The present invention relates to an ion trap mass spectrometer for use in analysis of organism-related materials, etc. More specifically, the invention relates to a technology for enabling only ions with their mass-to-charge ratios (m/z) within a predetermined range to be left in the ion trap of an ion trap mass spectrometer.
A quadrupole ion trap mass spectrometer enables ions to be trapped for a predetermined time period using an Rf electric field and enables the ions thus concentrated to be ejected sequentially from the ion trap depending on their mass-to-charge ratios (m/z) so as to be detected by a detector. In this manner, mass spectrometry can be achieved.
It is also possible to perform tandem mass spectrometry in which predetermined ions are dissociated and the mass spectrum of the dissociated ions (i.e., fragment ions) are obtained. More specifically, ions of two or more species are first accumulated within the ion trap, and precursor ions to be analyzed by the tandem mass spectrometry are then selected from among the accumulated ions.
Thereafter, isolation is performed by ejecting all the ions other than the selected precursor ions from the ion trap so that only the precursor ions are left in the ion trap.
The isolated precursor ions are then dissociated by a dissociating method, such as CID (Collision-Induced Dissociation), IRMPD (InfraRed Multi Photon Dissociation), ECD (Electron Capture Dissociation), or ETD (Electron Transfer Dissociation), so that the dissociated ions thus generated are accumulated in the ion trap.
The dissociated ions are then ejected from the ion trap depending on their m/z values to be detected by a detector, thus enabling the m/z values of the dissociated ions to be determined. It is also possible to perform MSn analysis (MS/MS/MS, MS/MS/MS/MS) in which isolation is performed so that predetermined dissociated ions are left as precursor ions and the precursor ions are then further dissociated.
A known isolation method used in a quadrupole ion trap will now be described.
Although quadrupole ion traps are classified into several classes, such as three dimensional quadrupole ion traps (3DQ) including a ring electrode and a pair of bowl-shaped electrodes and linear ion traps (LIT) including parallel pole electrodes, all of them operate on the same principle.
That is, while ions are trapped within a predetermined space in a quadrupole ion trap, they not only oscillate slightly due to an Rf voltage applied across electrodes facing each other at a frequency identical to the Rf frequency (micro motion) but also oscillate at a frequency that is lower than the Rf frequency (secular motion).
Here, the frequency of the secular motion varies depending on the m/z values of the ions. Therefore, if an AC electric field (supplemental AC) having the same frequency as the frequency of the secular motion corresponding to the m/z of a certain ion is applied to the space in which the ion is trapped, the amplitude of the secular motion of the ion is increased due to resonance.
As the potential of the supplemental AC is increased, the amplitude of the motion of the ion in resonance increases, and the ion will be eventually ejected from the ion trap due to collision with electrodes, dissociation through collision with a residual gas, etc.
In addition, Increasing the length of time for which the ion is exposed to the supplemental AC increases the possibility of the ion being ejected from the ion trap due to dissociation through collision with the residual gas, etc.
Ion isolation is typically performed on the basis of the above principle.
When ions of two or more species are trapped in a quadrupole ion trap, isolation in which all the ions other than the precursor ions are ejected leaving only the precursor ions can be achieved by applying a supplemental AC having frequencies corresponding to the m/z values of the other ions so that the other ions are resonance-ejected.
However, when the number of species other than the precursor ion species is very high or when their m/z values are unknown, it is advantageous to sweep (i.e., to vary) the frequency of the supplemental AC within a range in which the precursor ions do not come into resonance so that all the other ions are sequentially resonance-ejected. In that case, it is ideal that all the other ions be ejected completely with all the precursor ions retained as-is.
To this end, the Rf voltage needs to be increased so that the secular motion is stabilized when the precursor ions are trapped. The following values a and q are known as indicators associated with the stability of the secular motion.
Here, e denotes the elementary electric charge, U denotes the DC voltage applied to the ion trap, r denotes the radius of space formed by ion trap electrodes, mz denotes the m/z of the ion, F denotes the Rf frequency, and VRF denotes the Rf voltage.
As the DC voltage U is typically set to 0 volts, the a-value becomes zero. As a result, the stability of the secular motion is eventually represented by the q-value.
In that case, the secular motion may be considered stable if the q-value is equal to or less than about 0.908, and it is known that the higher the q-value becomes, the more the secular motion is stabilized and the more the resonance ejection is likely to occurs.
However, depending on the structure of the quadrupole ion trap or the m/z of the precursor ions, it may be difficult to vary the frequency of the supplemental AC due to constraints imposed by the power supply for generating the Rf voltage, etc.
Here, it is known that there exists a relationship represented by Expression 3 below between the q-value and the resonance frequency fr at which the ion is resonance-ejected.
Combining Expressions 2 and 3 reveals that the similar resonance ejection can also be achieved by sweeping the Rf voltage (VRF) with the frequency of the supplemental AC fixed (Patent Literature 1).
For example, isolation can be achieved by first applying a predetermined supplementary AC, then sweeping the Rf voltage so as to resonance-eject ions having their m/z values lower than that of the precursor ions, and finally sweeping the Rf voltage so as to resonance-eject ions having their m/z values higher than that of the precursor ions.
It is also possible to combine two or more supplemental AC components having different frequencies so that ions having different m/z values can be resonance-ejected at once. This is advantageous to increase the analytical throughput.
More specifically, if it is possible to generate a supplemental AC having various frequencies so that all the ions other than the precursor ions can be ejected at once, isolation can be completed in a short time. Methods referred to as FNF (Filtered Noise Field) (Patent Literature 3), SWIFT (Stored Waveform Inverse Fourier Transform), etc. operate on this principle. A waveform generated in this manner is a typical broadband waveform, and is configured so that only the amplitudes of components having frequencies at which the ions to be isolated come into resonance are reduced to zero.
Such a waveform is actually generated by combining multiple supplemental AC components having regularly spaced frequencies. For this reason, for ions that come into resonance at a frequency located in between any two adjacent frequencies, the resonance ejection efficiency is not necessarily high because the amplitude of the supplemental AC is relatively low.
In view of the foregoing problem, it is advantageous to apply a broadband waveform having a relatively high potential for a predetermined time period or to sweep the Rf voltage (q-value) as described above.
It is also possible to perform isolation by sweeping the Rf voltage for ions having m/z values lower than the m/z value of the precursor ions with a fixed supplemental AC applied so that the ions in the lower m/z range are ejected and applying a broadband waveform having a corresponding frequency range for the ions in the higher m/z range for a relatively short time period.
Using such an approach can prevent harmonics generated by the broadband waveform from affecting the analytical result. On the other hand, when a narrow m/z range having a width of 1 Da (dalton) or less is isolated, it is advantageous to sweep the Rf voltage (q-value) taking into consideration the fact that the frequency of the supplemental AC is close to the resonance frequency corresponding to the central m/z of the isolation.
In this manner, depending on the situation, a supplemental radio frequency (Supplemental Rf), such as a supplemental AC having a single frequency only, a combination of supplemental AC components having different frequencies, or a broadband AC in which various frequencies are combined, may be used in addition to the original Rf, so that the amplitude of the secular motion is increased thus enabling the resonance ejection to Occur.
In addition, because three dimensional quadrupole ion traps have holes formed through their electrodes having curved surfaces so as to eject ions, the quadrupole electric field inside the ion trap may be distorted. Therefore, one or more external electrodes may be disposed to correct the field distortion, so that high accuracy isolation can be achieved.
When isolation is performed, tuning may need to be carried out depending on the measurement purpose by e.g., increasing the throughput, removing ions other than the precursor ions thoroughly, minimizing the ejection and dissociation of the precursor ions, and defining the isolation width in a more accurate manner (Patent Literature 4).
In order to increase the overall analytical throughput and sensitivity of mass spectrometry using an ion trap, isolation needs to be performed at a high speed. The accumulation time in which ions are introduced may often be on the order of a few milliseconds although it varies depending on the amount of ions to be introduced into the ion trap. In view of such a short accumulation time, it is preferable that the length of time required to perform isolation be equal to or less than the accumulation time. Typically, it is preferable that isolation be completed within five milliseconds
Furthermore, in order to increase the throughput, it is necessary to use a broadband supplemental Rf obtained by combining multiple frequencies so that the frequency components corresponding to a certain mass range are reduced to provide a frequency window, instead of the approach in which a supplemental Rf having a single frequency is applied and the frequency thereof or the Rf voltage is swept. However, in that case, because the ions in a mass range higher than the ions to be isolated are less likely to be resonance-ejected than the ions in a mass range lower than the ions to be isolated, there may be caused a problem in that the ions on the higher mass side cannot be thoroughly ejected if the length of time allocated for the resonance ejection is reduced in a uniform manner.
Furthermore, there is another problem in that unstable ions may be dissociated because the frequency components corresponding to the frequency window cannot be eliminated completely. More specifically, with advances in the soft ionization technology, an increasing number of very unstable ions are starting to be analyzed. Typical examples of such ions include glycosylated peptides, protonated molecules of some low molecular weight compounds, etc. However, when such unstable ions are selected as the precursor ions, a large amount of precursor ions may be lost during the isolation process in the ion trap, thus reducing the analytical sensitivity. For this reason, in order to achieve high throughput and high sensitivity analysis, it is important to avoid loss of ions during the isolation process not only for relatively stable ions but also for relatively unstable ions.
An object of the present invention is to provide a method for mass spectrometry using an ion trap that enables unnecessary ions to be ejected thoroughly and enables high speed isolation to be performed while sufficient sensitivity for ions to be left is maintained.
An aspect of the present invention uses an ion isolation method comprising: an introduction step for introducing a plurality of ions into an ion trap having a plurality of electrodes; a trapping step for applying an RF voltage to at least one of the plurality of electrodes at a first potential to trap the plurality of ions within the ion trap; a first isolation step for applying a supplemental RF voltage to the electrode to which the RF voltage is applied, increasing the RF voltage above the first potential, and continuing the application of the RF voltage at the increased potential for a first time period such that ion isolation is performed; a second isolation step for, with the supplemental RF voltage applied to the electrode to which the RF voltage is applied, reducing the RF voltage below the first potential and continuing the application of the RF voltage at the reduced potential for a second time period longer than the first time period such that ion isolation is performed; and an ejection step for ejecting the ions remaining in the ion trap.
Another aspect of the present invention uses a mass spectrometer comprising: an ion source unit for generating a plurality of ions by ionizing a sample; an ion trap unit including an ion trap having a plurality of electrodes, an AC power supply for applying an AC electric field to the plurality of electrodes, and a controller for controlling the AC power supply; and a detector unit for detecting the plurality of ions depending on their mass-to-charge ratios. The mass spectrometer is characterized in that the controller controls the AC power supply to perform ion isolation by applying an RF voltage to at least one of the plurality of electrodes at a first potential to trap the plurality of ions, applying a supplemental RF voltage to the electrode to which the RF voltage is applied, increasing the RF voltage above the first potential, continuing the application of the RF voltage at the increased potential for a first time period, reducing the RF voltage below the first potential, and continuing the application of the RF voltage at the reduced potential for a second time period longer than the first time period.
An exemplary mass spectrometric method disclosed herein can complete isolation of precursor ions within a very short time.
In doing so, the method solves the problem that the ions on the higher mass side are less likely to be ejected when compared to the ions on the lower mass side. Furthermore, another exemplary mass spectrometric method according to the invention enables loss of ions during the isolation process to be suppressed to a very low level even if not only relatively stable ions but also relatively unstable ions are selected as the precursor ions.
As a result, high throughput and high sensitivity tandem mass spectrometry can be performed even for a sample including relatively unstable ions, such as glycosylated peptides.
The present invention achieves a configuration in which the Rf voltage (q-value) can be swept with the supplemental Rf applied irrespective of the type of ion trap.
First, the user of the mass spectrometer 1 may input parameters for isolation via the user interface unit 2. This, user interface unit 2 enables the user to specify parameters for not only the case in which ions of predetermined species are isolated but also the case in which the precursor ions are automatically selected and analyzed such as when a data-dependent analysis is performed.
When ions of predetermined species are isolated, a plurality of ion species can be set as the ions of interest and a plurality of values can be set for each parameter accordingly.
When automatic analysis is performed, on the basis of the information stored in the parameter storage unit 7, past records obtained by analyzing specific ions can be retrieved, previously set tables can be used, previously set functions regarding m/z and electric charge can be used, or any combination thereof can be carried out.
More specifically, the user interface unit 2 may be used to input specific parameters, i.e., the m/z of the precursor ions, parameters for the supplemental Rf, parameters specifying the shift amount of the supplemental Rf toward the precursor ions during isolation (in Da), and sweeping parameters for each of the lower and higher mass sides.
As the parameters for the supplemental Rf, when a waveform obtained by combining one or more frequencies is used as the supplemental Rf, their frequencies may be specified, and when a broadband waveform in which multiple components are combined is used as the supplemental Rf, the width of the frequency window including the m/z of the precursor ions may be specified.
It is also possible to separately set the supplemental Rf parameters for the lower mass side and the higher mass side of the precursor ions.
As the sweeping parameters, parameters specifying the shift amount of the supplemental Rf toward the target mass range, parameters specifying the range in which the Rf voltage is swept therefrom, and parameters specifying the gradient of the Rf voltage sweeping may be specified for Mode 1. For Mode 2, any arbitrary function for the Rf voltage may be set as the sweeping parameter. This function needs to be defined as a function of time. All the parameters can be stored in the parameter storage unit 7. The stored parameters can be retrieved via the user interface unit 2 for later use, while it is also possible to combine the retrieved parameters to generate a new parameter.
Instead of the m/z of the precursor ions, it is also possible to specify a list of the m/z values of the precursor ions, a list of the valences of the precursor ions, a list of combinations of the m/z value and valence of the precursor ions, the ranges of the precursor ions, a list of combinations of the range and valence of the precursor ions, and a list of any combination thereof can also be specified to be used as the parameters for automatic analysis. When liquid chromatography is included and used in the ion source unit 11, the retention time (hereinafter abbreviated as RT) for the precursor ions within the liquid chromatography also can be specified in combination with the m/z of the precursor ions or in combination with a combination of the m/z and valence of the precursor ions.
When the RT is specified in combination with the m/z, even if the m/z is matched, the ions are distinguished from the precursor ions if the RT is not matched. When the RT is specified in combination with both the m/z and valence, even if both the m/z and valence are matched, the ions are distinguished from the precursor ions if the RT is not matched.
It is also possible to specify parameters for ions that do not correspond to the specified m/z or m/z list of the precursor ions (i.e., default parameters) by specifying no m/z values of the precursor ions.
Furthermore, when ions previously included in the sample have a certain characteristic tendency, as is the case with glycosylated proteins or peptides, it is possible to set parameters adjusted to the specific characteristic, and it is also possible to prepare, edit, and store calculation formulae for automatically setting parameters on the basis of a calculation method that enables parameters to be set depending on the charges and mass-to-charge ratios of ions and to retrieve the stored formulae to set parameters.
More specifically, once the m/z and valence of the precursor ions and the degree of dissociatability thereof are selected and the width (in Da) of a range in which ions including the precursor ions are isolated is specified, parameters for the supplemental Rf, parameters specifying the shift amount of the supplemental Rf toward the precursor ions during isolation (in Da), and sweeping parameters for each of the lower and higher mass sides are automatically set.
It is also possible to manually modify the parameters automatically set in this manner in whole or in part.
Although the dissociatability of ions is basically divided into two levels, i.e., likely to be dissociated and unlikely to be dissociated, it is also possible to increase the number of levels and to set the associated parameters accordingly.
The control unit 3 transmits and receives signals to and from the mass spectrometer unit 10, the ion source unit 11, the ion trap unit 12, and the detector unit 13, and transmits signals to the AC circuit unit 8 and the DC circuit unit 9, thereby controlling them.
On the basis of the input parameters, the control unit 3 can not only perform analysis in which only ions of certain species are analyzed using the parameters set for the ion species but also perform analysis by automatically selecting ions and automatically setting parameters for them. The control unit 3 can also perform analysis in which the function for specifying certain ion species and the function for automatically setting parameters are combined, i.e., if certain ion species is detected, analysis can be performed on the basis of the parameters set for the ion species, otherwise, parameters can be automatically set to perform analysis. Furthermore, the control unit 3 can also set parameters in a real-time manner on the basis of information obtained by the detector unit 13 during analysis, and can perform further analysis.
More specifically, when analysis is performed, with liquid chromatography included in the configuration of the ion source unit 11, each ion species is typically measured using a separate time width. Therefore, it is possible to first perform analysis using specified parameters in the early part of the time width and then reset the parameters in the control unit 3 on the basis of the information obtained by the detector unit 13, such as the m/z, valence, and cleavage pattern in tandem mass spectrometry of the ions thereby performing analysis again under better conditions in the rest of the time width.
Furthermore, when the information obtained by the detector unit 13 corresponds to a list previously set via the user interface unit 2, it is possible to make use of the past records by performing analysis on the basis of the set values.
On the basis of the information input via the user interface unit 2, the internal parameter calculation unit calculates internal parameters for generating an ion trap control sequence using input information, past records, feedback information based on detected information, etc. by e.g., referring to the parameter storage unit 7 as needed.
The control sequence preparation unit 5 calculates an ion trap control sequence with respect to time such as shown in
The control sequence execution unit 6 controls the AC circuit unit 8 and the DC circuit unit 9 on the basis of the ion trap control sequence generated by the control sequence preparation unit 5.
The parameter storage unit 7 stores previously set information, past records, and method for automatically calculating internal parameters that may be used when parameters are input.
The AC circuit unit 8 and the DC circuit unit 9 transmit signals to the ion trap unit 12 under the control of the control sequence execution unit 6.
The detector unit 13 detects ions ejected from the ion trap and transmits information about the detected ions to the control unit 3.
All the ions are introduced into a linear ion trap 15 via a gate 14. The ions are ejected out of the ion trap via an end cap 16 after necessary operations are performed in the linear ion trap 15.
The gate 14 controls the introduction of ions from outside the ion trap on the basis of signals from the DC circuit unit 9, while the end cap 16 controls the ejection of ions out of the ion trap on the basis of signals from the DC circuit unit 9.
The behavior of ions within the linear ion trap 15 is controlled by signals from the AC circuit unit 8. In this example, mass spectrometry is performed by an external device.
Although in this exemplary linear, ion trap, ions are introduced and ejected in the axial direction of the ion trap, the directions of introduction and ejection are not limited to the axial direction.
Although the cross-units 17 of the linear ion trap are circular, any cross-sectional shape can be used as long as ions can be trapped using an Rf signal and resonance ejection can be performed using a supplemental Rf signal. Furthermore, the ion trap may have one or more apertures formed in the middle thereof for introduction and ejection of ions, and may also have additional devices attached for tandem mass spectrometry.
All ions are introduced via the center of an end cap A18, and are ejected via the center of an end cap B20 after necessary operations are performed in the ion trap formed by a ring electrode 19 and a space surrounded by the end cap B20.
The cross-unit 21 of the end cap A, the cross-unit 22 of the ring electrode, and the cross-unit 23 of the end cap B may take any cross-sectional shape as long as ions can be trapped using an Rf signal and resonance ejection can be performed using a supplemental Rf. Furthermore, the ion trap may have one or more apertures formed in the middle thereof for introduction and ejection of ions, and may also have additional devices attached for tandem mass spectrometry.
Specifically, various devices, such as a quadrupole mass filter, a TOF, an orbitrap, an FTICR, etc. may be connected to the ion trap and used to enable tandem mass spectrometry to be performed. Even in that case, the embodiments of the present invention can be used in a similar manner (see description referring to
The pre-isolation time T2 may be eliminated by reducing the time width to zero. The post-isolation time T4 may include a time required for performing tandem mass spectrometry, such as CID, and a time required for cooling the thermal energy given to ions. It is also possible to increase the measurement throughput by reducing the time width of the post-isolation time T4 to zero.
In
When ions are introduced, an appropriate accumulation time can be set taking into consideration the space charge effect inside the ion trap. That is, on the basis of the past records stored in the parameter storage unit 7 and feedback information such as the detected amount of ions obtained by the detector unit 13, the total amount of ions trapped inside the ion trap can be estimated in a real-time manner to set the accumulation time so that the space charge effect does not occur.
Rf voltage S2 controls the q-value of all the ions introduced into the ion trap, thereby controlling how the ions inside the ion trap are exposed to the supplemental Rf.
End cap voltage S3 serves to control the ejection of ions at the outlet of the ion trap. Reducing this voltage ejects ions out of the ion trap, while increasing this voltage halts the ejection of ions.
Supplemental Rf voltage S4 controls the exposure of ions inside the ion trap to the supplemental Rf during the isolation time T3. Supplemental Rf S5 is the supplemental Rf to which ions are actually exposed.
It is possible to learn how these steps S1 to S5 are performed in the mass spectrometer by using a device, such as an oscilloscope, to check signal lines connecting the control unit 3 to the AC circuit unit 8 and the DC circuit unit 9 and wiring connecting the AC circuit unit 8 and the DC circuit unit 9 to the mass spectrometer unit 10 in the mass spectrometer 1.
Since such signals are typically prepared at low voltages, and are then amplified with amplifiers to be transmitted to the ion trap, etc., the signal lines before amplified by the amplifiers may be checked in that case.
The higher the mass of ion is, the lower the q-value becomes, and the lower the mass of ion is, the higher the q-value becomes. In
With respect to the trapped ions 27 before application of the supplemental Rf, the supplemental Rf is first set at a position spaced apart from the target precursor ions on the lower mass side thereof (28). A sweeping operation 29 is performed by gradually increasing the Rf voltage to sweep the q-value, thereby sequentially resonance-ejecting ions that come into resonance with the frequency set for the supplemental Rf (30).
In a similar manner, the supplemental Rf is then set at a position spaced apart from the target precursor ions on the higher mass side thereof (32), and the sweeping operation is performed by gradually reducing the Rf voltage to sweep the q-value, thereby sequentially resonance-ejecting the ions that come into resonance with the frequency set for the supplemental Rf (33). As a result, only the ions of interest are left from among the trapped ions (34).
In ion traps, a phenomenon referred to as the space charge effect is known to occur, in which the apparent mass is increased when an excessive amount of ions are introduced.
The occurrence of the space charge effect may prevent accurate isolation from being performed. However, performing isolation from the lower mass side as shown in
Although in this example, a supplemental Rf including only one frequency is used in the isolation process for both the lower and higher mass sides, the same principle can be basically applied even when frequencies for the lower and higher mass sides are combined, a plurality of frequencies are combined, or a broadband signal is used for either the lower mass side only or the higher mass side only, or for both of them.
In addition: as can be understood from the definition of Expression 2, the fact that the q-value is relatively low on the higher mass side when compared to the lower mass side can be cited as an important characteristic.
Although in this embodiment, the case in which a=0 is described, the q-value can represent the stability of ions even when a≠0, and the same advantages of the invention can still be achieved in a similar manner by evaluating the q-value using a curve based on the a-value in that case.
However, when such an approach using an FNF is found to adversely affect the precursor ions, a supplemental Rf obtained by combining one or more frequencies may also be used.
When the control sequence execution unit 6 included in the control unit 3 executes a control sequence such as shown in
The Rf voltage may be not only a continuous function with respect to time but also a piecewise continuous function with respect to time. In addition, it may vary either linearly or nonlinearly with respect to time and may include both linear and nonlinear segments.
In this example, the supplemental Rf is brought into the proximity of the precursor ions in an instantaneous manner, and is kept in that state for a predetermined time. In order to increase the throughput, it is preferable that the length of time for which the supplemental Rf is kept constant be reduced as much as possible. However, uniformly reducing the isolation time in a simple manner may cause a problem in that isolation can be only performed insufficiently for the higher mass side when compared to the lower mass side.
For this reason, it may be necessary to increase the length of time for which the ions on the higher mass side are exposed to the supplemental Rf (exposure time) when compared to the lower mass side so that unnecessary ions are removed thoroughly, within a range that enables a required minimum scanning time to be set.
When sufficient resonance ejection is possible for the higher mass side, the length of time for the higher mass side may be the same as the lower mass side, and when no ions exist on the higher mass side, the length of time may be reduced to zero. Conversely, when the amount of ions on the lower mass side is high thus disabling sufficient resonance ejection from being performed, the length of time for the lower mass side may be increased, and when no ions exist on the lower mass side, the length of time for the lower mass side may be reduced to zero.
As the supplemental Rf, it is also possible to use a broadband signal for both the lower and higher mass sides, and when sufficient resonance ejection is possible, such as when the number of ion species is low, a combination of one or more frequencies may be used for either the lower mass side only or the higher mass side only, or for both of them.
Although the present example can address the problem that resonance ejection is less likely to be performed for the higher mass side, when an FNF is used, a gap is actually generated between any two adjacent frequency components as shown in an enlarged manner in
However, when such a problem does not occur, this approach has an advantage in that the isolation time can be reduced substantially, e.g., it can be reduced even to about 1 ms.
Before and after the isolation, there is provided a time zone in which the supplemental Rf is not applied. This particularly has an advantage in that the thermal energy of ions after the isolation is reduced, thereby enabling the ions to be stabilized and unintended dissociation of the ions to be prevented.
Furthermore, by adjusting the width and gradient of the sweeping operation during isolation, it is possible to secure a longer sweeping time for the higher mass side than the lower mass side, thus enabling the exposure time to the supplemental Rf to be increased for the higher mass side. As a result, the problem that resonance ejection is less likely to be performed for the higher mass side can also be addressed as in the example shown in
When sufficient resonance ejection is possible for the higher mass side, the length of time for the higher mass side may be the same as the lower mass side, and when no ions exist on the higher mass side, the length of time may be reduced to zero. Conversely, when the amount of ions on the lower mass side is high thus disabling sufficient resonance ejection from being performed, the length of time for the lower mass side may be increased, and when no ions exist on the lower mass side, the length of time for the lower mass side may be reduced to zero.
As the supplemental Rf, a broadband signal may be used for both the lower and higher mass sides, and when sufficient resonance ejection is possible, such as when the number of ion species is low, a combination of one or more frequencies may be used for either the lower mass side or the higher mass side, or for both of them.
For this reason, in contrast with ions that are less likely to be dissociated, such as reserpine, instable ions, such as glycosylated peptides and protonated molecules of some low molecular weight compounds, may be resonance-ejected by such frequency components remaining in trace amounts or may be dissociated by the thermal energy, thus resulting in the number of the ions being reduced.
In particular, in portions close to the upper and lower edges of the frequency window, the signal intensity of the frequency components is higher than the central portion of the frequency window. Therefore, keeping the edge portions close to the target precursor ions for a long time may cause the precursor ions to be resonance-ejected or dissociated.
Actually, performing the scanning operation in this manner enables the required length of time to be reduced; sufficient isolation efficiency and increased sensitivity for weak ions can be achieved in a length of time of about 5 ms.
As an exemplary sample that imitates samples actually analyzed in the field, there was prepared a sample in which reserpine (unlikely to be lost), Substance P (RPKPQQFFGLM) (very likely to be lost), and a mass marker (Ultramark) (likely to be lost) are mixed taking into consideration the extent to which they are lost during isolation.
This is because some of samples actually analyzed in the field may be unlikely to be lost during isolation but others may be likely to be lost depending on the molecules included therein, and the above sample was prepared to reproduce such a situation. In addition, for ease of the experimental reproduction, the above sample was prepared using materials that are commonly distributed and easily available.
In general, some of biomolecules, such as peptides and post-translationally modified peptides, are known to have different likelihoods of being lost during mass spectrometry. From among the above three materials, Substance P having an amino acid sequence of RPKPQQFFGLM can be considered to represent molecules that are likely to be lost. When isolation is performed, the reduction of survival rate of molecules other than the molecules to be isolated may be sometimes considered important to achieve accurate analysis, but in other cases, the sensitivity for the molecules to be isolated may be considered more important than the survival rate reduction of molecules other than the molecules to be isolated. Therefore, it is necessary to modify the parameters for isolation so as to suit the specific purpose.
Typically, for analysis such as MS/MS and MS/MS/MS, it is important to reduce the survival rate of the other molecules to zero percent because they may affect the analytical result if they survive the isolation. In contrast, when the molecules to be isolated are likely to be lost during isolation, the other molecules may be allowed to survive to some extent so that the survival rate of the molecules to be isolated can be increased so as to increase the sensitivity.
Focusing on the sweeping time for the higher mass side when compared to the lower mass side as an isolation parameter, setting the sweeping time for the higher mass side at 1.2 times the sweeping time for the lower mass side enables the survival rate of molecules other than the molecules to be isolated to be suppressed to 20% or less when each of the three molecular species is isolated.
In order to reduce the survival rate of the molecules other than molecules to be isolated to zero percent, it was necessary to set the sweeping time for the higher mass side at 1.4 times the sweeping time for the lower mass side.
The above condition, i.e., the condition with which the survival rate of molecules other than the molecules to be isolated can be reduced to zero percent, may be set and commonly used as one of normal measurement modes.
For Substance P (RPKPQQFFGLM) representing molecules that are likely to be lost, it is possible to increase the ion survival rate by setting the sweeping time for the higher mass side at a value lower than 1.4 times the sweeping time for the lower mass side so as to increase the survival rate of the molecules to be isolated so that the sensitivity is increased, even though the other molecules may be also allowed to remain to some extent. More specifically, focusing on the divalent ions (674.86) of Substance P (RPKPQQFFGLM), while the survival rate was 30% for the above setting of 1.4 times, setting the sweeping time for the higher mass side at 1.2 times the sweeping time for the lower mass side not only increased the survival rate of the neighboring ions (685.90) to about 20% but also increased the survival rate of the divalent ions of Substance P (RPKPQQFFGLM) to 70%. Therefore, this setting is advantageous for soft ions, i.e., ions that are likely to be lost.
For reserpine, even if the sweeping time for the higher mass side is set at two times the sweeping time for the lower mass side, the survival rate of reserpine itself could be kept at 99%. Therefore, this setting is advantageous when the isolation capability is preferred.
In theory, it is also possible to set the sweeping time for the higher mass side at any value higher than two times the sweeping time for the lower mass side. For example, it is even possible to perform the sweeping operation for a length of time required for completely removing ions existing on the higher mass side if no consideration needs to be given to the throughput. However, portions of the device configuration other than the ion trap may sometimes impose constraints. In the present embodiment, the overall isolation time width is limited to 100 ms, taking into consideration MS/MS analysis by the following ECD and timing adjustment for tandem mass spectrometry by TOF. As a result, the sweeping time for the higher mass side is limited to being equal to or less than 50 times the sweeping time for the lower mass side. This means that if the sweeping time for the higher mass side is set at 50 times the sweeping time for the lower mass side and a sweeping operation of about 2 ms is performed for the lower mass side, the overall isolation time width becomes about 100 ms.
For a sample including ions having more or less the same likelihood of being affected during isolation, i.e., of being lost during isolation, it is possible to increase the sensitivity by modifying the sweeping time setting so as to suit the sample.
As the supplemental RF, a broadband signal may be used for both the lower and higher mass sides, and when sufficient resonance ejection is possible, such as when the number of ion species is low, a combination of one or more frequencies may be used for either the lower mass side or the higher mass side, or for both of them.
Here, the Rf voltage shown in
As another example, the RF voltage may also be achieved as follows. That is, when isolation is performed on the lower mass side, the RF voltage has a maximum value, the differential coefficient of a curve followed by the RF voltage with respect to time before the RF voltage reaches the maximum value is always positive or zero except for breakpoints, and the differential coefficient of the curve followed by the RF voltage with respect to time after the RF voltage reaches the maximum value is always negative or zero except for breakpoints, and when isolation is performed on the higher mass side, the RF voltage has a minimum value, the differential coefficient of a curve followed by the RF voltage with respect to time before the RF voltage reaches the minimum value is always negative or zero except for breakpoints, and the differential coefficient of the curve followed by the RF voltage with respect to time after the RF voltage reaches the minimum value is always positive or zero except for breakpoints.
As a result, although the Rf voltage is changed abruptly after the introduction of ions and the q-value of ions is also abruptly changed accordingly, the stability of ions is not affected even in that case.
In this case, although more time is consumed than
This example is advantageous for the case in which when an FNF is used as the supplemental Rf, ions are left at several regions because the scanning range is insufficient.
As in
Furthermore, using the fact that each potential is maintained for a predetermined time, it is also possible to perform resonance ejection in an effective manner by calculating the length of time corresponding to one period of the frequency used for the resonance ejection and setting the duration on the basis of the calculation result. In practice, it is possible to achieve sufficient resonance ejection by setting the duration of each potential so as to correspond to a length of time of about 4 to 5 times one period.
As the supplemental Rf, a broadband signal may be used for both the lower and higher mass sides, and when sufficient resonance ejection is possible, such as when the number of ion species is low, a combination of one or more frequencies may be used for either the lower mass side or the higher mass side, or for both of them.
In the present example, the manner in which the supplemental Rf is brought close to precursor ions on the lower mass side is modified so that not only the length of time for which the supplemental Rf is positioned close to the precursor ions can be reduced as much as possible but also the necessary range can be still scanned.
As the supplemental Rf, a broadband signal may be used for both the lower and higher mass sides, and when sufficient resonance ejection is possible, such as when the number of ion species is low, a combination of one or more frequencies may be used for either the lower mass side or the higher mass side, or for both of them.
In this case, the RF voltage may be applied as follows. That is, the RF voltage may be applied so that the RF voltage has an extreme value with respect to time, the RF voltage varies nonlinearly with respect to time, and the rate of change of the RF voltage is increased as it approaches the extreme value.
In this example, it is possible to sufficiently remove the other ions present immediately close to the precursor ions on the lower mass side by bringing the supplemental Rf close to the precursor ions and making the sweeping gradient more moderate. As a result, accurate tandem mass spectrometry can be performed after the isolation.
As the supplemental Rf, a broadband signal may be used for both the lower and higher mass sides, and when sufficient resonance ejection is possible, such as when the number of ion species is low, a combination of one or more frequencies may be used for either the lower mass side or the higher mass side, or for both of them.
In this case, the RF voltage may be applied as follows. That is, the RF voltage may be applied so that the RF voltage has an extreme value with respect to time, the RF voltage varies nonlinearly with respect to time, and the rate of change of the RF voltage is reduced as it approaches the extreme value.
From among the two ion species on the lower mass side, the ion species that is away from the ion species of interest has a large amount of ions, and therefore, they are exposed to the supplemental Rf for a relatively long time. In contrast, the ion species on the lower mass side that is close to the ion species of interest and the ion species on the higher mass side are substantially the same in quantity, and because the supplemental Rf is fixed, the q-values for these two species are the same during resonance ejection. Therefore, the same exposure time is used for both of these two species.
From among the two ion species on the lower mass side, the ion species that is away from the ion species of interest has a large amount of ions, and therefore, they are exposed to the supplemental Rf for a relatively long time. On the other hand, although the ion species on the lower mass side that is close to the ion species of interest and the ion species on the higher mass side are substantially the same in quantity, the q-value during resonance ejection is lower on the higher mass side. Therefore, the exposure time is set longer for the higher mass side.
Substance P (RPKPQQFFGLM) is ion species that is relatively likely to be dissociated. However, the ratio of ion intensity between
Furthermore, another ion species 24 shown in
In view of the fact that 99% of reserpine, i.e., ions less likely to be dissociated, could survive the isolation, it can be understood that a similar level of isolation efficiency was achieved also for ions that are likely to be dissociated.
The specific parameters set for measuring Substance P (RPKPQQFFGLM) were as follows. The m/z of the precursor ions was 450.4, the valence was 3, an FNF was used as the supplemental Rf, the width of the frequency window was a total of 40 Da, i.e., 20 Da on the lower mass side and 20 Da on the higher mass side, the sweeping operation was performed in Mode 1, and the sweeping parameters were such that on the lower mass side, the sweeping operation is performed up to a position that is 1.7 Da away from the precursor ions and the gradient of the Rf voltage is set so that the sweeping width is 5 Da, and on the higher mass side, the sweeping operation is performed up to a position that is 3 Da away from the precursor ions and the gradient of the Rf voltage is set so that the sweeping width is 7 Da.
In the present embodiment, the Rf voltage is controlled digitally. The time width for which each potential is maintained is 12 micro seconds. As the resonance frequency is at about 400 kHz, this time width may correspond to 4 or 5 times the period of an oscillation motion having such a frequency. The ratio of sweeping width between the higher mass side and the lower mass side corresponds to the ratio of sweeping time between them. This is because the sweeping operation is performed in a stepwise manner on the basis of a fixed, uniform time width. As a result, the sweeping time for the higher mass side is 1.4 times the sweeping time for the lower mass side in this case. The above described parameters achieved an overall isolation time of about 5 ms.
As shown in
Number | Date | Country | Kind |
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2010-067205 | Mar 2010 | JP | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/JP2010/072331 | 12/13/2010 | WO | 00 | 8/16/2012 |