Patent Publication Number: US-6707040-B2

Title: Ionization apparatus and method for mass spectrometer system

Description:
RELATED APPLICATIONS 
     This application is a continuation-in-part of the U.S. patent application Ser. No. 10/105,172, filed Mar. 21, 2002, entitled “Ionization Apparatus and Method for Mass-spectrometer System”. 
    
    
     FIELD OF THE INVENTION 
     This invention relates generally to the field of mass spectrometry, and more particularly to sample ionization for mass spectrometer system. More particularly, this invention relates to an ionization apparatus and method for connection to a mass analyzer to improve mass analysis by seamlessly combining sample ionization and sample analysis. 
     BACKGROUND OF THE INVENTION 
     Mass analysis of any sample in a mass spectrometer requires sample ionization as a first step. Sample ionization can be performed under either vacuum or atmospheric pressure. Vacuum ionization techniques include electron impact ionization, fast ion bombardment, secondary ion ionization, and matrix-assisted laser deposition/ionization. Vacuum ionization occurs inside a mass spectrometer instrument under vacuum conditions. A disadvantage of vacuum ionizations is that a sample support must be inconveniently introduced into the vacuum via vacuum locks, making the linking of mass spectrometry with chromatographic and electrophoretic separation methods difficult. 
     Atmospheric pressure ionization takes place outside of the low pressure components of a mass spectrometer instrument. To sample atmospheric pressure ions, a mass spectrometer must be equipped with an atmospheric pressure interface (API) to transfer ions from an atmospheric pressure region to the mass analyzer under high vacuum. Atmospheric pressure ionization techniques include atmospheric pressure chemical ionization and electrospray ionization (ESI) among others. One problem of many prior art atmospheric pressure ionization techniques is the low transmission efficiency of sample ions to a mass analyzer due to ion losses and low throughput of ions for mass analysis due to non-seamless connection of atmospheric sample ionization and sample analysis under high vacuum. 
     U.S. Pat. No. 5,663,561 describes a device and method for ionizing analyte molecules at atmospheric pressure by chemical ionization. According to this method, the analyte molecules deposited together with a decomposable matrix material are first decomposed in the surrounding gas under atmospheric pressure to produce neutral gas-phase analyte molecules. Then these neutral gas-phase analyte molecules are ionized by atmospheric pressure chemical ionization. This method requires that the desorption of the analyte be carried out as a separate step from the ionization of the analyte. 
     U.S. Pat. No. 5,965,884 describes an atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) ion source. The AP-MALDI apparatus contains an atmospheric pressure ionization chamber hosting a sample to be analyzed, a laser system outside the ionization chamber, and an interface that connects the ionization chamber to the spectrometer. While this AP-MALDI apparatus combines analyte desorption and ionization in a single step, it cannot be operated at an optimum pressure regime, and ion transmission from the ionization chamber to the spectrometer is low. Moreover, analyte adducting is high and undesired molecular clusters are formed during the ionization process. 
     EP 0964427 A2 describes a MALDI ion source operating at pressures greater than 0.1 torr. While the claimed ion source may be operated at a greater pressure range, it has the same problems as U.S. Pat. No. 5,965,884: low ion transmission, high adducting among analytes and other molecules and undesired cluster formation. 
     WO 99/38185 and U.S. Pat. No. 6,331,702 B1 describe a spectrometer provided with a pulsed ion source and transmission device to damp ion motion and method of use. This design requires a sample loading chamber or lock chamber and a low pressure MALDI ion source, and has limited throughput. 
     WO 00/77822 A2 describes a MALDI ion source that is enclosed in a chamber and operated under a low pressure and has a limited throughput. 
     U.S. Pat. No. 6,331,702 B1 describes a MALDI ion source that is disposed in a vacuum chamber and has a limited throughput. 
     OBJECTS AND SUMMARY OF THE INVENTION 
     Accordingly it is an object of the present invention to provide an ionization apparatus for connecting to a mass analyzer to seamlessly combine sample ionization and sample analysis. 
     It is another object of the present invention to provide an ionization apparatus for fast sample scanning to increase throughput of mass analysis. 
     It is a further object of the present invention to provide an ionization apparatus which allows sample preparation at atmospheric pressure to increase reliability and reduce construction cost of mass analysis systems. 
     In accordance with the invention, there is provided an ionization apparatus for connection to a mass analyzer. The ionization apparatus comprises a sample slide having at least two sample spots containing analytes to be analyzed by a mass analyzer, means for delivering energy to one of the sample spots to release and ionize the sample analytes to form sample ions, and an interface for supplying the sample ions to the mass analyzer. The interface comprises a chamber having an orifice in close proximity to the irradiated sample spot and defining a first region encompassing the irradiated sample spot. An ion guide is disposed in the chamber and leads to the mass analyzer in a second region. Means for sustaining a pressure substantially lower than atmospheric within the first region is provided for capturing the ions while other sample spots are maintained at atmospheric pressure. Means for sustaining a pressure within the second region substantially lower than the pressure within the first region is provided. 
     The means for delivering energy is disposed such that the energy irradiates one of the sample spot through the orifice in front of the irradiated sample spot. Alternatively, the means for delivering energy is disposed such that the energy irradiates one of the sample spots from the back of a transparent sample slide. 
     The ionization apparatus may comprise a motorized stage for moving the sample slide to sequentially present sample spots to the first region. The motorized stage can be computer controlled and moveable in three dimensions. The sample slide is preferably disposed in proximity of about from 50 to 100 microns to the interface. 
     The ionization apparatus may comprise a cover slide that seamlessly takes place of the sample slide with the same proximity to the orifice when the sample slide moves away during sample change. 
     The means for sustaining a pressure substantially lower than atmospheric within the first region can maintain a pressure from few torr to few tens torr. The means for sustaining a pressure within the second region can maintain a pressure from about 0.001 to about 0.1 torr. 
     In another embodiment of the present invention, there is provided an ionization apparatus further comprising an external groove surrounding the orifice to stabilize the pressure within the first region. This ionization apparatus may further comprise spacing balls for engaging the sample slide and the interface to accurately space the slide from the orifice. 
     In another aspect of the present invention, there is provided a method for ionizing analytes in a sample for mass spectrometer analysis. The method comprises providing a sample slide having at least two sample spots containing analytes to be analyzed by a mass analyzer and providing an interface connecting one of the sample spots to the analyzer. The interface is provided with a chamber having an orifice in close proximity to one of the sample spots and defining a first region encompassing the sample spot. An ion guide is disposed in the chamber leading to the mass analyzer in a second region. Energy is delivered to one of the sample spots to release and ionize the analytes to form ions. A pressure substantially lower than atmospheric is sustained within the first region while maintaining atmospheric pressure at other sample spots. A pressure within the second region substantially lower than the pressure within the first region is provided. 
     In another embodiment of the present invention, the ionization apparatus comprises a sample slide that is provided with at least two channels therethrough. Samples are deposited on the inner surfaces of the channels. Means for delivering energy such as a laser irradiates the sample in one of the channels and ionizes the sample to form ions. An interfacial orifice is aligned with and in close proximity to the channel and collects ions formed in the channel. Preferably the sample slide is provided with a plurality of channels, and each channel is sequentially brought in registration with the interfacial orifice by moving the sample slide in three directions. The ionization apparatus may further comprise means for applying a voltage between the sample slide and the orifice for accelerating ion flow. The energy delivery means is disposed such that energy is directed to the samples. The energy delivery means may include a focusing lens aligned with and movable along the axis of the channel to deliver energy to the entire inner surface of the channel. Alternatively, the energy delivery means may include an optical fiber having an end movable along the axis of the channel to deliver energy to the entire inner surface of the channel. 
     In still another embodiment, the ionization apparatus includes a spacer attached onto the sample slide on the side facing the orifice. The spacer is provided with holes that have the same pattern and dimension as and in registration with the channels in the sample slide. The spacer can be made of electrically non-conductive materials. In operation, the sample slide-spacer assembly can be brought in tight contact with the orifice to increase suction force of gas flow and provide electrical insulation between the sample slide and the orifice. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The foregoing and other objects of the invention will be more clearly understood from the following description when read in conjunction with the accompanying drawings in which: 
     FIG. 1 is a schematic view of an ionization apparatus including a laser source delivering energy to a sample spot through an orifice in front of a sample slide. 
     FIG. 2 is a schematic view of an ionization apparatus including a laser source delivering energy to a sample spot from the back of a transparent sample slide. 
     FIG. 3 is a schematic view of an ionization apparatus having an interface including a groove and spacing balls at an orifice in front of the sample slide. 
     FIG. 4 is a schematic view of an ionization apparatus including a sample slide provided with a plurality of sample channels. 
     FIG. 5 is a schematic view of an ionization apparatus including a spacer attached to the sample slide illustrated in FIG.  4 . 
     FIG. 6 is an exploded view illustrating depositing samples into channels in a sample slide and attaching a spacer to the sample slide. 
     FIG. 7 is a partial sectional view of an ionization apparatus illustrating an orifice in form of a truncated cone in contact with a spacer attached to a sample slide provided with channels. 
     FIG. 8 is a partial sectional view of an ionization apparatus illustrating an orifice in form of a tube in contact with a spacer attached to a sample slide provided with channels. 
     FIGS. 9 and 10 are partial sectional views of ionization apparatus illustrating that energy beam irradiates samples in a channel at an angle with respect to the axis of the channel. 
     FIGS. 11 and 12 are partial sectional views of ionization apparatus comprising a focus lens movable along the axis of the channel. 
     FIGS. 13 and 14 are partial sectional views of ionization apparatus comprising an optical fiber having an end movable along the axis of the channel. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     FIG. 1 shows an embodiment  10  of an ionization apparatus of the present invention. This ionization apparatus  10  comprises a sample slide  101  having at least two sample spots  100  containing sample analytes to be ionized, a laser source  104  for delivering energy  112  to one of the sample spots  100  through a focus lens  105 . The energy  112  ionizes the sample at the irradiated sample spot  100 . An interface  15  collects ions generated at the irradiated sample spot  100  and delivers them to a mass analyzer (not shown) as indicated by arrow  103 . The mass analyzer  103  can comprise a time of flight (TOF) mass analyzer, an ion trap mass analyzer, an orbitrap mass analyzer, a magnetic sector mass analyzer, or a Fourier transform mass analyzer. 
     The sample slide  101  is maintained at atmospheric pressure and brought in close proximity to the interface  15  by a motorized stage  111 . The motorized stage  111  is computer controlled and movable in three dimensions (x, y, z). A plurality of sample spots  100  are provided on the sample slide  101  so that they are brought sequentially into position for ionization and analysis. Each individual sample spot  100  is brought sequentially in registration with the interface  15  by driving the motorized stage  111  controlled by a computer (not shown). Materials that can be used for the sample slide  101  include electrically conductive metals such as stainless steel, insulating polymers such as teflon, and porous silica. It is apparent that the sample can be deposited together with a decomposable matrix material at the sample spot  100  and the sample slide can be moved in the x-y-z directions to bring the spot in registration with the orifice  102  of the interface  15 . A cover slide (not shown) seamlessly takes place of the sample slide with the same proximity to the orifice during sample change. 
     The walls of interface  15  form a chamber  118  having an orifice  102  which captures ions generated at the irradiated sample spot  100 . An ion guide  106  is disposed in the chamber  118  to transport ions to the mass analyzer as indicated by arrow  103 . Preferably, the orifice  102  is in the shape of a truncated cone and is brought into a close proximity to the sample slide  101  so that the irradiated sample spot  100  is located opposite the opening of the cone. The distance between the irradiated sample spot  100  and the front surface of the orifice  102  can be precisely controlled by moving the motorized stage  111  in the x direction. Preferably, the distance is within from about 50 to 100 microns. A wall  17  is spaced from the end of the interface walls to define a subchamber  16  adjacent to the orifice  102 . A pump (not shown) is connected to port  108  which communicates with the subchamber to sustain a pressure within the region  107  of the orifice  102  which is higher than the pressure in chamber  118 . The pump can be a rotary vacuum pump and sustain a pressure from few torr to few tens torr at the sample spot  100  being ionized. Accordingly, the region surrounding the sample spot  100  being ionized can be sustained a pressure substantially lower than atmospheric while other sample spots  100  outside the region  107  encompassed by the orifice  102  are maintained at atmospheric pressure. 
     An ion guide  106  is disposed inside the chamber  118  and extends from the orifice  102  to a mass analyzer  103 , forming a multipole region  109  through which sample ions are transported by combination of gas flows and electric fields. The ion guide  106  can be any transmission or trapping device. Preferably the ion guide  106  is a RF-only multipole and can be heated. A turbo pump (not shown) is connected to a port  110  for sustaining a vacuum within the chamber  118 . A valve (not shown) is also equipped at port  110  so that the pressure within the multipole region  109  can be adjusted from 0.001 to 0.1 torr for optimal performance. 
     A laser source  104  delivers energy such as a UV light, visible light, or IR light  112  through a lens  105 , which focuses the energy on one of the sample spots to release and ionize the sample. The laser source  104  can irradiate pulsed or continuous energy to at least one sample at a time. In this embodiment  10  of the ionization apparatus, the laser source  104  and the lens  105  are disposed such that laser energy  112  is delivered to one of the sample spots  100  through the orifice  102  in front of the sample spot  100 . 
     FIG. 2 shows another embodiment  20  of the ionization apparatus of the present invention. The laser source  104  and the lens  105  are disposed such that the laser energy  112  is delivered to one of the sample spots  100  from the back of the sample slide  101 , either through a transparent slide, or the sample can be on the end of a transparent optical fiber. Preferably the sample slide or optical fiber is made of quartz. 
     FIG. 3 shows another embodiment  30  of the ionization apparatus of the present invention. In comparison with embodiments  10  and  20 , embodiment  30  has an external groove  113  surrounding the orifice at the end of the chamber  118 . The groove  113  is evacuated through the chamber passage  116  connected to port  108 , preferably by a rotary pump connected to the port  108 . This increases robustness of the differential pumping and stability of the pressure in the orifice region  107 . To further increase stability of the pressure in the orifice region  107 , the gap between the sample slide  101  and the orifice  102  is fixed by introducing spaced ball bearings  114 . This design provides a greater precision and accuracy for the gap between the sample slide  101  and orifice  102 . The ball size can be chosen large enough, so that the balls roll over the sample spots  100  without reaching the bottoms of the wells  100  in which the samples are located. This embodiment  30  can use either front or back laser irradiation as illustrated in embodiments  10  and  20 . 
     One advantage of the present invention is that sample analysis may be seamlessly combined with sample ionization that makes the system ideal for high-throughput proteomics. Ion losses on the orifice are low. Another advantage is that vacuum seals are not needed between the sample spot being ionized and other spots. The motorized stage moving the sample slide can be operated at atmospheric pressure. This results in higher reliability and lower construction cost of ionization system. Moreover, the present ionization apparatus can increase throughput up to 1 second per sample due to fast sample scanning and no time losses on sample introduction. The ionization system of the present invention is also advantageous in that it is easy to automate and interchangeable with ESI ion source, thus both proteomic tools can be used in parallel for the same sample. 
     FIG. 4 shows another embodiment  40  of the ionization apparatus of the present invention. In this embodiment  40 , the sample slide  101  is provided with at least two channels  119 . Samples  100  to be analyzed are deposited on the inner surfaces of the channels  119 . Preferably a plurality of channels  119  are provided in the sample slide  101  to increase throughput of mass analysis. The sample slide  101  can be moved in three directions (x-y-z) by the motorized stage  111  controlled by a computer to sequentially bring each channel  119  in registration with the orifice  102 . The gap between the sample slide  101  and the orifice  102  is controlled by moving the sample slide  101  in x direction until it is closely adjacent the orifice  102 . In operation, one channel is aligned with the orifice  102  and laser energy  112  irradiates sample  100  to form ions which are captured at region  107  and guided to the mass analyzer  103  by combination of electrical field and gas flow. This embodiment  40  is advantageous in that the channels  119  in the sample slide  101  enhance the air-dynamic properties of gas flow and improve ion entrainment at the entrance to the orifice  102 . 
     FIG. 5 shows another embodiment  50  of the ionization apparatus of the present invention. In this embodiment  50 , a spacer  120  provided with channels or holes  121  is attached to the sample slide  101  on the side that faces the orifice  102 . The holes  121  have substantially the same dimensions and patterns as the channels  119  in the sample slide  101 . When laser energy  112  irradiates the sample  100  in operation, all three of the channel  119  in the sample slide  101 , the hole  121  in the spacer  120 , and the orifice  102  are aligned on one axis. The sample slide  101  and spacer  120  assembly can be moved in three directions (x-y-z) by the motorized stage  111  to bring each channel  119  and hole  121  in registration with the orifice  102 . Preferably the sample slide and spacer assembly is brought in tight contact with the orifice  102  and slides across the orifice  102 . This embodiment  50  is advantageous in that the spacer  120  increases suction force of gas flow through the channel  119  to the orifice  102  and reproducibility of sample positioning with respect to the orifice  102 . The spacer  120  also provides electrical insulation between the sample slide  120  and the orifice  102  when a voltage is applied. In addition, the spacer protects orifice  102  from sample carryover and prevents sample cross contamination. 
     In the embodiments  40  and  50  of the present ionization apparatus illustrated in FIGS. 4 and 5, the laser source  104  and lens  105  are disposed such that laser energy  112  is delivered to one of the channels  119  from the back of the sample slide  101 . Alternatively, the laser source  104  can be disposed such that laser energy  112  is delivered to one of the channels  119  through the orifice  102  in front of the channel  119 , as illustrated in FIGS. 2 and 3. 
     More detail structure of embodiments  40  and  50  of the ionization apparatus of the present invention are now described with reference to FIGS. 6 to  14 . 
     FIG. 6 schematically shows channels  119  in sample slide  101  and deposition of samples  100  in the channels  119 . While FIG. 6 shows the channels  119  in shape of a cylinder for illustration purpose, other shapes of channel can also be used as long as they increase gas flow in the channels and improve ion entrainment at the orifice. For example, the channels can also be shaped in a truncated cone. The channels  119  can be fabricated in an array on one plate  101  to enhance throughput of sample deposition. The prior art methods of depositing samples on a flat surface can be used in depositing samples  100  in the channels  119 . For instance, the samples  100  can be mixed with an MALDI matrix and deposited in the channels  119  using known deposition protocols and robots. The samples  100  are sucked inside the channels  119  by capillary force. For example, a channel having a diameter of 0.65 mm and a length of 3 mm can accommodate 1.0 μl of samples. After drying for a few minutes, only solid residue remains in the channels  119 . In the embodiment where a spacer  120  is attached onto the sample slide  101  as illustrated in FIG. 5, the sample slide  101  provided with an array of channels  119  can be covered by an electrically insulating plate or spacer  120 . The electrical insulating plate  120  is provided with a plurality of holes  121  that are of the same dimension and pattern as the channels  119  in the sample slide  101 . The holes  121  in the insulating plate  120  are in registration with the channels  119  in the sample slide  101 . The insulating plate  120  can be made from electrically nonconductive materials, such as glass, teflon, and plastic. Preferably the insulating plate  120  has smooth surfaces for tight attachment to the sample slide and better sliding across the orifice  102 . The insulating plate or spacer  120  provides electrical insulation between the sample slide  101  and orifice  102  and also protects the orifice  102  from cross contamination from different samples. The sample slide and spacer assembly so prepared can be stored in an autosampler waiting for analysis. After analysis, the sample slide  101  and spacer  120  can be washed and reused. 
     The channels  119  in the sample slide  101  preferably have a diameter that is substantially same as or similar to the diameter of the orifice  102  at the interface, preferably ranging from about 0.2 mm to 2 mm. The length of the channels  119  can be several millimeter, preferably ranging from 0.5 mm to 20 mm. Preferably, a plurality of channels  119  are provided in the sample slide  101  to increase analysis throughput. In operation, each individual channel  119  is sequentially brought in registration with the orifice  102  for ionization and analysis. The distance between the sample slide  101  and the orifice  102  is preferably within from about 50 to 100 microns for easy access of laser radiation to sample  100  and efficient collection of ions. In the embodiment where a spacer  120  is attached to the sample slide  101  as illustrated in FIG. 5, the sample slide  101  and spacer  120  assembly is preferably brought in tight contact with the orifice  102 . Any gap between the spacer  120  and the orifice  102  is defined by the surface roughness and tolerances of the spacer  120  and orifice  102 , and is much smaller than the diameter of the channel  119 , allowing the main gas stream flows through the channel  119 . 
     FIGS. 7 and 8 schematically show the orifice  102  that is in alignment with an individual channel  119 . In FIGS. 7 and 8, an insulating spacer  120  is disposed between the channel  119  and the orifice  102 . Though the sample slide  101  and spacer assembly is shown as in contact with the orifice  102 , this is not required. A small gap between the orifice  102  and sample slide  101  allows a faster sample changeover and therefore improve throughput of analysis. In the embodiment where spacer is not used as shown in FIG. 4, the gap between the sample slide  101  and the orifice  102  is preferably controlled within 50 to 100 microns for better access for laser irradiation of samples and better gas flow and ion entrainment at the entrance to the orifice  102 . 
     The orifice  102  can be in form of a skimmer as shown in FIG. 7, or a tube as shown in FIG.  8 . In both embodiments of skimmer and tube, the orifice  102  has a diameter substantially same as the diameter of the channel  119  in the sample slide  101 , or the hole  121  in the spacer  120 , at the interface between the orifice  102  and the channel  119  or the hole  121 . Preferably the diameter of the orifice  102  at the interface is from 0.2 mm to 2 mm. 
     To facilitate gas flow of ions formed in the channel  119  to the orifice  102 , and eventually to the mass analyzer  103  through an ion guide, the pressure in the channel  119  can be controlled. In the embodiment of a skimmer orifice  102  as shown in FIG. 7, the pressure in the channel  119  is preferably maintained from a few Torr to a few tenths of Torr. In the embodiment of a tube orifice  102  as shown in FIG. 8, the pressure in the channel  119  is preferably maintained from below atmosphere to 10 Torr. In one embodiment, a voltage  122  is applied between the channel  119  and the orifice  102  to facilitate gas flow of ions, as shown in FIGS. 7 and 8. Ions of one polarity are accelerated towards the orifice  102  by electrical field, while ions of opposite polarity are prevented from entering the orifice  102  by the voltage  122 . 
     FIGS. 9 to  14  illustrate various means for delivering energy to the channel  119  to release and ionize samples  100 . To better irradiate samples  100  on the inner surface of the channel  119 , the laser beam  112  is preferably non-parallel to the axis of the channel  119 . In one embodiment illustrated in FIGS. 9 and 10, the laser beam  112  irradiates the sample  100  at a small angle with respect to the axis of the channel  119 . Preferably the angle ranges from 5 to 85 degrees with respect to the axis of the channel  119 . In another embodiment illustrated in FIGS. 11 and 12, a focus lens  105  is used where the laser beam  112 , the focus lens  105 , and the channel  119  are aligned on one axis. The laser beam  112  is focused in a focal point  124  in front of the channel  119 . Modern nitrogen lasers can be focused in a spot of 0.1 mm in diameter. After the focal point  124 , divergent beam  126  irradiates entire channel  119 . To reach deeper into the channel  119 , the focusing lens  105  is preferably movable along the axis of the channel  119  in x direction. In another embodiment illustrated in FIGS. 13 and 14, an optical fiber  130  is used to create a symmetrical, divergent laser beam  132  with point source in front of the channel  119 . To reach deeper into the channel  119 , the end  131  of the optical fiber  130  is preferably movable along the axis of the channel  119  in x direction. 
     One advantage of the ionization apparatus comprising a sample slide provided with channels is that during sample preparation steps, the channels can be used for fraction collection from HPCL, for automatic sample deposition by an autosampler, for mixing sample and MALDI matrix solutions, and for sample purification and affinity separation. 
     The foregoing description of specific embodiments and examples of the invention have been presented for the purpose of illustration and description, they are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously many modifications, embodiments, and variations are possible in light of the above teaching. It is intended that the scope of the invention encompass the generic area as herein disclosed, and by the claims appended hereto and their equivalents.