Patent Publication Number: US-2010123073-A1

Title: Electron capture dissociation in a mass spectrometer

Description:
FIELD 
     This invention relates to a mass spectrometer, more particularly a quadrupole/time-of-flight mass spectrometer, with capabilities to study daughter or secondary ions generated by, for example, electron capture dissociation (ECD). 
     BACKGROUND 
     The following paragraphs are not an admission that anything discussed in them is prior art or part of the knowledge of persons skilled in the art. 
     Recently developed electron capture dissociation (ECD), and electron transfer dissociation (ETD) techniques are important complements to the usual collision-induced dissociation (CID) method for the production of structurally-informative ions. These new methods tend to cleave peptides along their backbones, leaving the side chains intact, so they are especially useful for the analysis of post-translational modifications. Under some conditions (“hot electron capture dissociation”—HECD), they also have the ability to distinguish between isomeric leucine and isoleucine residues. Moreover, they are often more effective than CID in breaking up large parent ions, since a recombination energy of 4 to 7 eV per parent ion is available, (unlike CID, which relies on the collision energy in the centre-of-mass system). 
     A recent proposal by Syka et al implements ETD in a quadrupole ion trap (J. E. Syka, J. J. Coon, M. J. Schroeder, J. Shabanowitz and D. F. Hunt,  Proc Natl Acad Sci USA  101 9528-33 (2004)), but practical applications of ECD have until recently been restricted to FTICR spectrometers (H. J. Cooper, K. Hakansson and A. G. Marshall,  Mass Spectrom Rev  24 201-22 (2005)) because of the perceived need for a long interaction time between the necessary low energy electrons (&lt;0.3 eV), and the parent ions. However, this interaction time (originally ˜sec) can be reduced by several orders of magnitude (to ˜ms) by supplying high electron density with improved electron sources (such as dispenser cathodes), so the very long storage times provided by FTICR are no longer necessary. In addition, there are reasons why FTICR spectrometers are still far from an ideal solution to the problem: 
     FTICR instruments are presently an expensive and complicated variety of mass spectrometer, so the number of laboratories able to implement the technique in this way is likely to remain limited. 
     Even in FTICR spectrometers the ECD fragmentation efficiency is not very high, at least for doubly charged parents (e.g. ˜10% at 1570 Da as reported by M. A. McFarland, M. J. Chalmers, J. P. Quinn, C. L. Hendrickson, A. G. Marshall,  J Am Soc Mass Spectrom  16 1060-1066 (2005)).). This is probably caused by the low electron density achieved in this configuration, the high degree of coherence in the FTICR cyclotron/magnetron motion of the ions, and the confinement of an electron very close to a given line of force by the strong magnetic field; it has been suggested in this paper that once “the electrons . . . bleach a hole through the ion cloud”, the undissociated ions “ . . . never come into contact with the electrons” The coherence could probably be modified by gas collisions, but admission of gas to the FTICR chamber is a time-consuming and inefficient process. 
     It would be useful to make these methods available for use in other types of instrument if reasonable sensitivity can be achieved, and particularly if the measurements can be carried out on a time scale compatible with HPLC (High Performance Liquid Chromatography). Considerable progress in this direction has recently been reported. Zubarev has observed ions formed by ECD in a 3-D quadrupole ion trap (D. A. Silivra, I. A. Ivonin, F. Kjeldson and R. A. Zubarev, 52 nd  ASMS Conference, Nashville Tenn., May 2004), (O. A. Silivra, F. Kjeldsen, I. A. Ivonin, R. A. Zubarev,  J Am Soc Mass Spectrom  16 22-27 (2005), although at fairly low intensity. Baba et al, injected electrons and ions into a linear quadrupole ion trap with a superimposed magnetic field, (T. Baba, Y. Hashimoto, H. Hasegawa, A. Hirabayashi and I. Waki,  Anal Chem  76 4263-6 (2004) and see also published U.S. Patent Application 2005/0178955]. They used a TOF mass spectrometer to observe the daughter ions produced by ECD, including the characteristic c and z ions, and their ion production efficiency was comparable to Zubarev&#39;s (˜4% fragmentation efficiency for Substance P, 1347 Da). However, this group has recently employed a different configuration of the ion trap/TOF instrument with improved results (H. Satake, H. Hasegawa, A. Hirabayashi, Y. Hashimoto, T. Baba, and K. Matsuda,  Anal. Chem.  79 8755-8761 (2007)). This device is claimed to have much higher efficiency, and also to be compatible with the HPLC time scale. 
     Nevertheless, this design may not represent the optimum configuration for an ECD quadrupole/TOF instrument. It appears to have a number of disadvantages including: 
     A complicated instrument geometry that requires the ion beam to be deflected 90 degrees by a quadrupole deflector in order to enter and leave the ECD cell. This probably introduces problems in alignment. 
     There may also still be the problem of electron heating produced by the quadrupolar electric field, which tends to move the electrons away from the axis, and shifts the electron energy to higher values, where the cross section for interaction is much smaller. The magnetic field exerts a constraining effect on the electrons, but this may not always be sufficient to keep them well focused on the axis (see calculations below in  FIG. 5   b ). 
     INTRODUCTION 
     The following introduction is intended to introduce the reader to this specification but not to define any invention. One or more inventions may reside in a combination or sub-combination of the apparatus elements or method steps described below or in other parts of this document. The inventor does not waive or disclaim his rights to any invention or inventions disclosed in this specification merely by not describing such other invention or inventions in the claims. 
     The present invention can use a geometry similar to a QqTOF spectrometer, for example a Sciex QqTOF spectrometer, but with an additional CID/ECD cell on the opposite side of the TOF section from the other quadrupoles. In the present invention, mass-selected and cooled ions are injected into this cell through the storage region of the accelerating column. An innovative circuit to drive the collision cell quadrupole with minimal electron excitation is also provided, in case the magnetic field is inadequate to provide sufficient electron confinement. 
     In accordance with a first aspect of the present invention, there is provided a mass spectrometer comprising: 
     a source of ions having desired characteristics; 
     a time-of-flight mass spectrometer section including a modulator having a storage region, and first and second apertures on opposite sides of the storage region of the modulator, the first aperture providing a connection to the source of ions; and 
     a cell, for at least one of collisional-induced dissociation and electron capture dissociation, connected to the storage region of the modulator of the time-of-flight mass spectrometer section by the second aperture, whereby, in use, ions from the source of ions can pass through the first aperture, the modulator and the second aperture into the cell, for at least one of collisional-induced dissociation and capture of electrons, in order to generate daughter ions, and the daughter ions are passed back into the time-of-flight mass spectrometer section for analysis. 
     The source of ions can comprise an electrospray ion source, by itself, or with the addition of a mass selection device comprising at least one mass selection quadrupole rod set. Alternatively, the ion source can comprise an electrospray or other source and a quadrupole or other multipole rod set configured to focus the ions (it is here noted that while the invention is generally described as using quadrupole rod sets, for some purposes other multipole rod sets could be used.) while they are being cooled by collisions with a gas. 
     In accordance with another aspect of the present invention, there is provided a mass spectrometer comprising a source of ions of a desired mass, a mass analysis device having first and second connection apertures, with the first connection aperture providing a connection to the source of ions, and the second aperture providing a connection to a cell, for at least one of collision-induced dissociation and electron capture dissociation, The cell is connected to the mass analyzer by the second aperture, whereby ions from the source of ions can be passed through the mass analyzer into the cell to generate secondary ions, and the secondary ions are passed back into the mass analyzer for analysis. 
     The present invention also provides a method of mass analysis of ions, the method comprising:
         (i) providing a supply of ions having desired characteristics;   (ii) passing the ions through the modulator storage region of a time-of-flight mass analysis section;   (iii) passing the ions into a cell at energies suitable for at least one of collision-induced dissociation and electron capture dissociation to produce secondary ions;   (iv) for either collision-induced dissociation or electron capture dissociation supplying a collision gas to the cell, and for electron capture dissociation supplying electrons to the cell; and   (v) passing the secondary ions into the time-of-flight mass spectrometer section for analysis.       

     Another aspect of the method of the present invention comprises:
         providing a supply of ions having desired characteristics;   (ii) passing the ions through a mass analysis section;   (iii) passing the ions into a cell and supplying at least one of electrons to the cell for electron capture dissociation whereby the ions capture electrons to generate secondary ions, and a collision gas whereby the ions collide with the gas to generate secondary ions; and   (iv) passing the secondary ions into the mass analysis section for analysis.       

     The present invention also provides an electron capture cell comprising an electron source, a multipole rod set, and inlet aperture at one end for ions, a cathode for generating electrons at another opposite end thereof, a solenoid around the multipole rod set and a device for imparting an axial electric field along the rod set whereby, in use, an axial electric field can be established tending to drive electrons away from the inlet aperture and to drive positive ions generated in the electron capture cell towards the inlet aperture. 
     An additional aspect of the method of the present invention comprises effecting electron capture dissociation, the method comprising: 
     a) providing an electron capture cell with a multipole rod set to guide ions; 
     b) supplying positive ions at one end of the capture cell, and supplying electrons from another opposite end of the cell in the opposite direction to the supply of the ions; 
     c) providing an electric field along the electron capture cell tending to drive the positive ions towards one end thereof and to drive the electrons towards the other end thereof. 
    
    
     
       DETAILED DESCRIPTION OF THE DRAWINGS 
       For better understanding of the present invention and show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: 
         FIG. 1  is a schematic diagram of a mass spectrometer in accordance with the present invention; 
         FIG. 2  is a graph showing a waveform for excitation of a quadrupole for performance of ECD in the mass spectrometer of  FIG. 1 ; 
         FIG. 3  shows a possible synchronization of pulses applied to a dispenser or field effect cathode with a waveform applied to the quadrupole in the electron collision cell; 
         FIG. 4  is a graph showing synchronization of the extraction voltage on a quadrupole within an upstream mass selection section with the extraction voltage in a time-of-flight section of the mass spectrometer of  FIG. 1 ; 
         FIG. 5   a  is a simulation of ion trajectories in a quadrupole rod set under different driving voltages, and 
         FIG. 5   b  is a simulation of electron trajectories in the quadrupole rod set under different driving voltages; 
         FIG. 5   c  is a simulation showing ions and electrons traveling in opposite directions; 
         FIG. 6  is a schematic diagram of a driver circuit for generating the square waveform of  FIG. 2 ; and 
         FIG. 7  is a schematic diagram of another driver circuit for generating the square waveform of  FIG. 2 . 
     
    
    
     DETAILED DESCRIPTION 
     Various apparatuses or methods will be described below to provide an example of an embodiment of each claimed invention. No embodiment described below limits any claimed invention and any claimed invention may cover apparatuses or methods that are not described below. 
     The claimed inventions are not limited to apparatuses or methods having all of the features of any one apparatus or method described below or to features common to multiple or all of the apparatuses described below. It is possible that an apparatus or method described below is not an embodiment of any claimed invention. The applicants, inventors and owners reserve all rights in any invention disclosed in an apparatus or method described below that is not claimed in this document and do not abandon, disclaim or dedicate to the public any such invention by its disclosure in this document. 
     Additionally, reference is made, in the detailed description and elsewhere, to various prior publications, in the patent and non-patent literature, and the contents of all of these are hereby incorporated by reference. 
       FIG. 1  shows an embodiment of the invention, for producing either CID or ECD in a relatively simple configuration derived from a QqTOF spectrometer, for example a Sciex QqTOF spectrometer. One aim is not to do “top-down” sequencing, for which the “unlimited” m/z range of the TOF spectrometer is suitable, but for which the FTICR instrument has unique advantages in resolution. Instead, the inventors plan to improve “bottom up” sequencing by exploiting the ability of ECD to break up large ions. Thus the sample will be digested, as in conventional “bottom up” sequencing, but with more selective proteases, such as LysC, (instead of trypsin), and/or with shorter digestions (in order to create additional missed cleavages). This will produce considerably larger proteolytic peptides, which can still be broken up by ECD because of the large amount of recombination energy available. Further breakups of the daughter ions by successive electron captures may follow, as shown by Satake et al (citation above), thus producing lower charge ions, and simplifying the spectrum, The combination of a large peptide (which makes it easier to fit the daughter ion sequences together), the relatively simple spectrum produced from a low-charge ion, and the high absolute mass accuracy obtainable in a TOF instrument for daughter ions with m/z values up to a few thousand, should be a potent recipe for bottom-up sequencing. This procedure also avoids the need to carry out a separate set of reactions (proton transfer) to simplify the spectrum, as is often necessary in ETD. The geometry of the mass spectrometer, indicated generally at  10  in  FIG. 1 , is similar to that in the QqTOF (electrospray version) (A. Loboda, A. Krutchinsky, M. Bromirski, W. Ens, and K. G. Standing,  Rapid Commun. Mass Spectrom.  14 1047-1057 (2000)). However, an additional (CID/ECD) collision cell  12 , or q 3 , has been inserted on the other side of a TOF section  50  of the TOF instrument. 
     An ion source  14  is an electrospray source, which provides parent ions with charge  2  or more. An intermediate pressure chamber  16  of the mass spectrometer receives the ions, and from this chamber  16  the ions pass through into a chamber  18  with a first quadrupole rod set, commonly designated q 0 . From the chamber  18 , the ions pass through into a chamber  20  having a second quadrupole rod set, again by common convention often designated as Q 1 . As indicated, this rod set can be provided with a short set of rods, often designated as “stubbies” and providing a Brubaker lens. From the chamber  20 , the ions pass through into a chamber  22  provided with a third quadrupole rod set, again by common convention often designated q 2 . 
     As shown for the chambers  16 ,  18 , and  22 , connections can be provided to gas sources and to vacuum pumps to maintain desired pressures within these chambers. Chambers  16 ,  18 , and  22  are supplied with a chemically non-reactive gas (nitrogen, helium, argon, xenon, or similar) maintained at intermediate pressures (˜10 −2  Torr−1 Torr) to provide collisional cooling, while chamber  20  should have a good vacuum (˜10 −5  Torr). 
     Additionally, in known manner connections to the rod sets would be provided to AC and DC voltage supplies. For simplicity, vacuum and voltage supplies and other conventional peripherals are not shown. 
     The additional CID/ECD cell  12  provides a collisional dissociation/electron capture chamber  30  including another quadrupole rod set q 3 . It is attached to an electron source chamber  32  including a cathode  34  providing a source of electrons. An aperture  36  is provided between the chambers  32  and  12 . The electron source chamber  32  has a connection  38 , for connection to a vacuum source to maintain a desired low pressure (˜10 −5  Torr). Due to the possible high gas loads imposed by the collision gas in the enclosure around the quadrupole for ECD, a hafnium carbide cathode may be used. It is more durable than other possible cathodes (tungsten filament, lanthanum hexaboride, and others) in the presence of higher pressures and can tolerate pressures of up to 10 −4  Torr. The electron collision chamber  32  is provided with a connection  40  for supply of chemically non-reactive gas (nitrogen, helium, argon, xenon, or similar) to create a pressure of between 1 and 100 milliTorr. A solenoid  44  capable of generating an axial magnetic field of greater than 100 Gauss for guiding electrons is provided around the chambers  30 ,  32 . Ions to or from the ECD cell pass through a pair of apertures (typically 2 mm diameter from the output of the quadrupole; a horizontal rectangular aperture typically 1.5 mm by 6 mm) and various ion optical elements, connected to the TOF modulator region. These are designed to minimize vertical spread in the ion beam and thereby improve resolution. 
     The TOF section  50  includes a modulator  52  with a storage region  51 , and an acceleration column or region  54  extending upwards in this view from the storage region  51 , including an orthogonal pusher electrode in known manner. The acceleration region  54  of the modulator  52 , in use, causes ions to travel towards an ion mirror  56  through a field free drift region  58 , in which ion separation can occur. In known manner, the ion mirror  56  reverses the motion of the ions and directs them towards a four anode detector  60 . 
     The other unmodified sections of the mass spectrometer are operated in the conventional manner. There may be the need for additional ion focusing elements at the exit of q 2 . 
     In use, ions generated from the electrospray source  14  pass through the intermediate chamber  16  and are cooled within the first quadrupole rod set q 0  in the chamber  18 . The second quadrupole rod set Q 1  is operated to mass select ions of interest, and the mass selected ions pass into the chamber  22 , where the third rod set q 2  is operated to focus the ions while cooling is accomplished through collisions with the bath gas. 
     In accordance with the present invention, the selected, cooled and focused ions from the chamber  22  then pass through a first connection aperture  26  into the storage region  51  of the time-of-flight modulator  52  At this time, no orthogonal extraction pulses are applied to the pusher electrode. Rather, the ions are permitted to travel through the storage region  52  and through a second set of apertures and ion optics  42  into the CID/ECD chamber  30 . 
     It can be noted that the quadrupole q 2  in chamber  22 , usually operated as a collision cell in the conventional qQTOF configuration, now simply serves to provide additional cooling and pulsing for the mass selected ions; as is explained below, q 2  and the chamber  22  can be used to store ions and permit them to pass through the aperture  26  in pulses. 
     Electrons are emitted from the dispenser cathode  34  in a fairly high vacuum (less than ˜10 −4  Torr) and then pass into the electron capture chamber  30 , of the ECD cell  12 , through an axial aperture at the end of the quadrupole. These low energy electrons are confined by a longitudinal magnetic field produced by the solenoid  44  surrounding the quadrupole rod set q 3  and the chambers  30 ,  32 , so that they spiral along the axial magnetic lines of force. As detailed below, the electron beam may be pulsed so as to coincide with the zero-field part of the TOF waveform, or operated continuously. 
     This physical arrangement has several advantages, including: 
     The simple geometry, providing direct axial intersection of the ion and electron beams, and making it much easier to provide the required spatial overlap; 
     A simple magnetic field configuration (solenoid), with easy adjustment of magnetic field strength. Once optimum magnetic field conditions are determined, the solenoid could possibly be replaced by a permanent magnet configuration; 
     Positioning of the electron source within the magnetic field of the solenoid, thus minimizing the effects of space-charge blowup and allowing the use of high electron currents; 
     Separation of the functions of parent ion selection (in Q 1 ), and collisional cooling of the selected ions (in q 2 ), thus providing a high quality beam for transport between q 2  and the ECD cell; 
     Minimal exposure of the electron source to the most serious contaminants, such as oxygen; and 
     The ability to use alternate modes of operation (see below). 
     The quadrupole excitation can be modified by the use of a square waveform, instead of the conventional sinusoidal waveform. A square wave excitation yields performance comparable with that delivered by the usual sinusoidal excitation, as well as additional flexibility. However, the present inventors have realized that it also has an additional advantage in this particular case. This is the ability to tailor the waveform so as to provide zero quadrupole field for a reasonable fraction of the RF cycle, during which time the electrons are not accelerated by the RF field (H Wang, Y Wang D. Kennedy, Y Zhu and K Nugent, 53 rd  American Soc. for Mass Spectrometry, San Antonio Tex. June 2005, Poster TP 23, Berkout U.S. Pat. No. 6,858,840). Other methods for generating a zero-field interval in the quadrupole excitation waveform are also envisioned, including incorporating a zero-field interval into every period of a triangular or sinusoidal waveform, intermittently shutting off the excitation field, or forming the waveform from mixing of two sine waves with slightly different frequencies. However, these other solutions introduce greater complication and less flexibility than the method of rectangular waveform excitation. 
     For example, a quadrupole driver constructed for this purpose (and discussed in greater detail below) can be programmed to produce the idealized voltage shown at  72  in  FIG. 2 . The superimposed and conventional sinusoidal waveform normally used in such quadrupoles is indicated at  70 . The amplitudes of the signals  70 ,  72  in  FIG. 2  are chosen so that their integrated positive voltages are equal. 
     In addition, the quadrupole rod set in the electron collision chamber  30  is provided with extra electrodes between the quadrupole rods, in order to provide an axial field, or the quadrupole rods ion can be configured to generate the field. These additional electrodes or modifications can be in accordance with the U.S. Pat. No. 6,111,250, hereby incorporated by reference, although the configuration may be that described in A. Loboda, A. Krutchinsky, O. Loboda, J. McNabb, V. Spicer, W. Ens, and K. G. Standing, “Novel Linac II Electrode Geometry for Creating an Axial Field in a Multipole Ion Guide”,  Eur. J. Mass Spectrom.  6 531-536 (2000). 
     In use, ions from the ion source  14  pass into the quadrupole q 0  in the first chamber  18 , and then pass through the quadrupole Q 1  in the second chamber  24  for mass selection. The mass selected ions are then cooled and focused on the axis by the quadrupole q 2  in chamber  22 . 
     As shown in  FIG. 4 , a small pulsed DC offset  80  is provided at the outlet of the chamber  22 , as an ion shutter. As detailed below, this offset voltage  80  is synchronized with an orthogonal extraction pusher voltage  78 . This synchronization is such that the voltage  80  is high, preventing passage of ions into the time-of-flight section  50 , when ions leaving q 2  would be accelerated into the field free drift region  58  by the extraction pulse intended for secondary ions emerging from the ECD cell. As indicated, the period for each cycle is approximately 300 microseconds. After each pulse applied as the extraction voltage, the voltage on q 2  goes low, to permit ions to pass through the storage region of the modulator when no extraction field is present. 
     The effect of this is that ions pass out of the rod set q 2  into the storage region of the modulator  52 , and then pass straight through into the electron capture chamber  30  with no loss of ions into the TOF spectrometer. 
     In the electron collision chamber  30 , a pulse of electrons is injected at the beginning of the zero field interval. It then slows down gradually and finally reverses direction. This is caused by the presence of a small DC axial voltage gradient that may be generated by specially shaped LINAC (linear accelerator) electrodes such as those described in (A. Loboda, A. Krutchinsky, O. Loboda, J. McNabb, V. Spicer, W. Ens, and K. G. Standing, “Novel Linac II Electrode Geometry for Creating an Axial Field in a Multipole Ion Guide”,  Eur. J. Mass Spectrom.  6 531-536 (2000). The polarity is chosen such that the ion aperture end of the quadrupole for ECD is at a lower potential than the electron aperture end. This arrangement causes both ions and electrons entering from their respective ends to slow down and reverse direction. The electrons always have very low energy, and in fact reach zero axial velocity when they turn around, thus maximizing the cross section for capture. This process is repeated, with a pulse of electrons injected once in every q 3  RF cycle, as shown in  FIG. 3 . Electrons, for example, may have initial energies of the order of 10 eV or a few 10&#39;s of electron volts. It is not practical to predict interaction cross sections for the large biomolecules envisioned, but calculations on simpler systems are given by [D. R. Bates, Adv. Atomic Molec. Physics 34, 427-486 (1994) and C. Rebrion-Rowe, Int Rev. Phys. Chem 16, 201-213 (1997)]. They indicate that the cross section for electron capture increases significantly as the electron energy decreases. 
       FIG. 3  shows at  74  the waveform applied to the quadrupole in the electron capture chamber  30 , as shown in greater detail in  FIG. 2 . There are two envisioned methods of operation for the electron source: pulsed emission and continuous emission. With continuous emission, the electron source produces a continuous stream of free electrons that are able to penetrate into the ECD quadrupole and interact with ions during the field-free fraction of the quadrupole waveform. During the positive and negative parts of the quadrupole waveform, the electrons are deflected from the quadrupole axis by the field from the quadrupole rods, so they do not interact with the ions in the cell. This method of operation is believed to be more appropriate for thermionic cathodes. For a pulsed source, this may be effected by providing a grid immediately adjacent to the actual source, and applying a control voltage to it to control emission of electrons. During the positive and negative parts of the quadrupole waveform, no voltage is applied to the control grid and hence no electrons are emitted. This method of operation is more appropriate for electron sources that can produce increased current densities when operated in a pulsed fashion. This is illustrated in  FIG. 3 . At the start of each RF cycle, indicated at  74 , the voltage is applied to the grid, and the electron current is indicated by the waveform  76 . 
     The electrons trapped radially by the magnetic field, and longitudinally by the electric field of the LINAC, make a double pass, forward and back, through the ion distribution in the quadrupole in the electron capture chamber  30 . Calculations below show that the electron density can be high before space charge becomes a limiting factor. 
     The injected parent ions from the chamber  22  will slow down to thermal energies in the ECD cell or chamber  30  mainly by collisions with the gas, as in the present QqTOF spectrometer. The energies of the ion and electron beams entering the ECD cell will be set to give optimum overlap between the distributions. 
     That is, the region in which the ions lose their kinetic energy to the electric field and bath gas collisions, and then reverse direction, will coincide with the region in which the electric field causes electrons to lose their longitudinal velocity and finally reverse direction. Having electrons and ions intersect with low kinetic energy, as is done here, maximizes the efficiency of the ECD process. 
     After ECD, the daughter or secondary ions, being positive ions, will drift back to the entrance or second aperture  42  of the electron collision chamber  30  in response to the small axial electric field mentioned above, and will be injected into the TOF storage region for acceleration into the flight path of the TOF spectrometer. 
     Since the ion beams are so diffuse, there is little or no problem with individual ions traveling in opposite directions intersecting along the path from the TOF accelerating region to the quadrupole for ECD. However, gating of ions passing though the aperture of the ECD quadrupole toward the TOF region is possible and could be accomplished by applying a periodic additional positive voltage to the aperture through which ions depart the CID/ECD quadrupole for ECD on the way to the TOF region. When this additional positive voltage is applied, ions would be repelled from the aperture and so remain in the quadrupole. Such pulses would be timed so that the voltage would not be applied when the accelerating voltage is on. Thus ions would only be allowed into the TOF region during acceleration pulses causing them to be propelled into the field-free drift region and impact the detector. 
     Simulations preformed using SIMION 7.0 (simulation software for electron and ion behaviour) illustrate the marked improvement in electron stability resulting from the rectangular waveform. Ion and electron trajectories in the quadrupole of the electron collision chamber  30  under different waveforms are shown in  FIG. 5 . A uniform magnetic field of 500 Gauss is supplied by the solenoid, and a uniform voltage gradient of 0.2 V/cm is produced by the additional electrodes. The time axes are shown in different directions, because the ions and the electrons flow in opposite directions. 
     As shown in  FIG. 5   a , parts of the ion trajectory are shown expanded at  82  and  84 . The waveform  82  shows the motion of ions with a conventional sinusoidal excitation of the quadrupole, and the waveform  84  shows the ion motion with the modified square waveform  72  of  FIGS. 2 and 3 . As can be seen, ion stability is not significantly affected by the different waveforms. 
     On the other hand, referring to  FIG. 5   b , curves  86  show the electron motion with a conventional sinusodal waveform and line  88  shows motion with the modified square waveform  72 . It is clear that electron stability has vastly improved under zero-field conditions. 
     Mean free path calculations show that the average distance traveled by the electrons between collisions in 0.1 Torr He is about 4 cm, so for an 8 cm quadrupole, about one quarter of the electrons will remain undeflected. As indicated in  FIG. 5   b , the electron transit time through an 8 cm long quadrupole is about 0.3 μs—sufficiently short so that the electrons can traverse the quadrupole and interact with the ions within the 0.5 μs zero-voltage window (at 1 Mhz), i.e., half the period of the waveform  72  when no voltage is applied. 
     Since the ions are traveling much slower, (˜0.1 cm/μs for 10 eV for a typical Substance P, vs ˜60 cm/μs for 1 eV electrons), the ions see the RF field averaged over many cycles, and follow stable trajectories just as with sinusoidal excitation. In addition, the radial focusing force will be enhanced by the potential depression along the axis caused by the electron space charge, (not calculated here). The effects of ion and electron collisions with the buffer gas were modelled in a SIMION 8.0 simulation that was supplemented by a separate program the inventors have devised in Perl, which is based on our earlier simulation (A. Krutchinsky, I Chernushevich, V. Spicer, W. Ens, K. G. Standing, JASMS 9(6) 569-579 (1998)). In both simulations, ions that enter the quadrupole with large angular or velocity spreads are seen to be quickly focused onto the quadrupole axis as they lose energy to the bath gas. After traveling a few cm along the axis of the damping quadrupole, the majority of the ions have been focused into a region within a few mm of the axis, where the electron density is highest, so there should be good overlap between the ion and electron beams. The results of the SIMION buffer gas simulation are presented in  FIG. 5   c , where ions entering from the left hand side are indicated at  90  and electrons entering from the right hand side are indicated at  92 . 
     The invention, as described, has concentrated on the problem of coupling ECD to a TOF spectrometer. However, it is believed that the proposed configuration can be used for other measurements: 
     CID can be carried out in q 3  in the same way as is done in q 2  in the usual mode of qQTOF operation, i.e. with gas, but without an electron beam. It has been estimated that ˜60% of sequence information is obtained from ECD, and 40% from CID, when both modes of operation are available [Zubarev reference, mentioned above]; 
     CID can also be carried out in q 2  exactly as in the usual mode of qQTOF operation, but in this case the ions must be deflected (by the deflection plates in the TOF section) in order to hit the detector, with a consequent loss of resolution. This is because the ions will have a velocity transverse to the acceleration direction in the TOF section. As viewed in  FIG. 1 , this is in the horizontal direction; ions coming out of the cell will have a horizontal velocity that will give a desired trajectory in the TOF section, while ions coming out of q 2  will have the opposite velocity. However, this mode may be useful for comparison with the normal mode of operation Substitution of a negative ion source for the electron source should enable ETD in q 3 ; one could therefore compare ECD and ETD in the same geometry. 
     Although a spectrometer as described has not yet been completed, the inventors the inventors have assembled an ECD cell and are in the process of attaching it to a mass spectrometer as described herein. 
     It is believed that care needs to be taken in the construction of the rectangular voltage generator necessary to drive the quadrupole q 3 . Conventional sinusoidal generators use resonant circuits, but due to the rapid switching times necessary (less than 50 ns), the present design is based on power metal oxide field effect transistors (MOSFET&#39;s) as shown in  FIG. 6 . A simpler version, incorporating only one low-voltage transistor, may be used to pulse the electron source  34 . The overall circuit is indicated at  100  and includes three FET driver circuits indicated generally at  102 ,  104  and  106 , which have a generally similar configuration. 
     The desired rectangular wave is supplied from a programmable arbitrary waveform generator (Telulex Model SG-100, current equivalent now sold by Berkeley Nucleonics) and is transmitted to three transistor driving circuits, and its input is indicated at  108 . The first FET driving circuit  102  is triggered by the positive part of the signal, the second FET driving circuit  104  is triggered by the negative part of the signal, and the third FET driving circuit  106  is triggered by a supplemental output that is turned on whenever the signal voltage is at 0V. The optocouplers for the positive ( 102 ) and negative ( 104 ) halves of the circuit trigger on the positive and negative portions of the square wave from the Telulex SG-100. The clamping portion of the circuit ( 106 ) is triggered by a secondary output from the Telulex SG-100, programmed to occur immediately after the signal which triggers portions ( 102 ) and ( 104 ). 
     In order to make the rise and fall times as short as possible, we have chosen components that minimize the effective RC time-constant of the circuit. This necessitates using low-value resistors, which in turn increases the power drawn by the circuit. When working in operation range, both halves of the device will generate a total of 100 W of heat, which will be dissipated by forced air cooling. 
     A common DC bench power supply  100 , a 13.8 volt supply is connected to the three FET driving circuits,  102 ,  104  and  106 . The three circuits  102 ,  104  and  106  are generally similar, and for simplicity are described in relation to the circuit  102 ; it being understood that the other circuits correspond. 
     The FET driving circuit  102  has a DC to DC converter  112  that converts the DC voltage to a floating 12 volts, so that the ground of each circuit portion is no longer tied to the Earth ground of the power supply. This is connected to a DC regulator  114  (part number LM7805) that converts the voltage to 5 volts to power the optocoupler. 
     The input from the Telulex at  108  is connected across an LED to provide electrical isolation. The output from the LED  116  is received by its corresponding transistor  118 , so as together to form an optocoupler. The output from the transistor  118  is connected to a driver circuit  120 . This package essentially provides dual inverting amplifiers connected between the pin pairs 2, 7 and 4, 5. 
     The output from the driver circuit  120  is connected through a resistor  122  to the gate of an FET  124 . 
     Corresponding final drive FET&#39;s  126  and  128  are provided for the other drive circuits  104 ,  106 . As shown, these FET&#39;s  124 ,  126 ,  128  are connected to a connection  130  that provides the input to the quadrupole. They are arranged in an H-bridge arrangement, as detailed below. Thus, the FET  124  has a connection to a 500 volt positive source  132 , while the FET  126  is shown connected to a negative 500 volt source  134 . The FET  128  provides a connection through to ground indicated at  136 . 
       FIG. 6  shows a circuit for generating a positive-negative waveform, while  FIG. 7  shows a circuit for generating negative-positive waveform. For simplicity as many components are common between the two figures, the same reference numerals are used for the two figures and the description above applies to both figures. 
     In  FIG. 6 , the polarity-form of the output waveform a P-channel FET 128  pulls the quadrupole signal from −HV to “up” ground with a diode and 51-ohm resistor only permitting conduction from −HV up to zero volts. Conversely in the negative-positive waveform circuit, shown in  FIG. 7 , an N-channel FET 128  pulls the quadrupole signal from +HV “down” to ground with the same diode and 51-ohm resistor, but the diode configured to only permit conduction from +HV down to zero volts.” 
     In a quadrupole, diagonally opposite rods are connected together. Here each of the circuits of  FIGS. 6 And 7  is connected to one pair of rods to give the desired field. 
     It can be noted that the capacitors in the circuit denoted by A will; have a capacitance of 0.1 μF and are placed as close as possible to the supply pins on the integrated circuits to minimize unwanted oscillations. The DC to DC converters  112  are preferably ASTEC AEE 00B12-49 converters, the optocouplers are preferable HCPL2611 and FET drivers  120  are preferably TI (Texas Instruments) UCC27323 with the final drive FET&#39;s  124 ,  126  and  128  preferably being IRF 830, and the P-channel MOSFETS preferably being MTP 2P50E.