Patent Application
20070138386
The present invention relates to a method and apparatus for ion fragmentation by electron capture.
Mass spectrometry is a well-known analytical technique in which ions of sample molecules are generated by a number of different techniques, and are then analysed according to their mass to charge (m/z) ratios. There are several ways to do this, including trapping ions (such as in the well-known Paul ion trap, or in a Fourier Transform Ion Cyclotron Resonance (FT-ICR) cell, for example) or by allowing the ions to fly through to a detector, such as in a Time of Flight (TOF) device.
One technique that is particularly useful in analysing larger molecules is tandem mass spectrometry, in which ions of a large sample molecule are broken into smaller, fragment ions for subsequent analysis. This procedure may provide detailed structural information on the original sample molecules.
Various techniques are known for inducing dissociation of the parent ions. The most common of these is collisionally induced dissociation (CID), where gas atoms or molecules such as argon, helium or nitrogen are employed to cause fragmenting through collisions with the sample ions. Other techniques, using infrared photon irradiation, for example, are also known for fragmenting ions. There are a number of problems with such techniques. The occurrence of internal fragmentation may complicate interpretation, and it is usual for the weakest bonds in a parent ion to be cleaved so that the same mass products are yielded in similar abundance.
In recent years, techniques involving dissociation through the use of electrons have been disclosed. One particular dissociation technique involving electrons is known as electron capture dissociation (ECD) and is described in, for example, Zubarev R. A., Kelleher N. L., McLafferty F. W., J. Am. Chem. Soc., 1998, 120: 3265-3266; McLafferty F. W., Fridriksson E. K., Horn D. M., Zubarev R. A., Science, 1999, 284: 1289-1290; and Haselmann K. F., Budnik B. A., Olsen J. V., Nielsen M. L., Reis C. A., Clausen H., Johnson A. H. Zubarev R. A., Anal. Chem. 2001, 73: 2998-3005. Here, low energy electrons are captured by parent ions (at least doubly protonated) resulting in fragmentation of the bonds in that ion to produce fragment ions. Compared to traditional techniques such as CID, for example, ECD has the major benefit that cleavage is of different and often analytically more helpful bonds. For example, in analysis of polypeptides, ECD cleaves the N-Cα backbone bonds, disulfide bonds, and so forth, whereas the traditional CID or laser (photon) dissociation techniques mainly cleave the amide backbone bonds (i.e. the peptide bonds). The two techniques (CID or other similar techniques, and ECD) may be employed together to produce complementary data.
ECD has, to date, largely been limited to FT-ICR because, for successful electron capture, the electrons must be travelling slowly (energies only slightly greater than thermal energies), and must have a relatively long residence time in the vicinity of the ions by which they will be captured. Any increase in electron energy creates a dramatic decrease in the capture cross-section. FT-ICR allows low energy electrons to be injected into a trapped ion cloud because of the very strong magnetic field generated by the superconducting magnet of the FT-ICR; electrons simply drift along the magnetic field lines into the ion cloud. One such prior art arrangement is described in US-A-2003/0104483, in which a filament is employed to radiate electrons into a cell of an FT-ICR mass spectrometer containing ions generated by liquid chromatography (LC). In an alternative arrangement, shown in US-A-2003/183760, a hollow cathode and an infrared laser are employed simultaneously to allow traditional or ECD fragmentation of ions in an FR-ICR cell.
FT-ICR mass spectrometry is, nevertheless, typically the most expensive and bulky of the current commercially available mass spectrometry techniques. Attempts to expand ECD to other forms of mass spectrometry have been relatively limited due to the fundamental requirement for low energy electrons. For example, in US-A-2002/0175280, electrons are injected into a Paul ion trap. Since electrons injected during most of the duty cycle of the RF field in the trap will be accelerated by that field to unacceptably high energies, the electrons are allowed to enter the trap only during a very short period during the RF cycle where the electron source potential is not above the trap potential. At other times, the electrons are unable to climb the potential barrier and do not enter the trap at all. The problems even so are a very limited duty cycle, a poorly defined electron energy (resulting in excessive fragmentation in the trap) and deteriorated analytical performance due to space charge effects in the trap.
WO-A-02/078048 discloses a variety of embodiments for seeking to realize ECD in FT-ICR, in a quadrupole (Paul) ion trap, and in an RF-only linear multipole arrangement (triple quadrupole). In the case of the FT-ICR device in this document, the problems of cost and size outlined above exist. For the Paul trap embodiment, the problems of a reasonable duty cycle and the need to avoid undue acceleration of electrons are present. In the case of the triple quadrupole arrangement, there is a very limited residence time of ions in the multipole arrangement so that very high electron currents are needed if any ECD is to occur. As a result, severe space charge effects occur. The residence time in the multipole of the incident ions is also difficult to control, leading to poor fragmentation control. Moreover, the multipole arrangement means that RF fields will be present. Even small RF fields are capable of destabilising electron beams, especially when there is a severe space charge problem.
The problem of ion residence time is addressed in WO-A-03/102545. This document describes trapping ions in a linear multiple ion guide using RF fields. Electron or positron capture dissociation is carried out in the ion guide structures, either alone or in combination with conventional ion fragmentation methods. This document discloses the use of a magnetic field, but this is to enhance the axial capture of slow electrons/positrons introduced into the ion guide. It is stated that the ions are not affected by the magnetic field. The techniques described in this document still suffer from the problem of the RF fields used to trap the ions causing electron destabilisation. There is also a necessary compromise between the position of the electron generator and the ion transport and trapping optics.
Finally, WO-A-03/103007 shows still a further dedicated ECD chamber for use as a stage of, for example a Q/TOF mass spectrometer. In the ECD chamber of this disclosure, ions are introduced either orthogonally, or opposed to, electrons from an electron generator. The document does not, however, address the question of how electrons or ions might be confined in the ECD chamber. The arrangement of WO-A-03/103007 will accordingly suffer from interaction times which are too low and too poorly controllable to provide an adequate fragmentation.
Against the background set out above, the present invention provides an improved ECD method and apparatus. Ions are trapped in a storage device magnetically, so that no RF fields are allowed (under normal circumstances) within the storage device during fragmentation. Although an RF multipole may be employed, in this case, the RF voltage supply is switched off during fragmentation to maintain electron stability at that time.
Embodiments of the present invention provide for the trapping of ions in a storage device, with (unlike in prior art FT-ICR arrangements) the resultant ECD fragments being passed on to the separate mass analyser once they have been created, rather than being analysed in the storage device. This allows the stringent requirements for uniformity of magnetic field to be reduced significantly, which in turn permits the use of compact permanent magnet or Tesla coils.
Additionally or alternatively, the incident ions are kept away from the source of electrons, unlike in the above-referenced non-FT-ICR prior art where the electron source is typically so close to the ion flight path that significant ion loss and even thermal decomposition is likely.
In accordance with a first aspect of the present invention, therefore, there is provided a method of generating fragment ions by electron capture, comprising: (a) directing ions to be fragmented into a fragmentation chamber of a mass spectrometer arrangement; (b) trapping at least some of the ions to be fragmented in at least one direction of the fragmentation chamber by using a magnetic field, the ions being trapped within a volume V; (c) generating an electron beam using an electron source located away from the volume V; (d) irradiating the trapped ions in the volume V with the electrons generated by the electron source in the presence of the said magnetic field, so as to cause dissociation; and (e) ejecting the resultant fragment ions from the fragmentation chamber for subsequent analysis at a different location away from the fragmentation chamber.
In a further aspect of the present invention, there is provided a mass spectrometer comprising: an ion source for generating ions of molecules to be analysed; a fragmentation chamber downstream of the ion source, the fragmentation chamber comprising an ion entrance aperture for receiving ions from the ion source, an ion exit aperture for ejecting ions from the fragmentation chamber, a magnet, and an electron source arranged to generate electrons for direction into the fragmentation chamber, the fragmentation chamber being arranged to trap ions that have entered through the ion entrance aperture within a volume V, the electrons from the electron source being directed towards the volume V so as to irradiate the trapped ions in the presence of the magnetic field generated by the magnet, in order to cause dissociation; and; a mass analyser, arranged to receive the resultant fragment ions that have been ejected from the ion exit aperture thereof.
Further advantageous features are set out in the dependent claims.
The invention may be put into practice in a number of ways, and some specific embodiments will now be described by way of example only and with reference to the accompanying Figures in which:
Referring first to
Downstream of the ion source 10 is a linear trap (LT) 21, which, as will be well known, allows mass-selective radial or axial ejection. Ions from the ion source 10 typically contain a range of mass to charge ratios, and ions of only a single mass to charge ratio are passed by the linear trap 21.
Downstream of the linear trap 21 is a fragmentation chamber 40. A transport multipole 30 is located between the linear trap 21 and fragmentation chamber 40. The fragmentation chamber 40 comprises a front plate 41, an opposing back plate 43, and side walls 42. An ion entrance aperture 44 is formed in the front plate 41 of the fragmentation chamber 40, to allow ions from the linear trap 21, via the transport multipole 30 to enter. The fragmentation chamber 40 also includes an electron emitter 60 which, typically, is an indirectly heated cathode or the like which generates a continuous stream of electrons. Formed in the back plate 43 of the fragmentation chamber 40 is an electron entrance aperture 45 which permits electrons emitted by the electron emitter 60 to enter the inside of the fragmentation chamber 40. In the embodiment of
Surrounding the fragmentation chamber itself is a permanent magnet 50. The axis of the magnetic field along the bore thereof is parallel to the axis of the transport multipole 30 which guides ions from the linear trap 21 into the fragmentation chamber 40, and also parallel to the longitudinal axis of the fragmentation chamber 40 itself.
In use, precursor ions and which are preferably of a single mass to charge ratio isolated in the linear trap 21 and which are preferably injected into the fragmentation chamber 40 as a pulse of length 1-2 ms duration from the linear trap 21, through the transport multipole 30, and through the ion entrance aperture 44 in the front plate 41 of the fragmentation chamber 40. After all ions have passed through the ion entrance aperture 44, the potential of that aperture 44 is raised and ions are trapped in the axial direction of the chamber 40 by a DC voltage on the front and back plates 41, 43. In the embodiment of
After an exposure time of about 5-50 ms, electron capture dissociation has taken place and the resulting fragment ions, and any remaining precursor ions, are ejected from the fragmentation chamber 40 back out of the ion entrance aperture 44. As such, the ion entrance aperture 44 is also an ion exit aperture 44. This is done by lowering the voltage on the front plate 41. The electron emitter 60 may remain in continuous operation during this time period.
Upon ejection from the fragmentation chamber 40, fragment ions pass back through the transport multipole 30 to the linear trap 21. Subsequent mass analysis is then carried out in the usual manner.
Various options are contemplated with the arrangement of
The use of a linear trap 21 is preferable as opposed to, for example, a 3-D quadrupole (Paul) trap, due to the much higher trapping efficiency of the linear trap (up to 50-90% of incoming ions, compared to a few percent in a quadrupole trap), as well as higher space charge capacity.
It will be understood that the arrangement of
In
Upon exiting ms-1 20 , the precursor ions of the selected mass charge ratio enter a curved entrance multipole 31. This contains, in the preferred embodiment, a right-angled bend so that precursor ions exiting ms-1 20 in a first direction leave the curved entrance multipole 31 substantially at 90° to the direction of exit from the mass filter.
Upon exiting the curved entrance multipole 31, ions enter a fragmentation chamber 40′. This is similar to the fragmentation chamber 40 of
The electron entrance aperture 45 formed in the back plate 43 is generally coaxial with the ion entrance aperture 44 formed in the front plate. Thus, ions entering the fragmentation chamber 40′ are irradiated by electrons arriving along a broadly similar axis, but in the opposite direction.
Once fragments have been generated (as described in connection with
Aligned with the ion exit aperture 46 is a curved exit multipole 32. The curved exit multipole 32 has, like the curved entrance multipole, a 90° bend in it. Thus, fragment ions exit the fragmentation chamber 40 in a direction parallel with, but in the opposite direction to, the precursor ions arriving at the ion entrance aperture 44. They are then curved round in the curved exit multipole so that they arrive at a second stage of mass analysis (hereinafter referred to as ‘ms-2’) 70 which is separate from, but has an axis generally parallel with, ms-1 20.
As with the embodiment of
The ion entrance aperture 44 is formed within a front plate 41 of the fragmentation chamber 40″. This ion entrance aperture 44 is in turn coaxial with an ion exit aperture 46 within the back plate 43 of the fragmentation chamber 40″. Also formed in the back plate 43 is an electron entrance aperture 45 to allow injection of electrons from an electron emitter 60 outside of the back plate 43. The electron entrance aperture 45 is radially spaced on the back plate 43 from the ion exit aperture 46. Thus, there is a direct line of sight between the exit of ms-1 20, the entrance multipole 31, and the ion entrance and exit apertures 44, 46 within the fragmentation chamber 40″ of
In use, precursor ions enter the fragmentation chamber 40″ through the ion entrance aperture 44. As previously, the voltage on the front plate 41 is increased to generate a potential well in the axial direction for axial trapping. Radial trapping is, again as previously, achieved through the application of a magnetic field from permanent magnets 50. Once trapped, the precursor ions in the fragmentation chamber 40″ are displaced via magnetron motion off the axis defined between the ion entrance and exit apertures 44, 46, transversely across to a second axis defined perpendicular to the electron entrance aperture 45. Once resident on this second axis, the ions are irradiated by the incident electrons and electron capture dissociation occurs. After a suitable period of time, such as 1-2 ms again, the resultant fragment ions are displaced back onto the first axis defined between the ion entrance and ion exit apertures 44, 46. Once there, the voltage on the back plate 43 may be reduced to allow ejection of the fragment ions out of the ion exit aperture 46.
An exit multipole 32′ is preferably aligned with the ion exit aperture 46 so that the fragment ions are guided by the exit multipole 32′ from the ion exit aperture 46 to a mass analyser 70 downstream of the fragmentation chamber 40″.
A fourth embodiment of the present invention is shown in
The fragmentation chamber 40′″ comprises front and back plates 41, 43 with ion entrance and ion exit apertures 44, 46 respectively. Both the ion entrance aperture 44 and the ion exit aperture 46 are coaxial with one another and also with the entrance multipole 31′ and ms-1 20 . The fragmentation chamber 40′″ also comprises an electron emitter 60 and permanent magnets 50.
In the embodiment of
Downstream of the ion exit aperture 46 (which is also an electron entrance aperture, it will be understood) is an exit multipole 32′. In order to avoid scattering of the electron beam 60, the voltage on the exit multipole 32′ must be switched off whilst the electrons pass into the fragmentation chamber 40′″. Once fragments have been generated, voltages may be applied once more to the exit multipole 32′, along with a reduction in the voltage on the back plate 43, to allow the fragment ions to pass out of the fragmentation chamber 40′″ into the exit multipole 32′ and from there to a mass analyser 70.
Additional RF fields (especially those produced using hexapole or octapole devices, or using a set of apertures) may assist in the storage of high mass ions, by augmenting at higher radii the magnetic field which has a limited effect on high mass ions. The net result of the RF field is the same as employing a larger permanent magnet. At the same time, low mass fragments are kept near the axis by the magnetic field, so that the low-mass cutoff in RF fields (a known effect) does not result in ion ejection of these low mass ions. Such extension of the mass range both upwards and downwards is particularly important in electron-based dissociation, because fragments formed during such electron dissociation tend to have a lower charge state than their original pre-cursor ion, so that m/z of the fragment may also be much higher than the m/z of the precursor ion.
It is also possible to employ an RF voltage waveform which is pulsed, and where the duty cycle of that waveform is relatively low. For example, a 400 kHz waveform may be employed, with pulses having a 250 ns duration and with a 2000 ns (2 μs) gap between them. The electrons will enter the volume defined between the front and back plates and the storage multipole 48 throughout the cycle of the RF field. Whilst the voltage pulses are present, however, the electrons will not remain on the axis of the storage multipole 48 but will instead be pushed onto the poles themselves. This is why a relatively long period between pulses is desirable, since it is during that period that the electrons will reside amongst the ions on the axis to allow electron capture dissociation.
In the embodiment of
The final embodiment, shown in
Magnetic trapping alone has certain attractions, not least that, in the absence of any RF fields, the electrons should not be accelerated or dispersed, but should instead follow the magnetic field lines and drift at lower energies into the ion cloud trapped in the fragmentation chamber 40. The maximum m/z that may be trapped depends upon the magnetic field strength of the permanent magnet employed. With modern permanent magnets, a mass range up to about 2000-4000 Daltons may be stored. Obviously, by using superconductive magnets, larger mass ranges could be stored, but this results in a very expensive fragmentation chamber over all.
The use of an assisting RF field does allow much higher mass ranges to be trapped (as explained above) but means that there is the possibility of dispersal and/or acceleration of electrons at certain times.
Whilst a number of specific embodiments have been described, it will be appreciated that these are by way of example only and that various modifications could be contemplated. For example, the fragmentation chamber 40 could be formed from a quadrupole ion trap, a linear multipole ion trap with mass selective axial ejection, a linear multipole ion trap with mass selective radial ejection, an FT-ICR mass spectrometer, an ion tunnel trap comprising a plurality apertures connected to AC power supplies, or other devices.
Further activation methods may be employed to assist with electron fragmentation. For example, a collision or reaction gas may be added to the fragmentation chamber 40. Stored ions may be irradiated by pulsed or continuous laser radiation. The fragmentation chamber 40, or a part thereof, may be heated. As still a further alternative, ions of the opposite polarity to that of the ions of interest may be introduced from an additional ion source or created with the fragmentation chamber 40.
Moreover, whilst the foregoing preferred embodiments have been described in terms of electron capture dissociation (ECD), since the earliest publication in this field, it has been known that electrons may also cause other types of fragmentation. For example, ‘hot’ electron capture dissociation may occur at higher electron energies, and electron detachment dissociation may occur for negative ions. Accordingly, it is to be understood that the present invention is not limited to ECD, and that any form of dissociation that involves electrons is to be considered to fall within the scope of this invention.
Either ms-1 20, or ms-2 70, could be any of: a quadrupole ion mobility analyser, a quadrupole ion trap, a linear ion trap, a time of flight mass spectrometer, an FT-ICR mass spectrometer, a so-called orbitrap, as described in, for example, WO-A-02/078046, or any combination thereof. Instead of permanent magnets, Tesla coils may be employed. A high current electron emitter may be employed instead of an indirectly heated cathode, or an array of electron-emitting cathodes (including those made as an integrated circuit), or any other electron-emitting device may be contemplated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0407152.8 | Mar 2004 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB05/01198 | 3/29/2005 | WO | 9/13/2006 |
