Patent Application
20110204221
The present invention relates to a method of mass spectrometry and a mass spectrometer.
In mass spectrometry, a mass-to-charge ratio m/z (m: mass, z: number of charges) of target molecular ions is measured by: either ionizing sample molecules and introducing the sample molecular ions into a vacuum, or ionizing the sample molecules in a vacuum; and thereafter by measuring the motion of the sample molecular ions in an electromagnetic field. Since the obtained information is about the mass-to-charge ratio m/z, it is difficult to obtain information on the inner structure. For this reason, a method termed as tandem mass spectrometry is used. In the tandem mass spectrometry, sample molecular ions are specified or selected in the first mass analysis operation. The selected ions are called precursor ions. Subsequently, the precursor ions are dissociated in the second mass analysis operation by use of a given technique. The dissociated ions are called fragment ions. Then, the sequence structure of the precursor ions can be estimated by performing a mass spectrometry on the fragment ions.
Having dissociation patterns following their own specific laws, the dissociation techniques enable the estimation of the sequence structure of the precursor ions. The field of analysis of biomolecules having proteins as their skeletons, in particular, employs, as the dissociation techniques, charged-particle reactions using collision induced dissociation (CID), infrared multi-photon dissociation (IRMPD), electron capture dissociation (ECD), electron transfer dissociation (ETD), proton transfer charge reduction (PTR), and fast atomic bombardment (FAB).
In the field of the protein analysis, CID is a widely-used ion dissociation technique. Precursor ions are given kinetic energy and thereby are caused to collide with a buffer gas of He or the like introduced in a dissociation chamber. The collision induces molecular vibrations, and ions are thus dissociated at sites of the molecular chain which are susceptible to cleavage. Meanwhile, in IRMPD, precursor ions are irradiated with an infrared laser beam, and are made to absorb a large number of photons. This induces molecular vibrations, and ions are dissociated at sites of the molecular chain which are susceptible to cleavage. The sites susceptible to cleavage by CID and IRMPD are sites designated as a-x and b-y in the main chain consisting of amino acid sequence. It is known that a complete structural analysis cannot be carried out only by CID or IRMPD since even the a-x and b-y sites are sometimes hard to cleave depending on the kind of amino acid sequence pattern. For this reason, a pretreatment using enzyme or the like is necessary, but such a pretreatment hinders a fast analysis. Further, when CID or IRMPD is used for biomolecules having undergone a post-translational modification, side chains involved in the post-translational modification (modification molecules) tend to be susceptible to cleavage. Since the side chains are susceptible to cleavage, it is possible to determine on the basis of the lost mass what molecular species is involved in the modification, and whether or not the molecules are modified. However, important information on modification sites concerning which amino acid sites are modified is lost.
On the other hand, ECD, ETD and the like, which are dissociation techniques using electrons as an alternative dissociation means, are less dependent on amino acid sequence (as an exception, proline residue with a cyclic structure is not cleaved), and cleave only one c-z site on the main chain of the amino acid sequence. Accordingly, the main chain sequences of protein molecules can be analyzed by use of only a mass spectrometric technique. In addition, ECD and ETD are suitable as a means for research and analysis of post-translational modification owing to their characteristic of having side chains hard to cleave. For those reasons, the dissociation techniques, ECD and ETD, have been attracting particular attention in recent years. CID, IRMPD, ECD, and ETD can be used complementarily, because they provide pieces of sequence information that differ from one another.
The tandem mass spectrometry is widely used in mass spectrometers using an ion trap or a quadrupole, such as an ion trap mass spectrometer, an ion trap TOF (time-of-flight) mass spectrometer, a triple quadrupole mass spectrometer, and a quadrupole TOF mass spectrometer. The ion trap allows a tandem mass spectrometry multiple times, enabling an analysis of a sample whose sequence structure cannot be analyzed if a tandem mass analysis operation is performed only once. In the ion trap, the trajectories of ions are converged by applying a radio frequency voltage to a ring electrode or multipole rods (cylinder electrodes) in a three-dimensional ion trap. The quadrupole ion trap mass spectrometer includes: a Paul trap formed from a ring electrode and a pair of endcap electrodes; a linear quadrupole ion trap formed from four cylinder electrodes; or the like. When a radio frequency voltage having a frequency of about 1 MHz is applied to the ring electrode or the cylinder electrodes, ions in a certain mass range are put into a stabilized condition, and thereby can be accumulated. The triple quadrupole mass spectrometer and the quadrupole TOF mass spectrometer both include a quadrupole mass filter in a stage preceding the ion dissociation unit. The quadrupole mass filter plays a role of transmitting only ions having a specific mass-to-charge ratio and rejecting the other ions. Moreover, ions which the filter transmits can be changed from one kind to another by scanning the mass-to-charge ratio of ions to be transmitted.
Patent Documents 1 and 2 describe ECD methods performed in a radio frequency three-dimensional ion trap and a radio frequency linear quadrupole ion trap. A proposal is made on ECD methods in which: a magnetic field is applied to an ion trajectory in the three-dimensional ion trap and the linear ion trap; the trajectories of electrons are restricted by the magnetic field; and the heating of the electrons is avoided. For the configuration using the three-dimensional ion trap, a proposal has been made on a method in which: a magnet is disposed inside a ring electrode or outside end caps; and electrons are introduced from the outside of the ion trap. Moreover, for the configuration using the linear ion trap, descriptions are provided for a method in which: a magnetic field is applied to the center axis of the linear ion trap; and electrons are introduced onto the ion trajectory from the inside of the magnetic field.
Patent Document 3 describes an ECD method performed inside a radio frequency linear quadrupole ion trap. Descriptions are provided for the ECD method in which: a magnetic field is applied to an ion trajectory in the linear quadrupole ion trap; the trajectories of electrons are restricted; and the heating of the electrons attributable to a radio frequency voltage is avoided.
Patent Document 4 describes a configuration in which in a quadrupole to which a radio frequency voltage is applied, multipole rod electrodes disposed in an ion dissociation chamber or the like are tilted, or tilted electrodes are inserted between the multipole rod electrodes. With this configuration, an electrostatic field which urges ejection of ions toward the exit is generated on the center axis of the multipole, and thereby, the time needed to eject ions is decreased. Descriptions are provided for the apparatus configuration in which a mass filter and a quadruple ion dissociation chamber are connected together.
Patent Document 5 describes a triple quadrupole mass spectrometer system including an ion source, an ion trap (pre ion trap) configured to only accumulate ions, a quadrupole mass filter, an ion dissociation chamber, and an ion trap capable of mass-based selective ejection, the system in which sample ions coming out of the ion source are accumulated in the pre ion trap while an ion ejection operation is being performed in the ion trap capable of mass-based selective ejection.
Dissociation chambers include those of a non-ion-trap type and those of an ion-trap type. The non-ion-trap type has an advantage of achieving high throughput, but has a disadvantage of being incapable of a tandem mass spectrometry (MS/MS). On the other hand, the ion-trap type has a disadvantage of having low throughput, but has an advantage of being capable of adjust the dissociation reaction time freely, and performing a tandem mass spectrometry.
Non-ion-trap type ion dissociation chambers are used in triple quadrupole mass spectrometers and quadrupole TOF mass spectrometers. Triple quadrupole mass spectrometers are widely used for their capabilities to perform a high-throughput analysis and a quantitative analysis using a precursor scan and a neutral loss scan. Quadrupole TOF mass spectrometers are also widely used for their capabilities to perform a high-throughput analysis. For the ion dissociation chamber, CID or IRMPD has been widely used as the ion dissociation method. However, for the purpose of improving the efficiency in analyzing proteins, new ion dissociation methods such as ECD and ETD are expected to be employed in the future. Triple quadrupole mass spectrometers and quadrupole TOF mass spectrometers both include a quadrupole mass filter in a stage preceding the ion dissociation chamber. This quadrupole mass filter plays a role of transmitting only ions having a specific mass-to-charge ratio m/z and rejecting the other ions. The ions of the specific m/z passed through the mass filter enter the ion dissociation chamber, where an ion dissociation reaction operation is performed. Patent Document 4 states that time needed to eject ions is shortened by urging ion ejection in a non-ion-trap type dissociation chamber by means of slope electrodes.
Instead of a non-ion-trap type ion dissociation chamber, however, an ion-trap type ion dissociation chamber needs to be used when the reaction time needed for the ion dissociation is longer than 1 ms, or when a tandem mass spectrometry is intended to be performed multiple times. Patent Document 3 describes an ECD operation method performed in an ion-trap type dissociation chamber using a linear quadrupole ion trap. For the method, the ion-trap type ion dissociation chamber capable of securing a certain reaction time is used because the dissociation reaction time in ECD needs to be about 1 ms or longer. Meanwhile, a device termed as a travelling wave (a travelling-wave type dissociation chamber) has emerged in recent years. The device has a configuration in which multiple electrodes are aligned together, and is capable of adjusting the ion ejection rate by applying a DC electric field to a center axis. Unlike the ion-trap type dissociation chamber, this travelling-wave type dissociation chamber is not capable of performing a tandem mass spectrometry. However, like the ion-trap type dissociation chamber, the travelling-wave type dissociation chamber is capable of securing a certain ion reaction time.
The following problem occurs, nevertheless, in the case of a configuration where the ion-trap type or travelling-wave type ion dissociation chamber is installed in a stage following the quadrupole mass filter. Description is given below by taking the ion-trap type ion dissociation chamber as an example, but a similar problem will also occur in the travelling-wave type ion dissociation chamber. The quadrupole mass filter sequentially ejects only specific ions selected from incident ions. The time period in which ions stay inside the quadrupole mass filter is approximately 1 ms or shorter. Meanwhile, the ion dissociation chamber at the following stage operates with a cycle of ion accumulation, ion dissociation, and ion ejection in a normal tandem mass analysis operation. One cycle normally requires 10 msec or more. The difference in the ion stay time between the quadrupole mass filter and the ion dissociation chamber results in an ion loss.
Descriptions will be provided for an operation of the ion-trap type ion dissociation chamber and a problem with the ion-trap type ion dissociation chamber. Ions are supplied at a steady rate from the ion source to the quadrupole mass filter in the stage preceding the ion dissociation chamber. In contrast, the ions are allowed to enter the ion dissociation chamber, and are accumulated therein. Then, the dissociation chamber closes the gate by applying a voltage to an entry gate electrode, and thereby blocks the entry of the ions. Thereafter, operations of ion isolation, ion dissociation, and then ejection of the resultant ions for sending the ions to a detector are performed. The entry of ions into the ion dissociation chamber is not allowed during the ion isolation, ion dissociation, and ion ejection. Hence, ions from the ion source are discarded immediately before the ion dissociation chamber although passed through the quadrupole mass filter. This causes an ion loss. To be specific, in the one cycle operation in the ion dissociation chamber, ions can enter the ion dissociation chamber only during the accumulation, but cannot enter the ion dissociation chamber during any other time, i.e., the isolation, dissociation, or ejection time. Thus, ions coming out of the ion source during those other times have to be discarded. In this respect, the ratio of the accumulation time to a time needed for the one cycle (accumulation, isolation, dissociation, and ejection) is defined as transmittance of the ion dissociation chamber. A higher transmittance means a higher efficiency, and a lower transmittance means a greater ion loss. In the case where the accumulation, dissociation, and ejection respectively require 20 msec, 15 msec, and 5 msec in one tandem mass spectrometry, the transmittance is 50% (20/40).
Further, in a case where a tandem mass spectrometry is to be performed multiple times, that is, operations of ion dissociation, ion isolation, dissociation, ion isolation, dissociation, and ion isolation are to be performed in turn, or in a case where the dissociation time is as long as tens msec to over 100 msec as in ECD, ETD or the like, the time during which ions cannot enter there (time periods of dissociation, isolation, and ejection) becomes even longer, and thereby the amount of ion loss becomes larger. As a result, if the dissociation time exceeds, for example, 100 msec in the case described above, the transmittance falls to 20% or lower.
For the purpose of solving a problem of the ion loss, Patent Document 5 describes a configuration in which: the pre ion trap is disposed before the ion dissociation chamber to prevent an ion loss during the mass-based selective ejection. In this device configuration, however, a large variety and a large amount of ions obtained by ionization in the ion source are stored in the pre ion trap. Once the amount of stored ions exceeds its accumulation capacity, the trap cannot trap any more ions. For this reason, it is expected to be difficult to store a large amount of ions from the ion source in the pre ion trap for a long period of time. In other words, in the case of requiring a long ion dissociation time or using a highly-concentrated sample, it is expected that an ion loss still occurs and the problem cannot be fully solved even if the pre ion trap of Patent Document 5 is used.
For the purpose of solving the above-described problem, a mass spectrometer of the invention is characterized by including: an ion source for ionizing a sample; a mass filter, disposed at a stage following the ion source, for selectively transmitting ions falling within a specific mass number range; an ion trap unit, disposed in a stage following the mass filter, for accumulating ions; an ion dissociation unit, disposed at a stage following the ion trap unit, for accumulating ions and dissociating the ions thus accumulated; a detection unit, disposed at a stage following the ion dissociation unit, for detecting ions; and a controller for controlling the ion accumulation and ion ejection in the ion trap unit in accordance with an operation of the ion dissociation unit.
The controller causes the ion trap unit to accumulate ions passed through the mass filter, except while the ion dissociation unit is accumulating ions, or while the controller is applying a voltage to an electrode for controlling the entry of the ions into the ion dissociation unit in order to block the ions from entering the ion dissociation unit.
In addition, a method of mass spectrometry of the invention characterized by including the steps of: ionizing a sample; selecting first ions having a specific mass number range from the ions thus generated; accumulating the selected first ions in an ion dissociation unit; dissociating the first ions in the ion dissociation unit, and accumulating second ions having a specific mass number range in an ion trap unit provided at a stage preceding the ion dissociation unit; ejecting fragment ions generated by dissociating the first ions; detecting the ejected fragment ions, and accumulating the second ions, accumulated in the ion trap unit, in the ion dissociation unit; ejecting fragment ions generated by dissociating the second ions; and detecting the ejected fragment ions.
According to the disclosure concerned, it is possible to prevent an ion loss which may occur when connecting a quadrupole mass filter and an ion-trap type ion dissociation chamber, and thus to make the transmittance of the ion dissociation chamber closer to 100%. Accordingly, a high-throughput analysis can be achieved.
In a case where a long reaction time of 10 ms or longer is needed as in an ion dissociation reaction such as ECD or ETD, the conventional method makes an ion loss larger as the ion reaction time becomes longer; however, the disclosure concerned hardly causes the ion loss even when the reaction time is increased. As a result, the reaction time can be increased freely, whereby the dissociation reaction can be performed in an optimal reaction time depending on the dissociation method. Furthermore, in a case where an operation time of the dissociation chamber similarly becomes longer due to the multiple repetition of a tandem mass spectrometry, the conventional method resultantly loses ions; however, the disclosure concerned causes no ion loss no matter how many times the tandem mass spectrometry is performed in the ion dissociation chamber. The disclosure concerned is effective when devices having mutually different ion transmission rates are united together as in the case of a configuration where a quadrupole mass filter and an ion-trap type ion dissociation chamber are united together.
Moreover, the device configuration prevents an ion loss which is not expected to be solvable completely even by use of Patent Document 5, and is very effective when the ion dissociation time is desired to be made longer or when a highly concentrated sample is used.
As described above, the disclosure concerned can solve a problem with the ion-trap type and travelling-wave type ion dissociation chambers, which is the decrease in transmittance of the ion dissociation chamber, i.e., the decrease in throughput. Accordingly, the disclosure concerned can increase throughput of a structural analysis on a measurement sample.
A mass spectrometer of the present invention is capable of effectively using ions and accordingly performing a high-throughput analysis by accumulating ions, which would otherwise conventionally lose during ion dissociation, isolation and ejection times, in a pre ion trap by use of its configuration in which a mass filter, an ion trap (pre ion trap), and an ion-trap type ion dissociation chamber(s) are connected together in series.
In order to acquire the full mass spectrum, first of all, the quadrupole mass filter 3, the pre ion trap 4, and the ion dissociation chamber 5 are first set up such that all the ions can pass them. The ions passed through them are converged on a center axis in a collisional-damping chamber 6, and then the flight time of the ions is measured by the TOF mass spectrometers 31 to 33. Thereby, the full mass spectrum is acquired. From the full mass spectrum, two kinds of precursor ions are selected. Subsequently, the pre ion trap 4 is operated as described below, and the MS/MS spectra are acquired.
In order to acquire the MS/MS spectra, the quadrupole mass filter 3 allows only ions (precursor ions 1 or 2) having a specific, selected mass-to-charge ratio (m/z) to pass the quadrupole mass filter 3, and rejects ions having any other m/z. The ions passed through it enter the pre ion trap 4, and are accumulated there. The ions ejected from the pre ion trap 4 enter the ion dissociation chamber 5, and are subjected to a dissociation reaction operation such as CID or ECD. The fragment ions generated by the dissociation are detected by the detection system. The MS/MS spectra are acquired by repeating the above procedure one or more times. If a full mass spectrum is to be acquired again after obtaining the MS/MS spectra, the pre ion trap 4 is set up such that all the ions can pass the pre ion trap 4.
Using
A role of the pre ion trap 4 is to accumulate ions, which are discarded while the dissociation and ejection are performed in the ion dissociation chamber, in the pre ion trap 4. As shown in the middle section of
Description is given of the operation sequences of the voltages of the wall electrodes 23, 24, 25 shown in the bottom section of
In the method of the disclosure concerned, a certain amount of ions is already accumulated in the pre ion trap 4 during dissociation. Thus, unlike the conventional method, such a long accumulation time as 20 ms does not needs to be spent to accumulate a sufficient amount of ions in the ion dissociation chamber 5. Instead, only several milliseconds (3 ms in
In the meanwhile, the transmittance per unit time (the ratio of the ions to be used in the analysis to the ions from the ion source: accumulation time/1 cycle time) of the ion dissociation chamber 5 of the disclosure concerned is 87% (20/23). The transmittance of the ion dissociation chamber in the conventional method is 50% (20/40), as mentioned previously (
In this embodiment, the collisional-damping chamber 6 and the TOF mass spectrometers are used at the stage following the ion dissociation chamber 5, but a detection system capable of acquiring mass spectra, such as an ion trap, a mass filter, an orbitrap, a Fourier-transform ion cyclotron resonance mass spectrometer or a magnetic sector mass spectrometer, may be used there. Also, the pre ion trap 4 and the ion dissociation chamber 5 are illustrated using quadrupole electrode rods as an example, but some other multipole electrode rods, such as hexapole electrode or octapole electrode rods, may be used for the pre ion trap 4 and the ion dissociation chamber 5. The reaction to be performed in the ion dissociation chamber may be an ion reaction or a charged-particle reaction such as CID, ECD, ETD or IRMPD. In the case of performing ECD, an electron source such as a filament may be placed on a center axis that is slightly off the ion trajectory.
The pre ion trap 4 is placed at the stage following the quadrupole mass filter 3, but at the stage preceding the ion dissociation chamber 5. The pre ion trap 4 may be placed at a stage preceding the quadrupole mass filter 3. An advantage of placing the pre ion trap 4 after the quadrupole mass filter 3 as in the case of
As in the case of Embodiment 1, a detection system suffices if the mass spectrum can be acquired. Moreover, multipole electrode rods such as hexapole electrode rods or octapole electrode rods may be used for the pre ion trap 4 and the ion dissociation chamber 5.
The ion dissociation chamber 54 is located off the straight line joining the ion source 1 and the detection system. Hence, even when the ion dissociation is being performed in the ion dissociation chamber 54, ions newly coming out of the ion source 1 can travel to and be detected by the TOF mass spectrometers 31 to 33. That is, even while a dissociation operation or a tandem mass spectrometry is being performed in the ion dissociation chamber 54, a full mass spectrum or MS/MS spectra using the ion dissociation chamber 51 can be acquired. In a case where a long time is needed for the ion dissociation in the ion dissociation chamber 54, or when a long time is needed for multiple repetition of a tandem mass spectrometry or the like there, a full mass spectrum or MS/MS spectra using the ion dissociation chamber 51 may be acquired during that time-consuming operation there. Accordingly, an efficient measurement is feasible. In other words, a high-throughput analysis can be achieved.
It is also possible to perform ECD in the ion dissociation chamber 51. The ECD can be carried out when: a permanent magnet is placed in the ion dissociation chamber 51; and electrons from the electron source 42 are deflected by the quadrupole deflector 52, and are thus introduced into the ion dissociation chamber 51.
ETD can be carried out in the ion dissociation chamber 54 when: a negative ion source is used as the source 42; and a quadrupole filter or ion trap is placed between the negative ion source 42 and the ion dissociation chamber 54. ETD can be carried out in the ion dissociation chamber 51 as well. Moreover, IRMPD can be carried out in the ion dissociation chamber 54 when the negative source 42 is replaced with a laser source.
As in the case of Embodiment 1, the detection system suffices if the mass spectrum can be acquired. Furthermore, multipole electrode rods such as hexapole electrode rods or octapole electrode rods may be used for the pre ion trap and the ion dissociation chambers.
ECD can be carried out when: an electron source is used as the source 42; and a permanent magnet is placed in the ion dissociation chambers 51, 55, 56. ETD can be carried out in the ion dissociation chambers 51, 55, 56 when: a negative ion source is used as the source 42; and a quadrupole filter or ion trap is placed between the negative ion source 42 and the ion dissociation chamber 51, 55, 56. Moreover, IRMPD can be carried out in the ion dissociation chambers 55, 56 when a laser source is used as the source 42.
A configuration including no ion dissociation chamber 51 may be considered. In this case, a full mass spectrum is acquired from the straight line extending straight from the ion source to the detector, and MS/MS spectra are acquired from the ion dissociation chambers 55, 56 situated off that line. The time needed to acquire the full mass spectrum is often shorter than that needed to acquire the MS/MS spectra. Hence, there is an advantage that the full mass spectrum can be always acquired while no ions are present on the straight line, such as while ion dissociation is being performed in the ion dissociation chambers.
As in the case of Embodiment 1, a detection system suffices if the mass spectrum can be acquired. Moreover, multipole electrode rods such as hexapole electrode rods or octapole electrode rods may be used for the pre ion trap 4 and the ion dissociation chamber 5.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-264749 | Oct 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/067558 | 10/8/2009 | WO | 00 | 4/4/2011 |
