate the radio frequency voltage amplitude of a voltage applied to the electrodes, the two or more power supplies having different phases of a sine wave of the same frequency. The radio frequency voltage amplitude V0 is modulated and applied to the plurality of electrodes 1 each having a circular hole opened therein. Namely, the radio frequency voltage is applied so as to temporally change with a factor of cos Ωt. Moreover, to the adjacent electrodes, with regard to this factor of cos Ωt, the radio frequency voltage is preferably applied so as to have a phase difference of 2π/m (m is an integer). By setting the phase difference to 2π/m, the same voltage can be applied to every certain number of electrodes and therefore the present method can be implemented with fewer power supplies, which is efficient. Reference numeral m denotes the number of electrodes 1 per cycle, and the same voltage is applied to every m electrodes. Namely, in this disclosure, the radio frequency voltage is applied as shown in Equation 2.
V0 represents the amplitude of the radio frequency voltage, Ω represents the frequency of V0, m and n are integers, and t is time.
V40 is applied to the next electrode 6, V41 to an electrode 7, and so on. In this manner, in the case of m=4, a voltage having a different phase by πn/2 is repeated, and the same voltage is applied to every four electrodes.
When a maximum of the radio frequency voltage is applied to the electrode 2 (at the time of t=0 sec of
By applying a voltage as shown in Equation 2, such operation is repeatedly performed and the positive and negative ions are sequentially transferred from the left to the right of the view.
This transfer speed of ions, i.e., the transfer speed of the potential valley, is determined by the frequency Ω. Moreover, the transit time of the ions inside the plurality of electrodes is determined by the frequency Ω and the number of electrodes. For example, in the case of m=4, Ω=1 kHz, and 40 electrodes, an ion travels at the speed of 1 msec/four electrodes, and the residence time of an ion inside the plurality of electrodes 1 each having a circular hole opened therein is around 10 msec. Moreover, in this case, as shown in
Since these operations allow for the transfer of the positive ions and negative ions while these ions remain confined in the same area, the charged particle reaction of the positive ions and negative ions is able to occur during the transfer. Accordingly, the charged particle reaction such as electron transfer dissociation will proceed. In this way, the ions can transit therethrough while securing the reaction time without the ions having different masses being mixed to each other.
Moreover, in the same manner, PTR can be also performed using a negative ion source. Moreover, a particle reaction using FAB can be also achieved by replacing the negative ion source with an FAB ion source. However, if neutral particles are emitted from FAB, the neutral particles need to be directly introduced and collided because an optical system cannot be used.
With this configuration, as shown in Equation 2 and
The present invention can be applied to a method and device for the sequence structure analysis of biopolymers using a mass analysis method.
1 . . . a plurality of electrodes each having a circular hole opened therein, 2 to 7 . . . electrode having a circular hole opened therein, 8 . . . ion source, 9 . . . negative ion source, 10 to 13 . . . positive and negative ions, 14 . . . permanent magnet, 15 . . . filament, 16 to 17 . . . wall electrode, 18 . . . a plurality of quadrupole electrodes, 19 to 23 . . . quadrupole electrode, 24, 26, 28, and 29 . . . electrode having a hole opened therein, 25, 27, and 30 . . . quadrupole electrode, 31 . . . optical lens system, 32 . . . acceleration part, 33 . . . reflectron, 34 . . . detector, 35 . . . power supply.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-341365 | Nov 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/304498 | 3/8/2006 | WO | 00 | 5/27/2008 |
