Abstract:
Collisional cooling of ions in mass spectrometry has been known for sometime. It is known that collisional cooling can promote focusing of ions along the axis of an ion guide. A similar technique has been used to enhance coupling of a pulsed ion source such as a MALDI source to a Time of Flight instrument. It is now realized that it is desirable to provide, immediately adjacent to a MALDI or other ion source, a low-pressure region to promote ionization conditions most favorable for the particular ion source. Then, with the ions released and free, the ions are subjected to relatively rapid collisional cooling in a high pressure region adjacent to the ionization region. This will dissipate excess of internal energy in the ions, so as to substantially reduce the incidence of metastable fragmentation of the ions. The ions can then be subjected to conventional mass analysis steps.

Description:
FIELD OF THE INVENTION  
         [0001]    This invention relates to mass spectrometry. This invention more particularly relates to generation of ions with an ion source that produces internally excited or “hot” ions like MALDI (Matrix Associated Laser Desorption Ionization), and the problems of unwanted or premature fragmentation of ions.  
         BACKGROUND OF THE INVENTION  
         [0002]    Collision cooling of ions is now widely used for the purpose of improving the quality of the ion beams. Cooling can be accomplished in an RF only ion guide as disclosed in U.S. Pat. No. 4,963,736 to Douglas, et al. or in gas chamber, that do not include RF rods. Both these techniques provide a buffer gas, and the presence of the buffer gas slows down the ions and, in the case of the RF-ion guide, can lead to reduction of the size of the ion beam. The process may also cool down internal vibration and other degrees of freedom of the ions.  
           [0003]    In some cases the ions acquire a high degree of internal excitation during ionization or other processes. If left excited, the ions will eventually fragment; this process is called metastable fragmentation. Metastable fragmentation is one of the main reasons for poor quality spectra of large proteins and DNAs using MALDI (See, for example, A. V. Loboda, A. N. Krutchinsky, M. Bromirski, W. Ens, K. G. Standing, “A tandem quadrupole/time-of-flight mass spectrometer (QqTOF) with a MALDI source: design and performance”,  Rapid Commun. Mass Spectrom.  14, 1047 (2000))]. Some other ionization methods (surface ionization mass spectrometry SIMS, fast atom bombardment FAB, Laser ablation LA, electron impact EI, etc) have similar problems and the present invention is generally applicable to such other methods. However, the present invention is primarily intended for application to MALDI sources and the invention will be described primarily in relation to MALDI sources. Metastable fragmentation means that ions can spontaneously fragment at any time and at any location in a mass spectrometer instrument, and hence can give poor spectra.  
           [0004]    Because of this limitation, two types of axial MALDI TOF (Time of Flight) systems now exist on the market: linear MALDI TOF and reflectron MALDI TOF. In a linear MALDI TOF, ions are pulsed from an extraction region into a linear flight tube, and the ions are detected at the end of the flight tube. The time of flight through the flight tube depends upon the initial energy given to the ions in the extraction region and the ions&#39; mass to charge ratio. As ions have some energy and velocity before the extraction pulse is applied, this motion is reflected in the velocity of ions m/z ratio as they travel through the flight tube. The overall effect is to degrade the resolution and accuracy of a linear time of flight instrument. For this reason, reflectron MALDI TOF instruments were developed. In a reflectron MALDI TOF, ions are again pulsed out of an extraction region and are provided with a pulse of energy. However, after traveling through the first part of the flight tube, the ions enter a reflection region where a field is applied to reflect the ions back to a location beside the original extraction region. The overall effect, approximately, is to negate or at least reduce the effect of any original ion motion in the direction of ion travel, so that reflectron TOF instruments have excellent resolution and mass accuracy.  
           [0005]    Because of the different characteristics of linear and reflectron TOF instruments, metastable fragmentation has quite different effects in these two instruments. In a linear MALDI TOF instrument, although it has limited resolution and mass accuracy, it is much more tolerant of metastable fragmentation. This is because once the ions leave the short extraction region, they enter a field free drift chamber. If a metastable ion fragments in the drift tube the velocities of the fragments do not change significantly from the velocity of the original ion. Hence, the fragments will still arrive at the detector at the same time as the unfragmented ions, and there is little effect or degradation on the spectrum obtained.  
           [0006]    In contrast, in a reflectron instrument, if metastable fragmentation occurs before or in the reflector, this will cause the fragment to spend a different time in the drift chamber before reaching the detector, causing significant degradation of the spectrum. It is for this reason that linear MALDI TOF is used where metastable fragmentation is perceived to be a potential problem.  
           [0007]    As a first approximation, a linear MALDI TOF device can tolerate metastable fragmentation that occurs after a few microseconds (the time it takes for ions to leave the extraction region), while a reflectron MALDI device can only tolerate the metastable fragmentation that has a time scale of approximately 100 microseconds (the time when the ions leave the reflector); The time scale of metastable fragmentation usually depends on the level of internal excitation of the ions, the higher the degree of excitation the faster the ion will fragment.  
           [0008]    Collisional cooling of MALDI ions as disclosed in published International Patent Application No. WO99/38185 can cure the problem of metastable fragmentation to some extent. In one preferred embodiment the ions are cooled down at a pressure ˜10 mTorr. At this pressure the cooling time is about 100 μs. Thus, the fragmentation pattern in the spectra resembles the ones in Reflectron MALDI TOF, as some metastable fragmentation still occurs. The only difference is that the resolution and mass accuracy of the observed fragments in MALDI with collisional cooling stays the same as for the stable ions. Both fragments and primary ions leave the cooling stage cooled down and focused, prior to entry into the TOF section. As the ions are then cooled, no subsequent metastable fragmentation occurs in the TOF section.  
           [0009]    As the cooling time is inversely proportional to the pressure another arrangement was disclosed in published International Patent Application No. WO99/38185. That arrangement has a cooling stage at a pressure of ˜1 Torr. The cooling time in this case is ˜1 As and this is short enough that fragmentation is substantially reduced. The spectra observed resemble the spectra from a linear MALDI TOF.  
           [0010]    Unfortunately such a high pressure has the disadvantage that it can affect the ionization process resulting in cluster formation. Clusters of ions of interest with several matrix molecules begin to appear as the pressure increase. Since a typical MALDI sample has substances of interest embedded in the excess of the matrix molecules it has been speculated that the clusters represent the material that was cooled down too rapidly without allowing matrix molecules to “evaporate” from the analyte ions.  
         SUMMARY OF THE PRESENT INVENTION  
         [0011]    Therefore, the present inventors have realized that it is advantageous to have a low pressure in the ionization region to permit complete “evaporation” of the matrix material and release of desired analyte ions, a subsequent high-pressure region for rapid cooling of ions, and then again a low pressure region for mass analysis. Also, the first low pressure region and the high pressure region have to be close to each other because the velocity of the ions leaving the MALDI source is in the range of 1 mm/Is. Since the time interval between ionization and cooling has to be a few mircoseconds, the distance between the ionization surface and the high pressure region must be no more than a few millimeters. This invention proposes several embodiments of an apparatus to create such a sequence of low-high-low pressure conditions. In some other ionization sources (SIMS, FAB, EI, LA, for example) maintaining low pressure in ionization region can be vital for the source operation. Thus, maintaining low-high-low profile pressure profile can be important. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]    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 of the accompanying drawings which show, by way of example, embodiments of the present invention and in which:  
         [0013]    [0013]FIG. 1 is a schematic view indicating basic principles of generation of ions by MALDI;  
         [0014]    [0014]FIG. 2 is a schematic view showing an ideal pressure distribution along the axis from a MALDI ion source;  
         [0015]    [0015]FIG. 3 shows a first embodiment of the present invention including a double cone arrangement for providing cooling gas flow;  
         [0016]    [0016]FIG. 4 shows a second embodiment including the provision of a high-density gas intersecting the ion path at an angle;  
         [0017]    [0017]FIG. 5 shows a third embodiment including the separate high-pressure chamber with two outlets for gas;  
         [0018]    [0018]FIG. 6 shows a fourth embodiment including annular, ring-shaped outlet for cooling gas;  
         [0019]    [0019]FIG. 7 shows a gas dynamic simulation of the apparatus of FIG. 3;  
         [0020]    [0020]FIGS. 8 a ,  8   b  and  8   c  show three variants of a fifth embodiment of the present invention;  
         [0021]    [0021]FIGS. 9 a ,  9   b  and  9   c  show a further variant of the fifth embodiment of the present invention, showing multiple sample spots; and  
         [0022]    [0022]FIG. 10 a ,  10   b  and  10   c  are mass spectra of insulin, showing the effect of different ion source conditions. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0023]    Referring first to FIG. 1, this shows schematically the general arrangement for producing ions from a MALDI source indicated schematically at  10 . In known manner, the source  10  includes a target probe  12 , on which is located a MALDI sample  14 . In known manners the MALDI sample  14  comprises a sample of analyte molecules, or which usually are large molecules and exhibit only moderate photon absorption for molecule embedded in a solid or liquid matrix consisting of a small, highly absorbing molecular species.  
         [0024]    In use, a laser beam is provided as indicated at  16  and the laser is usually a pulsed laser. The sudden influx of energy, from each laser pulse, is absorbed by the matrix molecules of the sample  14 , causing them to vaporize and to produce a small supersonic jet of matrix molecules and ions in which the analyte molecules are entrained. Such a jet of material is indicated schematically at  18 . During this ejection process, some of the energy absorbed by the matrix is transferred to the analyte molecules.  
         [0025]    The analyte molecules are thereby ionized, but without excessive fragmentation, at least in an ideal case. As noted, this technique can result in the analyte molecules being over-excited and acquiring a high degree of internal excitation, which can result in metastable fragmentation.  
         [0026]    Referring to FIG. 2, this shows a variation of pressure on the vertical axis, with distance in the axial direction from the sample  14  indicated on the horizontal axis (the axial direction being a direction perpendicular to the plane of the target probe  12 ). As FIG. 2 shows, an ideal pressure profile has a first low pressure ionization region indicated at  20  where the pressure is relatively low (10 −7  to 10 Torr). This enables free expansion of the jet or plume  18  of vaporized material, permitting the ions to be released, and permitting the matrix material to evaporate and to dissipate, while minimizing formation of unwanted ions clusters. Immediately downstream from this region there is a high pressure, cooling region  22  maintained at a relatively high pressure (10 −2  to 1000 Torr), and configured to promote rapid cooling of analyte ions by collisional processes. The intention is to dissipate unwanted internal energy within the ions, so as to eliminate, or at least substantially reduce, the likelihood of metastable fragmentation.  
         [0027]    Further downstream there is a collisional focusing region indicated at  24 . The pressure here would be in the range of 10 −3  to 10 Torr, and would be provided, typically, within a quadrupole or other multipole rod set or double helix ion guide or a set of rings ion guide. This collisional focusing region is intended to collect, collimate and focus ions, for subsequent processing. After collisional focusing, ions could be passed into the usual processing section of a mass spectrometer e.g. a mass analyzer section, collision cell, time of flight section and the like.  
         [0028]    It will also be understood that while the pressure is shown as varying smoothly along the axis, this may not be the case and indeed may not be the best arrangement. For example, where anything in the nature of a lens or aperture in a wall is provided between two regions, this will eventually give a step-wise variation to the pressure profile and the pressure in each region may then be moved or less constant.  
         [0029]    Reference will now be made to FIGS.  3 - 6 , which shows different embodiments of an apparatus for implementing the present invention. All of these figures show the basic MALDI source, and for simplicity and brevity, the same reference numerals as used in FIG. 1 are used in FIGS.  3 - 6 , and the description of these common and basic elements of a MALDI ion source is not repeated. Also, the references  20 ,  22 , and  24 , where applicable, are used to indicate different pressure regions in FIGS.  3 - 6 , but it is to be understood that the pressure profile in each case will not correspond exactly with that shown in FIG. 2.  
         [0030]    Referring first to FIG. 3, a dual cone arrangement is provided, including an outer cone  30  and an inner cone  32 . The cones are closed off as indicated at  34 . A short cylindrical section  36  is attached to the outer cone  30 , so as to define between the cylindrical section  36  and the inner cone  32  and an annular outlet  38 . The cones  30 ,  32 , and the annular outlet  38  are all coaxial with an ion axis extending from the MALDI sample perpendicularly to the target probe  12 , and provide a wall around a high pressure region.  
         [0031]    Consequently, in use, as indicated by the arrows, an annular flow of gas is provided from the annular outlet  38  directed away from the jet or plume  18  of expanding, vaporized material. This ensures that adjacent the jet  18 , there is a low-pressure region, as indicated at  20 . The ions are liberated from the jet  18 , and they then pass axially downstream and are entrained by the jet of gas from the annular outlet  38 . This thus provides a cooling region  22  downstream from the outlet  38 , at a relatively high pressure, in which ions are subject to collisional cooling processes to reduce their internal energy and thereby to reduce the likelihood of metastable fragmentation.  
         [0032]    Referring to FIG. 4, in this embodiment, a cooling gas is supplied through a pipe or conduit  40 , which includes a bend  42 , that turns the gas flow through an angle towards an outlet  44 . As shown, the outlet  44  is directed at an angle to intersect an axis for the flow of ions, indicated at  46 .  
         [0033]    Again, as for FIG. 3, this enables initial expansion of a jet  18  to occur in a low-pressure region  20 . On the axis downstream from the jet  18 , the ions then encounter the flow from the gas outlet  44  to provide a high-pressure cooling region  22 , equivalent to the cooling region  22  of FIG. 2.  
         [0034]    Referring to FIG. 5, this shows a high-pressure chamber  50  which would be supplied with gas from an external source (not shown). The chamber  50  has first and second outlets indicated at  52  and  54 , and both are provided on the axis  56 .  
         [0035]    The arrangement of FIG. 5 provides a more controlled definition of the cooling region, equivalent to cooling region  22  of FIG. 2 and here indicated at  59 . Thus, the immediate surroundings outside of the housing  52 , as indicated generally at  55  would be pumped down to a suitable pressure. This then defines at least the pressure for the initial cooling region. Within the chamber  52 , the higher, cooling pressure  59  could be maintained, and gas would then flow axially out from the chamber  50  through the outlets  52 ,  54  as indicated by the arrows.  
         [0036]    Referring to FIG. 6, the fourth embodiment of the present invention provides inlets for gas indicated at  60  connected to an annular gas outlet indicated at  62 . This is directed inside a cylindrical sleeve  64 .  
         [0037]    Thus again in use, a relatively low-pressure region  20  would be provided around the jet  18 . Immediately downstream from the jet  18 , within the cylindrical sleeve  64 , the vaporized material and ions would be entrained with the gas flow from the gas outlet  62 , providing a cooling region  22  at a higher pressure. The flow of gas would then be drawn into a downstream region, e.g., the region  24  of FIG. 2, and where the pressure would be reduced and where collisional focusing could be provided. In some applications, the cylindrical sleeve  64  may be omitted if required pressure regimes and available pumping speed allow so.  
         [0038]    Also, the embodiments shown here (FIGS. 3, 4,  5 , and  6 ) have the pressure profile generating elements separate form the MALDI target. But, it is anticipated that in some circumstances the pressure profile generating elementscan be completely or partially associated with the target, i.e. more or less integral with the ion source.  
         [0039]    It should also be noted that, while the arrangements of FIGS.  3 , 4 , 5 , 6  show the axis of the ionization region coaligned with the axis of the elements determining the required pressure profile and with the axis which would define any following ion guide, this need not always be the rule; in some cases, there may be an advantage to have these axes tilted or even slightly offset with respect to each other, i.e. there could be a first ion axis portion extending from the ion source and a second ion axis portion extending at least through the high pressure region and preferably into a downstream ion guide, with these two ion axis portions at an angle to one another and/or offset relative to one another. Such an arrangement may facilitate separation of ions from neutrals and heavy charged clusters formed in the ion source. The ions will be drawn into the ion guide by the gas flow and/or electrostatic forces while neutrals and heavy clusters will pass away from the ion guide, generally along the axis of the first ion axis portion.  
         [0040]    Referring to FIG. 7. This shows the result of a direct gas dynamic simulations that shows gas density distributions in the apparatus of FIG. 3. For simplicity and brevity, the same components in FIG. 7 are given the same reference as in FIG. 3 and the descriptions of these components are not repeated. A low pressure region is visible at  20 ; the high pressure region  22  is indicated by the darker shading; and further downstream there is a lower pressure region, where collisional cooling occurs.  
         [0041]    Reference will now be made to FIGS. 8 a ,  8   b , and  8   c , which show a fifth embodiment of the present invention. This embodiment is based on the realization that, once the supersonic jet of matrix molecules and ions is formed, there is a tendency for the jet to expand or spread in all available directions, although the main trajectory tends to be orthogonal to the surface of the target probe. If the distance that the jet travels before it enters the cooling region is significant, or if the opening of the ion transmission path (skimmer orifice) is small compared to the diameter of the expanding jet, a significant portion of the analyte molecules may not be detected.  
         [0042]    Thus, in FIG. 8 a , to overcome this difficulty, a fifth embodiment of the invention, indicated generally at  90 , is shown. This embodiment includes a cone-shaped target probe  92 . The target probe  92  would, in a section perpendicular to the axis of the device, have a circular section. The probe  92  has a circular MALDI surface  94 , located coaxial with an opening or orifice  96  in a sampling cone or skimmer  98 , the cone  98  being similar to earlier embodiments. An ionization region P 1  outside the cone  98  has a pressure that is generally greater than the pressure P 2  within the cone. A MALDI sample is located on the MALDI surface  94  and is ionized with a laser  102 .  
         [0043]    Consequently, there is a flow of gas from the relatively high-pressure ionization region to the interior of the sampling cone or skimmer  98 , as indicated by the arrows  100 . These arrows  100  show, schematically, streamlines representative of gas flow, and indicate how the gas flow follows the profile of the target probe  92 . This gas flow entrains the jet of molecules and ions from the MALDI sample and transfers the plume through the skimmer opening or orifice  96  into the skimmer or cone  98 .  
         [0044]    The entrainment has the effect of confining the plume to prevent spreading of the plume. In contrast, in the earlier embodiments, the MALDI sample is on a flat surface so that there will be no strong confining flow immediately adjacent to the sample itself.  
         [0045]    [0045]FIG. 8 a  shows the MALDI sample surface  94  positioned outside of the sampling cone  98 , i.e. just upstream of the inlet  96 . It is possible that the MALDI sample surface  94  could be provided in different locations relative to the cone  94 , and alternative configurations are shown in FIGS. 8 b  and  8   c . For simplicity and brevity in these figures, the same reference numerals are used, with suffixes “b” “c”, to distinguish them from FIG. 8 a.    
         [0046]    Thus, a second variant,  90   b , in FIG. 8 b  has the target probe  92   b  positioned such that the sample surface  94   b  is now located generally coplanar with the opening or orifice  96 . In FIG. 8 c , the variant is indicated at  90   c  and here a cone-shaped target probe  92   c  has its MALDI sample surface  94   c  positioned just inside the opening  92 ,  
         [0047]    Streamlines are indicated in FIGS. 8 b  and  8   c  by arrows  100   b  and  100   c  respectively, to indicate gas flow. Again, these are schematic, and the detailed gas flows will vary slightly between the variants of FIGS. 8 a ,  8   b  and  8   c.    
         [0048]    A further, simple alternative is shown in FIG. 9 a . Here, the skimmer or cone is again indicated at  98  and has the opening or orifice  96 . The laser beam is again indicated schematically  100 .  
         [0049]    In FIG. 9 a , in place of the cone-shaped target probe, there is provided a post  102  mounted on a planar support  104 . The post  102  includes an end surface  106 , providing a MALDI support surface, for a MALDI sample. The post  102  is of sufficient length, to enable streamlines to develop to entrain the flow, as indicated by the arrows  108 . It is expected that this arrangement will give similar advantages to the configurations of FIGS. 8 a, b  and  c,  while providing a structurally simpler arrangement for the target probe.  
         [0050]    It is preferred for the post  102  to be generally circular, but it could have other profiles. For example, FIGS. 9 b  and  9   c  show generally elliptical cross sections for the post  102 . As indicated schematically in FIGS. 10 a  and  10   b , the MALDI support surface  106  can be used for just a single MALDI sample  108  or a number of separate samples  110 , as shown in FIG. 10.  
         [0051]    It is preferred for the post  102  and the end of the cone-shaped target probe  92  not to have any sharp edges, so as to permit continuous, smooth gas flow, without any unwanted turbulence. Thus, in FIGS. 9 a ,  9   b  and  9   c , the post  102  is shown with generally rounded edges to the surface  106 .  
         [0052]    Referring to FIG. 10, MALDI spectra of insulin are shown for different ion source conditions. FIG. 10 a  shows a low pressure of approx. 8 mTorr in the ionization region. FIG. 10 b  shows a high pressure of approx. 1 Torr in the ionization region  20 . while FIG. 10 c  shows a configuration, as in FIG. 2 (i.e, low pressure ionization region  20 , higher pressure cooling region  22  and low pressure collisional focusing region  24 ) and using the configuration of FIG. 3. In FIG. 10 a , fragment ions  82  are abundant, showing the benefit of higher pressure. In FIG. 10 b , analyte-matrix cluster ions  84  are abundant; emphasizing the necessity of low pressure during initial stage of MALDI. The flow of gas can be supplied to all of the above embodiments continuously or in a pulsed fashion. Pulsed gas introduction may be beneficial to reduce pumping speeds required for the setup because the average gas load will be reduced. Alternatively, higher peak pressures can be obtained with pulsed gas flow in the setup designed for continuous gas introduction. The pulse of gas will be provided the means of a pulsed valve or similar device. The opening of the valve will be synchronized with ionization event allowing certain delays for ionization to occur and for gas pressure to rise to a desired level.  
         [0053]    The pressures in sections  20  and  24  may not be equal. A wall can be added to separate the above sections for arrangements from FIG. 3, 5 and  6 . An extra pumping can be provided to obtain desired pressures in sections  20  and  24 .