Abstract:
A method of analyzing a substance comprises ionizing the substance to form a string of ions. The ions are then subject to a first mass analysis step. In one embodiment, the ions are accelerated into a collision cell in known manner to form primary fragment ions. These primary fragment ions are then accelerated into a downstream mass analyzer, to promote secondary fragmentation. In another embodiment of the invention, ions are passed through the collision cell, without fragmentation, and then accelerated from the collision cell into a low pressure section, which may be a mass analyzer or a rod set for collecting and collimating ions. This is done under conditions that promote fragmentation. The operating conditions of the low pressure section can be such as to promote collection or retention of ions depending upon their mass, and more specifically to reject low mass ions. This enables primary fragment ions to be cooled, and secondary fragment ions to be formed subsequently from these ions after they have disipated some of their energy. This enables control of secondary fragmentation processes, and offers numerous opportunities for analyzing complex ions.

Description:
FIELD OF THE INVENTION  
         [0001]    This invention relates to mass spectrometers. More particularly, this invention relates to tandem mass spectrometers, intended to perform multiple mass analysis or selection steps.  
         BACKGROUND OF THE INVENTION  
         [0002]    Presently, a variety of mass spectrometry/mass spectrometry (MS/MS or MS 2 ) techniques are known. These techniques provide for detection of ions that have undergone physical changes during residence in a mass spectrometer. Frequently, the physical change involves inducing fragmentation of a selected precursor ion and recording the mass spectrum of the resultant fragment ions. The information in the fragment ion mass spectrum is often a useful aid in elucidating the structure of the precursor ion. The general approach used to obtain an MS/MS spectrum is to mass select the chosen precursor ion with a suitable m/z analyzer, to subject the precursor ion to energetic collisions with a neutral atom or molecule that induces dissociation, and finally to mass resolve the fragment ions again with a m/z analyzer.  
           [0003]    Triple quadrupole mass spectrometers (TQMS) accomplish these steps through the use of two quadrupole mass analyzers separated by a pressurized reaction region for the fragmentation step. Since the three steps of the MS/MS process are carried out in different locations, MS/MS using a triple quadrupole mass spectrometer is referred to as “tandem in space”. MSIMS spectra with a TQMS can be quite complex in terms of the number of mass resolved features due to the tens of electron volts laboratory collision energies used and the fact that once a fragment ion is formed it can undergo further decomposition producing additional second generation ions and so on. The resulting MS/MS spectrum is a composite of all the fragmentation processes that are energetically allowed: precursor ion to fragment ions and fragment ions to other fragment ions. This spectral richness is often a benefit to compound identification when searching databases of MS/MS libraries. However, this same spectral complexity can make structural identification of a completely unknown compound difficult since not all of the fragment ions in the spectrum are first generation products from the precursor ion.  
           [0004]    There are also situations in which the MS/MS spectrum yields only one or two fragment ion features that correspond to loss of a structurally insignificant part of the precursor ion. The data from these MS/MS spectra are not particularly helpful for determining the structure of unknown precursor ions.  
           [0005]    An additional stage of MS applied to the MS/MS scheme outlined above, giving MS/MS/MS or MS 3 , can be a useful tool for both of the problems outlined above. When the MS 2  spectrum is very rich in fragment ion peaks the technique of subsequently mass isolating a particular fragment ion, dissociating a selected fragment ion, and mass resolving the resultant ions helps to clarify the dissociation pathways of the original precursor ion. It also aids in accounting for the mechanism of formation of all of the mass peaks in the MS 2  spectrum. In the case in which the MS 2  spectrum is dominated by primary fragment ions with little structural information, MS 3  offers the opportunity to break down these primary fragmentation ions, to generate additional or secondary fragment ions that often yield the information of interest.  
           [0006]    Three-dimensional ion traps provide the capability of multiple stages of MS/MS (often referred to as MS n  since n stages of MS can be carried out). Since the precursor ion isolation, fragmentation, and subsequent mass analysis is performed in the same spatial location, any number of MS steps can be performed, with the practical limitation being losses and diminution of the total number of ions retained after each step. Typically, an ion trap is operated to cause all of the unwanted ions to become unstable in the trapping volume, so as to isolate a precursor ion. Next, the trapping conditions are modified such that a range of fragment ions will be created and trapped in the device. For this purpose, the precursor ion is collisionally activated by application of an AC excitation frequency that increases the ion&#39;s kinetic energy in the presence of a neutral gas such as helium. These low energy collisions result in fragment ion generation. Finally, the fragment ions can be mass selectively scanned out of the three-dimensional ion trap toward an ion detector. Further stages of MS/MS are accomplished by simply repeating the mass isolation and collisional activation steps prior to scanning the ions out of the ion trap.  
           [0007]    True MS 3  experiments are difficult to accomplish with TQMS instruments since there are only two mass analyzers and one collisional activation region. Additional fragmentation steps can be carried out within the RF-only collision cell by applying an appropriate AC excitation frequency to the quadrupole rods such that a particular fragment ion is activated and dissociates further. But since TQMS instruments are normally operated as flow-through devices there is usually insufficient time to isolate a particular ion and to collisionally activate it during the brief time it is resident in the RF-only collision cell.  
           [0008]    An additional stage of fragmentation within a flow-through pressurized collision cell, but without the isolation step has been demonstrated for a QqTOF instrument as described by Cousins [47th ASMS Conference on Mass Spectrometry and Allied Topics, 1999]. Here, a precursor ion is selected within the first quadrupole mass analyzer, and then accelerated into the collision cell where primary fragment ions are produced. Further fragmentation of a selected primary fragmentation is induced by an appropriately chosen AC voltage source that is resonant with the particular, primary, fragment ion. This excited primary fragment ion then undergoes further collisions with background neutral species and dissociates, to generate secondary fragment ions. The result is a MS 3  spectrum superimposed upon the MS 2  spectrum, which complicates data analysis. This can be partially overcome by subtracting the MS 2  spectrum from the MS 2 +MS 3  spectra, but this approach can be time consuming and may discriminate against important low intensity MS 3  spectral features.  
           [0009]    An alternative approach is to trap the ions within the collision cell and this offers the opportunity to both isolate and fragment a chosen ion using techniques analogous to those used in a conventional three-dimensional ion trap. Theoretically, this should overcome the flow through characteristics, resulting in insufficient time for additional fragmentation, noted above. The problem with this approach is that once the ions are released from the collision cell the downstream mass spectrometer must perform the mass analysis step very quickly since the pulse of released ions is temporally very narrow. This requires that the downstream mass analyzer be a very fast scanning device, such as a TOF mass spectrometer.  
           [0010]    Thus, a conventional scanning quadrupole mass analyzer or the like is unsuited for processing a temporally narrow pulse of ions. If the ions could somehow be scanned out of the trap in some mass-dependent manner, this difficulty could be overcome.  
           [0011]    In earlier U.S. Pat. No. 6,177,668, also published international application WO 97/4702, there is disclosed a multipole mass spectrometer provided with ion trap and an axial ejection technique from the ion trap. The contents of these two applications are hereby incorporated by reference.  
           [0012]    The technique disclosed in those two applications, relies upon emitting ions into the entrance of a rod set, for example a quadrupole rod set, and trapping the ions at the far end by producing a barrier field at an exit member. An RF field is applied to the rods, at least adjacent to the barrier member, and the RF fields interact in an extraction region adjacent to the exit end of the rod set and the barrier member, to produce a fringing field. Ions in the extraction region are energized to eject, mass selectively, at least some ions of a selected mass-to-charge ratio axially from the rod set and past the barrier field. The ejected ions can then be detected. Various techniques are taught for ejecting the ions axially, namely scanning an auxiliary AC field applied to the end lens or barrier, scanning the RF voltage applied to the rod set while applying a fixed frequency auxiliary voltage to the end barrier and applying an auxiliary AC voltage to the rod set in addition to that on the lens and the RF on the rods.  
           [0013]    It has now been realized that this 2-dimensional linear ion trap mass spectrometer can be used to enhance the performance of a triple quadrupole to provide MS 3  capabilities.  
         SUMMARY OF THE INVENTION  
         [0014]    In accordance with a first aspect of the present invention, there is provided a method of analyzing a substance, the method comprising:  
           [0015]    (1) ionizing the substance to form a stream of ions;  
           [0016]    (2) subjecting the ions stream to a first mass analysis, to select ions having a desired mass to charge ratio, as precursor ions;  
           [0017]    (3) introducing the precursor ions into a collision cell to promote fragmentation of the precursor ions, thereby to generate primary fragment ions; (4) in the collision cell, selecting primary fragment ions having a desired mass to charge ratio, and rejecting other ions;  
           [0018]    (5) accelerating the selected primary fragment ions from the collision cell into a downstream mass analyzer, thereby to promote secondary fragmentation; and  
           [0019]    (6) mass analyzing the secondary fragment ions to generate a mass spectrum.  
           [0020]    In accordance with a second aspect of the present invention, there is provided a method of analyzing a substance, the method comprising:  
           [0021]    (1) ionizing the substance to form a stream of ions;  
           [0022]    (2) subjecting the ions stream to a first mass analysis, to select ions having a desired mass to charge ratio, as precursor ions;  
           [0023]    (3) introducing the precursor ions into a collision cell to promote fragmentation of the precursor ions, thereby to generate primary fragment ions;  
           [0024]    (4) in the collision cell, selecting primary fragment ions having a desired mass to charge ratio, and rejecting other ions by removing ions of a mass to charge ratio greater than the mass to charge ratio of the selected primary fragment ions and separately removing ions with a mass to charge ratio less than the mass to charge ratio of the selected primary fragment ion, the removal of the ions with mass to charge ratios higher and lower than the mass to charge ratio of the selected primary fragment ion being effected in either order;  
           [0025]    (5) causing the selected primary fragment ions to collide, to promote further fragmentation, generating secondary fragment ions; and  
           [0026]    (6) mass analyzing the secondary fragment ions to generate a mass spectrum. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0027]    For a better understanding of the present invention and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings which show a preferred embodiment of the present invention and in which:  
         [0028]    [0028]FIG. 1 is a schematic view of an apparatus in accordance with the present invention;  
         [0029]    [0029]FIG. 2 a  shows an MS/MS spectrum for mass  609  of reserpine;  
         [0030]    [0030]FIGS. 2 b  and  2   c  show the spectrum of FIG. 2 a , with high masses above mass  397  and low masses below mass  397  removed respectively;  
         [0031]    [0031]FIG. 2 d  shows the spectrum of FIG. 2 a  with both high and low masses above and below mass  397  removed;  
         [0032]    [0032]FIG. 2 e  shows an MSIMS/MS spectrum of mass  397  obtained by secondary fragmentation of mass  397  as shown in FIG. 2 d ;  
         [0033]    [0033]FIG. 3 a  shows the MS/MS spectrum of mass  609 , equivalent to FIG. 2 a ;  
         [0034]    [0034]FIGS. 3 b - 3   e  show MS/MS/MS spectra of the four major ions shown in the spectrum of FIG. 3 a ;  
         [0035]    [0035]FIG. 4 shows MS/MS/MS of the residual mass  609  ion obtained from the spectrum of FIG. 3 a ;  
         [0036]    [0036]FIG. 5 is an MS/MS spectrum of m/z  609  reserpine molecular ion;  
         [0037]    [0037]FIG. 6 is a further MS/MS spectrum of m/z  609  reserpine molecular ion with a different fill mass and fill time;  
         [0038]    [0038]FIG. 7 is a scan function which displays the timing of the various steps used to generate Q 2 -to-Q 3  MS/MS spectra;  
         [0039]    [0039]FIG. 8 is another MS/MS spectrum of m/z  609  reserpine molecular ion with a different fill mass and fill time;  
         [0040]    [0040]FIG. 9 is an MS/MS spectrum of the m/z  552  bosentan molecular ion obtained using conventional acceleration into the collision cell;  
         [0041]    [0041]FIG. 10 is an MS/MS spectrum of the m/z  552  bosentan molecular ion obtained with different acceleration conditions, and with a different fill mass and fill time;  
         [0042]    [0042]FIG. 11 is an MS/MS spectrum of the m/z  552  bosentan molecular ion obtained with the same acceleration condition as FIG. 10, and with a different fill time and fill mass;  
         [0043]    [0043]FIG. 12 shows MS/MS spectra of the doubly charged m/z  1094  ion from beta-casein digested by the enzyme trypsin obtained (a) by normal acceleration into the collision cell and (b) by acceleration out from the collision cell; and.  
         [0044]    [0044]FIG. 13 shows mass-to-charge scale expanded views of the same MS/MS spectra of the doubly charged m/z  1094  ion from beta-casein digested by the enzyme trypsin obtained (a) by normal acceleration into the collision cell and (b) by acceleration out from the collision cell. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0045]    Referring first to FIG. 1, an apparatus in accordance with the present invention is indicated generally by reference  10 . In known manner, the apparatus  10  includes an ion source  12 , which may be an electrospray, an ion spray, a corona discharge device or any other known ion source. Ions from the ion source  12  are directed through an aperture  14  in an aperture plate  16 . On the other side of the plate  16 , there is a curtain gas chamber  18 , which is supplied with curtain gas from a source (not shown). The curtain gas can be argon, nitrogen or other inert gas, such as described in U.S. Pat. No. 4,861,988, Cornell Research Foundation Inc., which also discloses a suitable ion spray device, and the contents of this patent are hereby incorporated by reference.  
         [0046]    The ions then pass through an orifice  19  in an orifice plate  20  into a differentially pumped vacuum chamber  21 . The ions then pass through aperture  22  in a skimmer plate  24  into a second differentially pumped chamber  26 . Typically, the pressure in the differentially pumped chamber  21  is of the order of 2 torr and the second differentially pumped chamber  26 , often considered to be the first chamber of mass spectrometer, is evacuated to a pressure of about 7 mTorr.  
         [0047]    In the chamber  26 , there is a standard RF-only multipole ion guide Q 0 . Its function is to cool and focus the ions, and it is assisted by the relatively high gas pressure present in this chamber  26 . This chamber  26  also serves to provide an interface between the atmospheric pressure ion source and the lower pressure vacuum chambers, thereby serving to remove more of the gas from the ion stream, before further processing.  
         [0048]    An interquad aperture IQ 1  separates the chamber  26  from the second main vacuum chamber  30 . In the main chamber  30 , there are RF-only rods labeled ST (short for “stubbies”, to indicate rods of short axial extent), which serve as a Brubaker lens. A quadrupole rod set Q 1  is located in the vacuum chamber  30 , and this is evacuated to approximately 1 to 3×10 −5  torr. A second quadrupole rod set Q 2  is located in a collision cell  32 , supplied with collision gas at  34 .The collision cell is designed to provide an axial field toward the exit end as taught by Thomson and Jolliffe in U.S. Pat. No. 6,111,250. The cell  32  is within the chamber  30  and includes interquad apertures IQ 2 , IQ 3  at either end, and typically is maintained at a pressure in the range 5×10 −4  to 8 ×10 −3  torr, more preferably a pressure of 5×10 −3  torr. Following Q 2  is located a third quadrupole rod set Q 3 , indicated at  35 , and an exit lens  40 . The pressure in the Q 3  region is nominally the same as that for Q 1  namely 1 to 3×10 −5  torr. A detector  76  is provided for detecting ions exiting through the exit lens  40 .  
         [0049]    Power supplies  36 , for RF and resolving DC, and  38 , for RF, resolving DC and auxiliary AC are provided, connected to the quadrupoles Q 1 , Q 2 , and Q 3 . Q 1  is a standard resolving RF/DC quadrupole. The RF and DC voltages are chosen to transmit only the precursor ions of interest into Q 2 . Q 2  is supplied with collision gas from source  34  to dissociate precursor ions or fragment them to produce fragment or product ions. Q 3  is operated as a linear ion trap mass spectrometer as described in U.S. Pat. No. 6,177,668, i.e. ions are scanned out of Q 3  in a mass-dependent manner, using the axial ejection technique taught in that earlier U.S. patent.  
         [0050]    In the preferred embodiment, ions from ion source  12  are directed into the vacuum chamber  30  where the precursor ion m/z is selected by Q 1 . Following precursor ion mass selection, the ions are accelerated into Q 2  by a suitable voltage drop into Q 2 , inducing fragmentation. These 1st generation fragment ions are trapped within Q 2  by a suitable repulsive voltage applied to IQ 3 . Once trapped the RF voltage applied to the Q 2  rods is adjusted such that all ions above a chosen mass are made unstable, that is there a,q values fall outside the normal Mathieu stability diagram. Removal of ions above the mass of a particular ion of interest is facilitated by the addition of a small amount of resolving DC voltage, here 1.8 volts, applied to the Q 2  rods. Next the RF is adjusted so that ions below a particular mass are made to be unstable. These two steps can be accomplished very quickly, on the order of 1-3 ms each. The result is a mass isolated ion population, which can be further collisionally activated.  
         [0051]    The subsequent collisional activation step can be accomplished as in a conventional three-dimensional ion trap, that is by application of an appropriate resonant AC waveform. This however requires sophisticated electronics and has the additional requirement that the trapping RF voltage be such that the lowest mass fragment ion and the precursor ion are simultaneously stable within Q 2 .  
         [0052]    An alternative technique is to simply accelerate the mass isolated ions in to the subsequent mass analyzer. Since Q 2  is operated at elevated neutral gas pressure, say 5×10 −3  torr, there is a neutral gas pressure gradient between IQ 3  and the subsequent mass analyzer. If the mass isolated ions within Q 2  are accelerated through this pressure gradient into the Q 3  linear ion trap there will be a sufficient number of collisions to induce further fragmentation. The result is a MS 3  mass spectrum.  
         [0053]    By way of example consider the following set of experimental results obtained using the apparatus in FIG. 1. A sample of 100 pg/mL of reserpine (MW=608) is introduced into the ion source  12  where it is ionized and directed into the vacuum chamber  30 . The RF and DC voltages of Q 1  are adjusted to transmit a 0.7 amu wide beam of the protonated reserpine ions at m/z 609 into Q 2 . The DC voltage offset of Q 2  relative to Q 1  is chosen to be 35 volts, which is sufficient to produce extensive fragmentation of the reserpine precursor ion. Q 2  is operated as a simple accumulation ion trap by adjusting IQ 3  to an appropriately repulsive DC voltage so that none of the entering precursor ions or fragment ion generated therein can exit. Q 2  is filled for 50 ms, after which the DC voltage applied to IQ 2  is raised to the same value as the trapping IQ 3  value. There is now a trapped population of primary fragment and residual precursor ions resident within Q 2 . If all the ions within Q 2  are now allowed into the Q 3  linear ion trap mass spectrometer and mass analyzed, the MS 2  mass spectrum displayed in FIG. 2 a  is obtained. To obtain MS 3  data of the m/z 397 ion), this fragment ion must be isolated and collisionally activated prior to mass analysis by the Q 3  linear ion trap mass spectrometer.  
         [0054]    Ion isolation of the m/z  397  fragment ion was accomplished in a step-wise fashion by first adjusting the RF voltage applied to the Q 2  rods such that ions above m/z ˜397 become unstable within Q 2  and are lost. The result of this step is displayed in FIG. 2 b . Here, one can see that the ion population within Q 2  has been modified such that there is little or no contribution to the MS 2  mass spectrum from ions m/z&gt;397.  
         [0055]    Low mass ions may be eliminated from the Q 2  ion population by adjusting the RF voltage such that the trapped ions with m/z below ˜397 become unstable in the Q 2  and are also lost. The result of this step prior to mass analysis is displayed in FIG. 2 c , which shows that low mass ions can be effectively eliminated from Q 2 .  
         [0056]    A combination of these two steps thus provides good mass isolation of the m/z 397 fragment ion within Q 2  as is displayed in FIG. 2 d , i.e. these two steps are performed sequentially in Q 2 . The time penalty for the mass isolation steps is approximately 2×2 ms or a total of 4 ms. As Q 2  is a high pressure collision cell, true mass filtering is not possible, and in particular it is not possible to get a sharp cutoff between selected or retained ions, and rejected ions, as is possible in a low pressure mass analysis section, such as Q 1 . For this reason, it is not possible to apply a narrow window selecting just the desired m/z 397. Any attempt to do this would result in significant loss of the 397 ion. Rather, it has been found that by sequential rejection of masses above and below the mass of interest, the bulk of the unwanted ions can be rejected. Note that in FIGS. 2 a - 2   e , the vertical scale indicates relative intensity with the most populous ion being indicated as 100%.  
         [0057]    Finally, the m/z 397 ions are accelerated into the Q 3  linear ion trap MS by increasing the relative DC voltage offset between Q 2  and Q 3  from 5 volts (used in FIGS. 2 a - c ) to 25 volts. Collisions at the exit of Q 2  and entrance of Q 3  lead to fragmentation of the m/z 397 ions and results in the MS 3  spectrum displayed in FIG. 2 d . As expected, a range of masses of secondary fragmentations, with masses below m/z 397, are present in the spectrum. Again, the vertical axis shows relative intensity, and as the residual primary fragment ion 397 is still the most populous, it is shown with an intensity of 100%, with the secondary fragment ions of low masses shown accordingly.  
         [0058]    This procedure can be carried out separately on the major fragment ions in the reference reserpine MS 2  spectrum of FIG. 2 a . The result is displayed in FIG. 3 where the highest mass peak in each spectrum corresponds to the isolated MS 2  primary fragment ion used to obtain the MS 3  spectrum. Thus, FIG. 3 a  again shows the complete MS 2  spectrum for m/z 609; FIGS. 3 b - 3   e  show the MS 3  spectra for the primary fragment ions  448 ,  397  (equivalent to FIG. 2 e ),  195  and  174 , respectively.  
         [0059]    For this technique to be widely applicable the collisional activation step must be sufficiently energetic to provide a wide range of MS 3  fragment ions. The ability to fragment the m/z  609  reserpine ion is a good measure of the energetics of fragmentation since approximately 30 eV lab  of energy is required to observe the m/z  174  and  195  ions.  
         [0060]    [0060]FIG. 4 shows the MS 3  mass spectrum obtained after isolation of the residual m/z  609  ions in Q 2 , i.e. here the residual precursor ions  609  were retained and all the primary fragment ions were rejected. These residual precursor ions  609  were then subjected to collisional activation using a 30-volt potential drop between Q 2  and Q 3 . One can see that all of the major fragments in the MS 2  spectrum (FIG. 2 a ) are present in FIG. 4, although the relative intensities differ, as the relative intensities, in known manner, will vary depending upon variations in the collision energy of the fragmentation process. This demonstrates that the method for obtaining MS 3  provides sufficiently energetic collisions to generate fragmentation for many potentially important compounds.  
         [0061]    It is understood that the ion isolation step can be accomplished via notched broadband isolation techniques. This entails subjecting the trapped ions to a plurality of excitation signals uniformly spaced in the frequency domain with a notch of no excitation signals corresponding to the resonant frequencies of the ions to be isolated within the ion trap as described by Douglas et al. in WO 00/33350.  
         [0062]    The present inventors have also discovered and identified that one of the important experimental parameters in the transfer of ions from the Q 2  linear ion trap to the Q 3  linear ion trap is the RF voltage value applied to the Q 3  linear ion trap during the Q 2 -to-Q 3  ion acceleration process. Ions received in Q 3  can only be successfully trapped within Q 3  if their associated q-value is less than ˜0.9. FIG. 5 shows that when the reserpine molecular ion at m/z  609  is accelerated from Q 2  into Q 3  while the RF voltage is set such that only ions with m/z&gt;350 have a q-value&lt;0.9, only product ions with mass-to-charge values greater than 350 are observed in the final mass spectrum. The m/z value associated with the q=0.9 RF voltage during the Q 3  fill step is referred to as the “Q 3  fill mass”; and while this suggests a single mass, as FIG. 5 shows it really defines a lower limit to a range of masses.  
         [0063]    The inventors have found that another important parameter is the time for which the Q 3  RF voltage is held at the fill mass, referred to as the “Q 3  fill time”. This Q 3  fill time is in general longer than the actual time required to empty the Q 2  ion trap. Ions can be removed from Q 2  very rapidly by using an axial DC field as taught by Thomson and Jolliffe in U.S. Pat. No. 6,111,250. At the pressures and voltages used in the current instrument all the ions within Q 2  should be transferred to the Q 3  ion trap in less than 2 ms, which can be identified as a “transfer time”. Any time in excess of this 2 ms or other transfer time but less than the Q 3  fill time is referred to as the “delay time”.  
         [0064]    The Q 3  fill time for the experiment that resulted in the spectrum displayed in FIG. 5 was 50 milliseconds (i.e. 2 ms transfer time and 48 ms delay time). If this value is reduced to 5 milliseconds (i.e. 2 ms transfer time and 3 ms delay time) then the mass spectrum in FIG. 6 results. The most obvious difference between the mass spectra in FIGS. 5 and 6 is the appearance of low mass product ions below the Q 3  fill mass in FIG. 6.  
         [0065]    It is necessary to consider the details of the scanning procedure to understand the reason for the appearance of the low mass-to-charge product ions in the FIG. 6 mass spectrum. The particular scan function employed here is shown in FIG. 7, which shows the timing steps from the Q 3  fill step onward. During the Q 3  fill step the value of IQ 3  is set to allow ions to flow from Q 2  into Q 3 , as indicated at  20 . Simultaneously, an RF voltage  22  is supplied to the rod set Q 3 . The value of the Q 2  to Q 3  DC voltage rod offset (not shown in FIG. 7) is simultaneously adjusted to the value of the desired laboratory reference frame collision energy. The exit lens  40  is provided with a high voltage, indicated at  24 , during the Q 3  fill step, so as to provide an appropriate trapping voltage. The drive RF voltage  20 , and thus Q 3  fill mass, is set to some optimum value during the Q 3  fill step, and at the end of the fill step, is then rapidly changed (in less than 100 microseconds as indicated at  26 ) to an RF voltage  28  to be used at the beginning of the mass scan.  
         [0066]    As indicated at  30 , at the end of the fill time, the voltage on the interquad aperture IQ 3  is increased to a potential indicated at  32 . Simultaneously, the voltage on the exit lens  40  is maintained, so that Q 3  then acts as an ion trap.  
         [0067]    At the end of the Q 3  fill time, the voltage on the exit lens  40  is dropped as indicated at  34  to a voltage  36 , and both the RF voltage and the AC excitation voltage for Q 3  are ramped up as shown at  38  and  40 , respectively. This then provides a mass spectrum of the ions trapped in the Q 3  linear ion trap. At the end of the scanning phase the voltage at IQ 3  drops at  42  to a lower voltage  44 . Simultaneously, the RF and AC voltages are dropped as shown at  46  and  48  respectively, to final voltages  50  and  52 .  
         [0068]    The inventors have found that a very important factor influencing whether or not ions with mass-to-charge ratios below that of the Q 3  fill mass are observed is the duration of the Q 3  fill step, i.e. the Q 3  fill time up to the voltage changes indicated at  26  and  30  in FIG. 7. This is shown by the differences between the product ion mass spectra for the protonated reserpine molecular ion at m/z  609  in FIGS. 5 and 6. The only differences between the spectra are the Q 3  fill time which is  50  ms (i.e.  2  ms Q 2 -to-Q 3  transfer time and 48 ms delay time) for FIG. 5 and 5 ms (i.e.  2  ms Q 2 -to-Q 3  transfer time and  3  ms delay time) in FIG. 6, all other parameters are the same: Q 2 -to-Q 3  acceleration energy=35 volts and Q 3  fill mass=350.  
         [0069]    It is believed that the reason for the observation of ions with q-values seemingly greater than the first stability region limit of ˜0.908 is the unique Q 2 -to-Q 3  fragmentation environment. The pulse of ions was introduced into the Q 3  linear ion trap at a translational energy of 35 eV lab . Since the neutral gas pressure within Q 3  is relatively low, approximately 3×10 −5  torr, the corresponding collision frequency is also low. Thus, in a short time frame there will be few momentum dissipating collisions within Q 3 , at least compared to the conventional high pressure collision cell (B. A. Thomson et al. Anal. Chem. 1995, 34, 1696-1704). A considerable amount of translational kinetic energy will remain in any unfragmented precursor ions after a short Q 3  fill time of  5  ms. The end of the Q 3  fill period is marked by a rapid reduction in the Q 3  RF voltage at  26 , i.e. a reduction in the lowest m/z ion that is now stable within the Q 3  linear ion trap. If any precursor ion within the Q 3  ion trap has retained sufficient internal energy, it may collide with a neutral gas atom or molecule to produce a product ion with a q-value that falls within the first stability region defined by the RF voltage during the cooling portion (shown at  28  in the FIG. 7 timing diagram), this product ion can be trapped and detected during the subsequent mass scan. The presence of low mass product ions in the 5 ms Q 3  fill time spectrum in FIG. 6 is clear evidence that sufficient energy was retained by the precursor ion population trapped within the Q 3  ion trap, so that when the RF voltage was reduced in the “cooling time” step, these precursor ions could provide efficient fragmentation and the fragment ions would then be stable in Q 3 . In contrast, the  50  ms Q 3  fill time spectrum in FIG. 5, shows that the amount of energy dissipated between the time ions are injected into Q 3  and the time when the Q 3  RF voltage is reduced to the lower level of the cool step is too long for a sufficient number of precursor ions to retain a high enough kinetic energy for the production of fragment ions. Also, if any fragment/product ions are generated during the fill time, the higher mass cutoff will cause them to be rejected. Consequently, with a long delay time, the precursor ions have experienced enough collisions within the Q 3  linear ion trap to preclude the formation of any significant quantity of low mass-to-charge product ions of reserpine. Thus, this method allows one to vary the average amount of internal energy deposited into a precursor ion and more significantly retained until the start of the cooling step when the lighter ions will be stable within Q 3 . This variation is effected simply by changing the delay time between the 2 ms Q 2 -to-Q 3  transfer time and the time at which the Q 3  RF amplitude is reduced, terminating the Q 3  fill time and starting the cooling time.  
         [0070]    One advantage to operating the instrument with a high Q 3  fill mass is a higher intensity product ion mass spectrum relative to that obtained with a low Q 3  fill mass. FIG. 8 shows the product ion mass spectrum of the protonated reserpine ion at m/z  609  obtained with a Q 3  fill mass of 180. Comparison of this mass spectrum with that in FIG. 6 (which was obtained under the same conditions except that the Q 3  fill mass was 350) shows that the higher Q 3  fill mass of 350 results in a sensitivity increase of about 20 X. The increased in sensitivity for the Q 3  fill mass of 350 mass spectrum is likely due to a larger radial well depth that better confines any scattered ions during the Q 3  fill step. Intensity is maximized when the Q 3  fill mass is approximately ½ that of the precursor ion mass-to-charge ratio, although the optimization characteristics are broad.  
         [0071]    A further advantage to the use of an elevated Q 3  fill mass is that the ions with m/z&lt;Q 3  fill mass are produced at a later time (after the cooling time) than those with m/z &gt;Q 3  fill mass, as they are products of precursor ions with lower kinetic energy since some collisional relaxation of the precursor ion during the delay time. That is, the energy of the precursor ion has been reduced by some of the relatively infrequent collisions within Q 3  during the fill time. Thus consecutive fragmentation processes producing these ions with m/z &lt;Q 3  fill mass are less favoured since the precursor ion has less internal energy at the time at which the lower mass product ions are collected. The resulting product ions in turn have less internal energy and thus reduced probability of further fragmentation, leading to suppression of second generation product ion precursor-to-product ion pairs. This can make it easier to identify first generation precursor-to-product ion pairs, which can be especially useful in the identification and differentiation of different dissociation pathways.  
         [0072]    An example is the mapping of the product ions of bosentan studied by Hopfgartner et.al. (J. Mass Spectrom. 1996, 31, 69-76). Hopfgartner et. al. found that the major m/z  280  product ions ion in the product ion spectrum of the m/z  552  bosentan molecular ion does not arise directly from the molecular ion, but rather from a two step process involving fragmentation of the m/z  508  ion to the m/z  311  ion and finally to the m/z  280  product ion. The product ion mass spectrum of the m/z  552  molecular ion is displayed in FIG. 9. This spectrum was obtained by mass selecting the m/z  552  precursor ion with Q 1  and accelerating this ion into the conventional Q 2  collision cell and trapping the resultant product and residual precursor ions in the Q 3  linear ion trap, from which they were mass selectively scanned out. This mass spectrum is virtually identical with that reported by Hopfgartner et al. Note the strong product ion feature at m/z  280 .  
         [0073]    A product ion mass spectrum for bosentan was obtained using the method described herein. Once again the precursor ion was mass selected by Q 1  and then, in accordance with the present invention, it was introduced into and trapped within Q 2 , this time at low energy in order to eliminate fragmentation. Next, the ions trapped within Q 2  were accelerated into the Q 3  linear ion trap at a laboratory collision energy of 30 eV, a Q 3  fill mass of 400,and a Q 3  fill time of 5 ms (i.e. 2 ms transfer time and 3 ms delay time). Thus, the only product ions that would be stable during the 5 ms fill time in the Q 3  ion trap have m/z&gt;400. Immediately after the Q 3  fill time (at  26  in FIG. 7) the Q 3  RF voltage was reduced to that corresponding to m/z  100 , which would allow trapping of any product ions with m/z&lt;400. As the delay time is short, precursor ions and first generation fragment ions should have retained sufficient energy, to collide and fragment, forming lighter ions which are now stable. The result is a somewhat different product ion mass spectrum from the one in FIG. 10, in that the relative intensity of the m/z  280  product ions ion is significantly reduced from that in FIG. 9.  
         [0074]    The product ion mass spectrum of the m/z  552  bosentan molecular ion obtained with the Q 3  fill mass set at  400  for a 10 ms fill time (i.e. 2 ms transfer time and  8  ms delay time) is displayed in FIG. 11, with the conditions otherwise being the same as in FIG. 10. The additional 5 ms spent at the Q 3  fill mass has a profound effect on the mass spectrum. This increased delay time allows the precursor ions time to dissipate some energy; thus residual precursor ions and first generation fragments, after commencement of the cooling time with the broader stability band, are much less likely to have sufficient energy for further fragmentation to occur. Most of the same product ions ion peaks are still distinguishable, but at much reduced intensity below the fill mass; note that intensities in the mass range to &lt;m/z 480 are shown magnified by a factor of 10. Notable also is that the mass spectrum shows virtually complete elimination of the m/z  280  product ions ion peak. This is strong evidence that the m/z  280  product ions ion is a secondary fragmentation product, or has a higher appearance energy (i.e. requiring a precursor ion to have a high energy than other product ions ions &lt;m/z 400. These results are in agreement with those of Hopfgartner et. al.  
         [0075]    The only limitation for the use of a variable Q 3  fill mass is that the precursor ion must be stable within the Q 3  linear ion trap, so the Q 3  fill mass must be less than the mass-to-charge ratio of the precursor ion.  
         [0076]    This method has also been found to be useful for the simplification of peptide product ion spectra as is demonstrated in FIG. 12. This figure displays two product ion spectra of a doubly charged peptide product ions at m/z  1094  from digestion of beta-casein in the presence of trypsin. FIG. 12 a  is the optimized product ion spectrum using conventional Q 1 -to-Q 2  acceleration and generation of fragment ions in the Q 2  collision cell with subsequent mass analysis using the Q 3  linear ion trap. The resulting spectrum is particularly rich in the low mass-to-charge region due to the presence of sequential fragmentation and internal product ions products. FIG. 12 b  is a Q 2 -to-Q 3  acceleration product ion mass spectrum of the doubly charged m/z  1094  ion from the same beta casein sample, i.e. with ions passed through Q 2  with substantially no fragmentation. FIG. 12 b  was obtained with a Q 3  fill mass of 600 and a Q 3  fill time of 7 ms. The two spectra are similar, however FIG. 12 b  is much less congested in the region below the Q 3  fill mass. FIG. 13 shows an expanded view of the lower mass-to-charge region of these product ion spectra. The assignments of the mass peaks in the product ion spectra have been included. FIG. 13 b  was obtained using the Q 2 -to-Q 3  acceleration method show only y-ions in this mass-to-charge region. The standard Q 1 -to-Q 2  acceleration data in FIG. 13 a  displays the same y-ions and many other fragmentation products including b-ions and internal product ions. The congestion in FIG. 13 a  makes identification of sequence specific product ions difficult if not impossible. However FIG. 13 b  contains only sequence specific y-ions. The discrimination against b-ion products and those resulting from internal fragmentation pathways has been found to be general phenomenon for Q 2 -to-Q 3  acceleration collisional dissociation of peptides resulting from trypsin digestion using an elevated Q 3  fill mass.  
         [0077]    The technique of ion isolation within a nominally RF-only collision cell and subsequent ion acceleration with concomitant fragmentation is also applicable to other Qq(MS) (where Q designates the mass selection step via a conventional RF/DC resolving quadrupole mass spectrometer and q the higher pressure nominally RF-only collision cell , here carried out in Q 1  and Q 2  respectively) instruments, where the MS stage can be another fast scanning mass spectrometer other than a linear ion trap mass spectrometer. One such device is a QqTOF tandem mass spectrometer. The TOF is particularly well suited to be used for the final mass analyzer since it is best used with a pulsed ion source, which is what emerges from the collision cell. Furthermore, a full mass spectrum can be obtained for each ion pulse, giving better overall efficiency.  
         [0078]    Additionally, it may in some circumstances be possible to eliminate the collision cell, and provide the collision gas by some other mechanism to the flow of ions into Q 3 . Additionally, the basic requirement for the section of containing Q 3  is that it will be a lower pressure section capable of collecting and collimating ions. It could include, for example, a multipole rod set that provides just this function without acting as a mass analyzer. Where it is desired to set a fill mass, the multipole rod set must be capable of defining this cut off mass with a required degree of precision. A mass analyzer can then be provided downstream.  
         [0079]    The final step of mass analyzing the MS 3  fragment ions can also be carried out using other mass analyzers that yield full mass spectra for a single pulse of ions such a 3-dimensional ion trap.