Abstract:
A system for determining the ratio of mass to charge of an ion including a pulsed ionizer, a high pressure co-linear ion guide/accelerator, and a mass analyzer. The pulsed ionizer generates intact analyte ions from a sample of matter to be analyzed. The high pressure co-linear ion guide/accelerator is interfaced with the ion source for receipt of the intact ions of the sample. The ion guide/accelerator simultaneously dampens and linearly accelerates the intact ions in the substantial absence of fragmentation of the ions to provide a substantially continuous beam of the intact ions for mass analysis. The mass analyzer is connected to the ion guide/accelerator for receipt of the beam of ions and determines the mass to charge ratio of the intact ions.

Description:
This application is a continuation application of U.S. patent application Ser. No. 10/657,580 filed on Sep. 8, 2003, now U.S. Pat. No. 6,809,318, which is a continuation of application Ser. No. 09/835,943 filed on Apr. 16, 2001, now U.S. Pat. No. 6,617,577, which is incorporated herein by reference. 
    
    
     GOVERNMENTAL SUPPORT 
     The research leading to the present invention was supported, at least in part, by NIH Grant No. RR 00862. Accordingly, the Government may have certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to the art of mass spectroscopy, and in particular, to a method and system for high sensitivity, rapid, high efficiency mass spectroscopy. 
     It is known in the field of mass spectroscopy to provide spectrometers with an elongated conductor having multipole conductors which act as ion transmitters. In PCT Publication WO 99/38185 (the contents of which is incorporated herein by reference), a method and apparatus are disclosed for providing ion transmission between an ion source and a spectrometer. The ion transmission device includes a multipole rod set and a damping gas which dampens spatial and energy spreads of ions generated by a pulsed ion source. The multipole rod set has the effect of guiding the ions along an ion path so that they can be directed to the inlet of a mass spectrometer. 
     The WO &#39;185 publication discloses a MALDI (matrix-assisted laser desorption/ionization) ion source for producing a small jet of matrix and analyte molecules and ions and which have a wide range of energy spreads. The ion transmission device of WO &#39;185 spreads out the generated ions along the multipole ion guide axis to provide a quasi-continuous beam while i) reducing the energy spread of ions emitted from the source and ii) at least partially suppressing unwanted fragmented analyte ions. These ions are delivered to a time-of-flight spectrometer or other spectrometers. 
     The apparatus described in WO &#39;185 provides that single multiple rod sets or two or more rod sets can be used. Regardless of the number of rod sets used or the number of rods provided therein, the conductors merely provide ion guidance and possible energy damping by way of collision with a damping gas within the ion guide itself. No provision is made to enhance the efficiency or improve the speed of movement while retaining integrity of the ion beam sent to a mass spectrometer. 
     Another disclosure, U.S. Pat. No. 6,111,250 to Thomson, et al., discloses a mass spectrometer which includes rod sets constructed to create an axial field, e.g., a DC axial field. The Thomson, et al. &#39;250 disclosure provides for speeding the passage of ions through an ion guide and causing the ions to be fragmented. The ion source is disclosed as being an electrospray or ion spray device such as those described in U.S. Pat. Nos. 4,935,624 and 4,861,988, or a corona discharge needle or a plasma, as shown in U.S. Pat. No. 4,861,965. The ions are directed and their speed controlled for introduction into a “time-of-flight” mass analyzer. In one embodiment, Thomson, et al. disclose the use of a set of auxiliary rods in combination with a set of quadrupole rods for the purpose of, among other things, introducing very low energy ions into a quadrupole mass analyzer. There is no disclosure by Thomson, et al. regarding transmitting intact analyte ions as a substantially continuous ion beam for highly sensitive, rapid mass analysis. 
     While there are numerous disclosures relating to the art of mass spectroscopy of analyte ions, there is an ever increasing demand for high speed and accurate mass spectroscopy of specimens, especially dilute specimens having only trace amounts of analyte ions. It is the purpose of the present invention to meet this and other needs in the art of mass spectroscopy. 
     SUMMARY OF THE INVENTION 
     The present invention is a method and system for determining the ratio of mass to charge of an analyte ion. According to the present invention, intact analyte ions are prepared from a sample by pulse ionizing using a pulse ionizer, e.g., preferably by matrix-assisted laser desorption/ionization (MALDI). 
     The present invention further includes simultaneously damping and linearly accelerating intact ions in a co-linear ion guide/accelerator to reduce the energy spread of the ions without fragmenting them and to linearly accelerate the ions to provide a substantially continuous beam of intact ions. This dual functionality step of the process in the system is implemented by co-linearly arranged multipole rods and accelerator rods which define an axial ion path along which the continuous ion beam travels. This step of the process and the system also includes a damping gas which acts to reduce the energy spread of the ions. While the pressure of the damping gas can range from 0.1 mTorr to 10 Torr, it is preferably from about 10 mTorr to about 1000 mTorr, and most preferably from about 50 mTorr to about 100 mTorr. 
     In a preferred embodiment of the present process and system, an additional ion guide can be provided for receipt of the ion beam resulting from the simultaneous damping and linear acceleration and further directing such beam to mass analysis. Preferably the additional ion guide is provided with a multipole ion guide having at least about eight ion guide rods. 
     Finally, the present invention includes a determination of mass to charge ratio of the substantially intact analyte ions provided from the previous step(s). In a preferred embodiment the determination of mass to charge ratio is conducted in an ion trap spectrometer. The invention is ideally suited for high-efficiency rapid ion trap spectroscopy. 
     The present invention provides a highly sensitive instrument for detection of analyte ions, e.g., peptides, in a concentration at the subfemtomole level. The present invention provides true MSMS capabilities which enable one to perform multiple MSMS experiments within very short periods of time. Moreover, the process and system of the present invention provide a high degree of accuracy even at extremely diluted levels and at unexpectedly high speed. 
     For a better understanding of the present invention, together with other and further objects, reference is made to the following description, taken in conjunction with the accompanying drawings, and its scope will be pointed out in the claims which follow. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Preferred embodiments of the invention have been chosen for purposes of illustration and description and are shown in the accompanying drawings, wherein: 
         FIG. 1  illustrates a block diagram of a system for mass spectroscopy in accordance with the present invention; 
         FIG. 2  is a schematic diagram of a first embodiment of the present invention; 
         FIG. 3  is an exploded view of the ionguide/accelerator of the present invention;  FIG. 4  is a cross sectional view taken along line  4 — 4  in  FIG. 3  showing a multipole rod set and an accelerator rod set; 
         FIG. 5  is a plan view of a sample introduction system for use with the present invention; 
         FIG. 6  is a schematic diagram of a second embodiment of the present invention; 
         FIG. 7  is an exploded schematic diagram showing the quadrupole positioned between the ion trap and the detector of the second embodiment of the present invention; 
         FIG. 8  illustrates a mass spectra of a six peptide mixture acquired in about 2 seconds for a sample amount of 100 fmole; 
         FIG. 9  illustrates a mass spectra of a six peptide mixture acquired in about 2 seconds for a sample amount of 10 fmole; 
         FIG. 10  illustrates a mass spectra of a six peptide mixture acquired in about 2 seconds for a sample amount of 1 fmole; and 
         FIG. 11  illustrates a MS/MS spectrum of an ion at m/z 1956.7 selected from the spectrum of the 1 fmole peptide mixture corresponding to  FIG. 10  that was acquired in about 2 seconds. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Referring now to  FIG. 1 , a system for mass spectroscopy  10  in accordance with the present invention is illustrated as a block diagram. The system for mass spectroscopy  10  includes a pulsed ionizer  12 , an ionguide/accelerator  14 , and a mass analyzer  16 . The pulsed ionizer  12  is preferably a matrix assisted laser desorption device that ionizes a sample to form analyte ions. The ionguide/accelerator  14  is interfaced with the pulsed ionizer  12  for receiving desorbed intact analyte ions from the sample to simultaneously dampen and linearly accelerate the intact ions in the substantial absence of fragmentation of the ions to provide a substantial continuous beam of the intact ions for mass analyses. Preferably the ionguide/accelerator  14  includes a multipole rod set  18  and an accelerator rod set  20  in a collinear arrangement in the presence of high pressure gas. The mass analyzer  16  is connected to the ionguide/accelerator  14  for receiving the beam of ions and to determine the mass charge ratio of the intact ions. 
     Referring now to  FIGS. 2 through 5 , a first preferred embodiment of the system for mass spectroscopy  10  according to the present invention is illustrated. The first embodiment includes a matrix assisted laser desorption ionization (MALDI) pulsed ionizer  12  and ionguide/accelerator  14  configured to cooperate with a mass analyzer  16 , such as the mass analyzer of a commercially available Finnigan LCQ ion trap mass spectrometer as shown in  FIG. 2 . While, the Finnigan LCQ mass spectrometer is generally equipped with an electro spray ionization device (ESI) when sold to consumers, in the first embodiment shown herein the ESI device was removed to accomodate the pulsed ionizer  12  and ionguide/accelerator  14 . It is also possible to configure the device to accommodate both ESI and MALDI. 
     Referring now to  FIG. 2 , the MALDI pulsed ionizer  12  includes a laser  21  configured to pulse a sample located on a substrate  22 . Any pulsed laser that can produce ions from a sample for mass spectrometry can be used. The laser  21  is preferably a nitrogen laser. As known in the art, the laser may be focused at the sample on the substrate  22  by various optical components, examples of which are shown in  FIGS. 2 and 6 . A suitable laser is the VSL-337 Nitrogen Laser manufactured by Laser Science, Inc. of Franklin, Mass. which operates at a repletion rate of 10–20 Hz. The laser  21  can also be a Nd:YAG laser. In  FIG. 2 , the laser  21  is focused on the sample through a lens  24  and a mirror  26 . Preferably the lens collimates the laser beam and has a focal length of about 1 mm to about 1 meter, preferably about 50 cm. The mirror  26  directs the collimated laser beam through a window  25  towards the surface of the substrate  22  at an angle of about 10 degrees to about 80 degrees, preferably about 60 degrees to the normal of the substrate  22 . Preferably the laser beam has a laser spot diameter on the surface of a sample from about 0.3 mm to about 0.5 mm. Preferably the power density of laser radiation in the spot is about 10 7  W/cm 2  . The mirror  26  is preferably configured to be “wobbled” in order to scan the sample with the laser beam. Alternatively as shown in  FIG. 6 , the laser  21  can be focused on the sample located on the substrate  22  through an optical fiber  28 . 
     The sample is supported on a substrate  22 . Various substrates are known in the art to be useful. For example, the substrate may be made of a plastic material, preferably a polycarbonate surface such as that found in a commercially available compact disc. 
     Referring now to  FIGS. 2 and 5 , preferably the first embodiment of the mass spectroscopy system  10  includes a sample introduction system  30  such as that disclosed in Andrew Krutchinsky&#39;s and Brian Chait&#39;s copending U.S. patent application Ser. No. 09/737,660 entitled “High Capacity and Scanning Speed System for Sample Handling and Analysis” filed on Dec. 15, 2000, the disclosure of which is incorporated herein by reference. The sample introduction system  30  generally includes a support plate  27  configured to support a substrate in the form of a compact disc  32  for holding a plurality of samples  34  as shown in  FIG. 5 . The sample introduction system  30  preferably includes a video camera  36  for monitoring the sample during the pulsed ionizing by the laser  21  as shown in  FIG. 2 . Preferably the sample introduction system  30  is connected to a pump (not shown herein) via vacuum line  38  which maintains a vacuum lock between the pump and the system  30  such as by use of an o-ring  40  shown in  FIG. 5 . 
     Referring to  FIG. 5 , the plurality of samples  34  located on the compact disc  32  are preferably formed by dissolving a compound to be analyzed in a solution containing a large molar excess of a matrix forming material that efficiently absorbs the light of the laser  21 . A small amount of the solution is then deposited on the compact disc  32  and dried to form a sample  34 . The samples  34  can be deposited on the compact disc  32  in a variety of known methods including spraying as an aerosol, ultrasonically, or by using a micropipette or fine needle. Preferably, the plurality of samples  34  are discretely deposited over the surface of the compact disc  32  as shown in  FIG. 5 . The location of each sample  34  can be tracked for use with a high speed compact disc drive to enable the analysis of an extremely large number of samples within a short period of time. During the analysis, the matrix absorbs the energy from the laser pulse resulting in the vaporization and ionization of the sample. 
     Referring now to  FIGS. 3 and 4 , the ionguide/accelerator  14  preferably includes a multipole rod set  18  and an accelerator rod set  20  in a collinear arrangement in the presence of high pressure gas. That is, both the multipole rod set  18  and an accelerator rod set  20  are preferably symmetrically arranged about an axis  54  of the ionguide/accelerator  14  as shown in  FIG. 4 . The high pressure gas is maintained generally from about 0.1 mTorr to about 10 Torr by a pump represented as arrow  45  in  FIG. 2 . Preferably the high pressure gas is maintained from about 10 m Torr to about 1000 m Torr, and most preferably from about 50 m Torr to about 100 m Torr. The presence of the high pressure gas provides collisional damping for reducing the energy spread of the desorbed ions without substantial fragmentation. Preferably the ionguide/accelerator  14  is arranged spatially at a distance, A, of not greater than about 2.0 cm from the source of ions for entry of analyte ions, which is generally measured from the substrate  22  as shown in  FIG. 2 . Preferably the spatial distance is from about 0.1 mm to about 1 cm, and most preferably from about 0.8 mm to about 1.2 mm. Referring to  FIG. 3 , preferably the ionguide/accelerator  14  includes a plate  44  at an opposite end of the source of ions formed with an aperture  46  having a dimension, e.g., a diameter, from about 0.1 cm and to about 2 cm. Preferably the dimension of the aperture  46  is from about 0.2 cm to about 1.0 cm, and most preferably is about 0.3 cm. Preferably the aperture  46  is circular. The ionguide/accelerator  14  preferably includes an ion guide screen  48 . 
     The multipole rod set  18  confines the ions. and preferably includes at least four (4) ion guide rods  40  symmetrically arranged about the axis  54 . The multipole rod set  18  can be configured to include more than four (4) ion guide rods  40 . For example, the multipole rod set  18  could include eight (8) ion guide rods  40  to be configured in a similar manner as an octopole. Preferably each ion guide rod  40  has a length in a range from about 1 cm to about 100 cm and has a largest cross-sectional dimension, e.g., a diameter, in a range from about 0.1 cm to about 2 cm. The length of each ion guide rod  40  is preferably from about 10 cm to about 40 cm and most preferably from about 18 cm to about 22 cm. The cross-sectional dimension of each ion guide rod  40  is preferably from about 0.2 cm to about 1 cm and most preferably from about 0.50 cm to about 0.8 cm. Preferably each ion guide rod  40  has a circular cross section. 
     The accelerator rod set  20  provides an electrical force to drag the ions towards the exit of the ion guide  14  and preferably includes at least four (4) accelerator rods  42  symmetrically arranged about the axis  54 . The accelerator rod set  20  can be configured to include more than four (4) accelerator rods  42 . For example, the accelerator rod set  20  could include eight (8) accelerator rods  42 . The accelerator rods  42  are arranged closer to the axis  54  of the ion guide  14  at the entrance  50  and further from the axis  54  at the ion guide  14  exit  52 . Preferably each accelerator rod  42  has a length in a range from about 1 cm to about 100 cm and has a largest cross-sectional dimension, e.g., diameter, in a range from about 0.1 mm to about 2 cm. The length of each accelerator rod  42  is preferably from about 10 cm to about 40 cm and most preferably from about 16 cm to about 20 cm. The cross-sectional dimension of each accelerator rod  42  is preferably from about 0.1 cm to about 1 cm and most preferably from about 0.25 cm to about 0.5 cm. Preferably each accelerator rod  42  has a circular cross section. 
     In operating the ionguide/accelerator  14 , the multipole rod set  18  is preferably driven by an independent RF power supply to generate a sine wave amplitude from about 1 V to about 10,000 V. Preferably the amplitude is in the range from about 100 V to about 1000 V, and most preferably from about 300 V to about 500 V. The power supply can include a 500 kHz crystal oscillator-controlled sine wave generator and a power amplifier such as Model No. 240L of ENI, Rochester, N.Y. The multipole rod set  18  can also be operated as a mass filter by applying DC voltages from about −50 V to about +50 V while providing the necessary offset from about 15 V to about 25 V. Both the plate  44  and ion guide screen  48  are grounded as shown in  FIG. 3 . The voltage applied to the accelerator rod set  20  creates a small electrical field along the axis  54  of the ion guide  14  because of the changing proximity of the accelerator rods  42  to the axis  54  of the ion guide  14  that drags the desorbed ions along the axis  54 . Preferably, a constant voltage is applied to the accelerator rod set  20  from about 1 V to about 10,000 V. The accelerator rod set voltage can be in the range from about of 100 V to about 1000 V, and preferably is about 100 V. Although MALDI spectra can be obtained when the substrate  22  is isolated and no potential is applied to the support plate  27 , preferably about 200 V is applied to the support plate  27  for the optimum recording of MALDI spectra. 
     Referring now to  FIG. 2 , the mass analyzer  16  preferably includes an ion trap  56  and a detector  58 . In the first embodiment of the present invention, the mass analyzer  16  utilizes the ion trap  56  and the detector  58  configuration of the commercially available Finnigan LCQ ion trap mass spectrometer (hereinafter “Finnigan mass spectrometer”). The Finnigan mass spectrometer also includes an octopole  60  which interfaces with the ionguide/accelerator  14 . 
       FIGS. 8 through 10 , illustrate the MALDI spectra of samples obtained from a mixture of six peptides at an equimolar concentration of 100 fmol/μl in a solution of 60/35/5 MeOH/water/acetic acid as well as dilutions thereof at respectively 10 fmol/μl and 1 fmol/μl. The sample analyzed for  FIGS. 8 ,  9 , and  10  respectively contained 100, 10 and 1 fmole of each peptide. The sample matrix solutions were prepared by depositing the solution onto the polycarbonate surface of the compact disc  32  and allowed to dry. The samples were bombarded with a collimated nitrogen laser beam having a diameter between 0.3 and 0.5 mm and a power density of about 10 7  W/cm 2  while applying about 200 V to the support plate  27 . The desorbed ions were introduced into the ion guide/accelerator  14  for simultaneously damping by high pressure gas at about 65 mTorr and dragging the ions with the accelerator rod set  20 . A constant voltage of about 100 V was applied to the accelerator rod set  20 , and about 400 V was applied to the multipole rod set  18 . The mass analyzer  16  of the Finnigan LCQ was operated in substantially the traditional intended manner for analyzing the ions. The MALDI spectra reproducibly exhibited ion signals from all six components of the peptide mixture, even for the sample having only 1 fmole of each peptide. All spectra were acquired in about 2 seconds. 
     Referring now to  FIG. 11 , the MS/MS spectrum of the peptide at 1956.7 m/z selected from the MALDI spectrum of the 1 fmole peptide mixture shown in  FIG. 10  is shown. This fragmentation spectrum was also acquired in about 2 seconds. Almost all major peaks in the spectrum can be identified as b or y-type fragments of the peptide. 
     Referring now to  FIGS. 6 and 7 , a second preferred embodiment of the system for mass spectroscopy  100  according to the present invention is illustrated. The second embodiment includes a matrix assisted laser desorption ionization (MALDI) pulsed ionizer  112 , an ionguide/accelerator  114 , and a mass analyzer  116  all in a substantially collinear arrangement. Both the ionguide/accelerator  114 , and a mass analyzer  116  are subjected to a vacuum as represented by arrows  145  in  FIG. 6 . Preferably the second embodiment of the system  10  also includes at least one additional multipole  118  located between the ionguide/accelerator  114  and the mass analyzer  116 . The multipole  118  can be any type including a quadrupole or an octopole. The matrix assisted laser desorption ionization (MALDI) pulsed ionizer  112  and the ionguide/accelerator  114  are preferably configured in a similar manner as described above with respect to the first embodiment  10 . The ionguide/accelerator  114  can be configured as a flexible device built from metallic springs or flexible metallized rods for use as a “sniffing” type of a sample scanning system as disclosed in U.S. application Ser. No. 09/737,660. The details of the mass analyzer  116  are shown in  FIG. 7  and will now be described below. 
     Referring now to  FIG. 7 , the mass analyzer  116  preferably includes a quadrupole ion trap  156  and a detector  158  interfaced by a second ionguide/accelerator  162 . The detector  158  includes a conversion plate  159  for converting ions to secondary charged particles received from the exit end  164  of the second ionguide/accelerator  162 . The secondary charged particles include electrons and ions. The second ionguide/accelerator  162  is configured in a similar manner as the first ionguide/accelerator  14  and includes a first end  166  that is preferably coupled to the exit of the quadrupole ion trap  156 . In this embodiment, the second ionguide/accelerator  162  provides for the efficient transport of ions from the quadrupole ion trap  156  to the detector  158 . The second ionguide/accelerator  162  can also be operated as a mass filter as described above with respect to the first ionguide/accelerator  14  for selecting a subset of ions ejected from the quadrupole ion trap  156  to the detector  158 . 
     The operation and advantages of the second ionguide/accelerator  162  will now be explained with reference to  FIG. 7  where the flow of ions is depicted by arrows. The ion trap  156  operates in its original mode admitting the injected ions and collisionally cooling them. After some time, the ejection process from the ion trap  156 . starts. The ejection of ions from the trap  156  is usually achieved by changing the amplitude of RF potential applied to the trap (by using a so called instability scan). The increased RF field inside of an ion trap makes the trajectory of some ions with a particular mass-to-charge ratio unstable such that these ions are caused to hit the walls or leave through one of the holes in the ion trap electrode. The process of ion ejection also causes the kinetic energy of the ejected ions to increase so that there is a greater chance that the ejected ions will fragment upon collision with buffer gas molecules present in the ion trap. With the second ionguide/accelerator  162  it is possible to select some particular fragment of the ejected ions. In this way only those ejected ions that produce a particular fragment will be capable of going through the second ionguide/accelerator  162  to the detector  158  using the well known “linked scan” mode of detection. Thus it may be possible to measure the spectrum of only those ions that undergo a particular fragmentation, but with very high efficiency. 
     Different types of so-called “link scans” can be performed with this instrument, including neutral ion losses scan, parent ion scan etc. In the proposed device, these types of scans can be performed with much greater efficiency compared with those carried out on existing instruments (e.g., the triple quadrupole mass spectrometer). Because only particular ions are ejected from the ion trap at a given ejection time, other ions are left in the ion trap to be ejected at different time. Thus no losses are expected because all ions undergo the same linked scan analysis during the total ion ejection analysis scan. 
     Thus, while there have been described what are presently believed to be the preferred embodiments of the invention, those skilled in the art will realize that changes and modifications may be made thereto without departing from the spirit of the invention, and is intended to claim all such changes and modifications as fall within the true scope of the invention.