Patent Publication Number: US-8119984-B2

Title: Method and apparatus for generation of reagent ions in a mass spectrometer

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the priority benefit under 35 U.S.C. §119(e)(1) of U.S. provisional patent application Ser. No. 61/057,751 by Earley et al., entitled “Method and Apparatus for Generation of Reagent Ions in a Mass Spectrometer”, the disclosure of which is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to ion sources for mass spectrometry, and more particularly to an ion source for generating reagent ions for electron transfer dissociation or other ion-ion reaction experiments. 
     BACKGROUND OF THE INVENTION 
     Mass spectrometry has been extensively employed for ion-ion chemistry experiments, in which analyte ions produced from a sample are reacted with reagent ions of opposite polarity. McLuckey et al. (“Ion/Ion Chemistry of High-Mass Multiply Charged Ions,  Mass Spectrometry Reviews , Vol. 17, pp. 369-407(1998)) discusses various examples of mass spectrometric studies of this type. It has been recently discovered that by selecting an appropriate reagent anion and reacting the reagent anion with a multiply charged analyte cation, a radical site is generated that induces dissociation of the analyte cation into product ions. This process, called electron transfer dissociation (ETD), is described by Hunt et al. in U.S. Pat. No. 7,534,622 for “Electron Transfer Dissociation for Biopolymer Sequence Mass Spectrometric Analysis”, as well as by Syka et al. in “Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry”,  Proc. Nat. Acad. Sci. , vol. 101, no. 26, pp. 9528-9533(2004), both of which are incorporated herein by reference. ETD is a particularly useful tool for proteomics research, since it yields information complementary to that obtained by conventional dissociation techniques (e.g., collisionally induced dissociation), and also because ETD tends to generate product ions having intact post-translational modifications. 
     Implementation of ETD or other ion-ion experiments in a mass spectrometer requires two ion sources: a first ion source for generating analyte ions from a sample, and a second ion source for generating reagent ions. Typically, the analyte ion source utilizes an ionization technique, such as electrospray ionization, that operates at atmospheric pressure. Atmospheric or near-atmospheric pressure ionization techniques have also been employed or proposed for production of reagent ions (see, e.g., Wells et al. “‘Dueling’ ESI: Instrumentation to Study Ion/Ion Reactions of Electrospray-Generated Cations and Anions”,  J. Am. Soc. Mass Spectrometry , vol. 13, pp. 614-622(2002), and U.S. Patent Application Publication No. 2008/0245963 by Land et al. entitled “Method and Apparatus for Generation of Reagent Ions in a Mass Spectrometer”). However, it has been found that atmospheric-pressure ionization techniques may not be well-suited to production of certain labile ETD reagent ion species, which tend to be neutralized within the environment of an atmospheric-pressure ionization chamber via loss of electrons to background gas molecules or form ion species (unsuitable for ETD) through reaction with species present in the background gas. 
     Generation of reagent ions using a conventional chemical ionization (CI) technique has been disclosed in the prior art (see, e.g., the aforementioned Syka et al. paper as well as U.S. Pat. No. 7,456,397 by Hartmer et al.), and has been implemented in at least one commercially-available ion trap mass spectrometer. In such sources, reagent ions are formed by reaction of reagent vapor molecules with secondary electrons. CI sources typically employ an energized filament to produce a stream of electrons that preferentially ionizes secondary molecules. Reagent ions formed in the CI source may be directed through a dedicated set of ion optics, and introduced into a two-dimensional ion trap for reaction with analyte ions via an end of the trap opposite to the end through which the analyte ions are introduced, as described in Syka et al. Alternatively, analyte and reagent ions may be sequentially passed into a common aperture or end of an ion trap by an ion switching structure, as described in the Hartmer et al. patent. 
     Mass spectrometer configurations utilizing a CI reagent ion source have been utilized successfully for ETD experiments, but present a number of operational and design problems. The filaments in the CI source may fail in an unpredictable manner and need to be replaced frequently. Cleaning and maintenance of the CI source may require venting of the mass spectrometer and consequent downtime. Further, the need to provide dedicated guides or switching optics to direct ions from the CI source to the ion trap complicates instrument design and may interfere with the ability to incorporate additional components, e.g., other mass analyzers, into the ion path. 
     SUMMARY 
     Embodiments of the present invention provide a reagent ion source for a mass spectrometer having a reagent vapor source that supplies gas-phase reagent molecules to a reagent ionization volume maintained at low vacuum pressure. A voltage source applies a potential across electrodes disposed in the reagent ionization volume to produce an electrical discharge (e.g., a glow discharge) that ionizes the reagent vapor to generate reagent ions. The reagent ions flow through an outlet to a reduced-pressure chamber of the mass spectrometer, and are thereafter directed to an ion trap or other structure for reaction with oppositely charged analyte ions. 
     In specific implementations, the reagent may take the form of a polyaromatic hydrocarbon suitable for use as an ETD reagent. The reagent vapor may be generated by heating a quantity of the reagent substance in condensed-phase form and transported to the reagent ionization volume by entrainment in a carrier gas stream. The ionization volume may be divided by an apertured partition into a discharge region extending between the electrodes and an exit region located adjacent to the outlet of the ionization volume. The pressure within the reagent ionization volume (or portion thereof in which the discharge occurs) may be maintained between 0.5-10 Torr. The potential applied to the electrodes may be pulsed on and off to control the production of reagent ions. The reagent vapor source may include first and second evaporation chambers respectively containing a first reagent substance (e.g., an ETD reagent) and a second reagent substance (e.g., a proton transfer reaction (PTR) reagent. The reagent ion source constructed in accordance with embodiments of the present invention may be combined with an atmospheric-pressure analyte ionization source, such as an electrospray ionization source, which produces analyte ions of opposite polarity to the reagent ions. In this configuration, the analyte ions traverse under the influence of a pressure and/or electrical gradient and pass into the reduced-pressure chamber of the mass spectrometer. The reagent or analyte ions are selectively admitted and transported through downstream ion optics to the ion trap by adjusting the polarities and amplitudes of the DC offset voltages applied to the ion optics. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       In the accompanying drawings: 
         FIG. 1  is a symbolic diagram of an ion trap mass spectrometer incorporating a front-end reagent ion source, in accordance with an illustrative embodiment of the invention; 
         FIG. 2  is a symbolic diagram showing details of the reagent ionization volume of  FIG. 1 ; 
         FIG. 3  is a symbolic diagram showing a reagent ionization volume constructed according to a different embodiment of the invention, having a discharge region oriented transversely to an ionization region; 
         FIG. 4  is a symbolic diagram depicting an alternative implementation in which the reagent ionization volume is located adjacent to the entrance to an RF ion transport optic constructed from a plurality of spaced ring electrodes (hereinafter referred to as an “S-lens”); 
         FIG. 5  is a symbolic diagram of a reagent vapor source configured to supply two different reagents to the reagent ionization volume; 
         FIG. 6  is a symbolic diagram depicting another embodiment of the invention, wherein the reagent ionization volume is located at the end portion of an ion transfer tube; and 
         FIG. 7  is a symbolic diagram showing a reagent ionization volume constructed in accordance with a variation of the  FIG. 3  design, wherein the reagent vapor and carrier gas are introduced along an axis transverse to the discharge region. 
     
    
    
     DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
       FIG. 1  schematically depicts a mass spectrometer  100  incorporating a front-end reagent ion source constructed according to an embodiment of the present invention. As used herein, the term “front-end” denotes that the ion source is configured to introduce reagent ions into a region located upstream in the analyte ion path relative to components of mass spectrometer  100  disposed in lower-pressure chambers (e.g., a mass analyzer), such that the analyte ions and reagent ions traverse a common path. Analyte ions (typically multiply-charged cations) are formed by electrospraying a sample solution into an analyte ionization chamber  105  via an electrospray probe  110 . Analyte ionization chamber  105  will generally be maintained at or near atmospheric pressure. The analyte ions, together with background gas and partially desolvated droplets, flow into the inlet end of a conventional ion transfer tube  115  (which may take the form of a narrow-bore capillary tube) and traverse the length of the tube under the influence of a pressure gradient. Analyte ion transfer tube  115  is preferably held in good thermal contact with a heated block (not depicted). As is known in the art, heating of the ion/gas stream passing through analyte ion transfer tube  115  assists in the evaporation of residual solvent and increases the number of analyte ions available for measurement. The analyte ions emerge from the outlet end of analyte ion transfer tube  115 , which opens to reduced-pressure chamber  130 . As indicated by the arrow, chamber  130  is evacuated to a low vacuum pressure (typically within the range of 0.1-50 Torr, and more typically between 0.5 and 10 Torr) by a mechanical pump or equivalent. 
     To produce reagent vapor for production of the requisite reagent ions (having a polarity opposite to that of the analyte ions), a reagent evaporation chamber  140  is provided having located therein a volume of a reagent substance  145  (for example and without limitation, a polyaromatic such as fluoranthene for ETD reagent ions, or benzoic acid for proton transfer reaction (PTR) reagent ions) in condensed-phase (solid or liquid) form. Reagent substance  145  is placed in thermal contact with a block  150  heated by a cartridge heater  155 . The reagent vapor pressure within chamber  140  is regulated by controlling the temperature (via adjusting power supplied to heater  155 ) of block  150 . A flow of generally inert carrier gas (such as nitrogen, argon or helium) is introduced at a controlled rate through inlet  160  opening to the interior of chamber  140  to assist in the transport of reagent vapor molecules. The carrier gas also functions to continuously purge the interior of chamber  140  to prevent the influx of oxygen or other reactive gas species, which can react with and destroy ions formed from the reagent vapor. 
     While the interior volume of reagent evaporation chamber  140  will typically be held at or near atmospheric pressure, embodiments of the invention should not be construed as limited to atmospheric pressure operation. In certain implementations, it may be advantageous to maintain evaporation chamber  140  at a pressure substantially above or below atmospheric pressure. It is noted, however, that the pressure of reagent evaporation chamber  140  will need to be elevated relative to the pressure within reduced-pressure chamber  130  to establish a pressure gradient that results in the forward flow of reagent molecules through reagent transfer tube  170 . 
     Molecules of reagent vapor entrained in the carrier gas enter an inlet end of reagent transfer tube  170  and traverse the length of the tube under the influence of a pressure gradient. Reagent transfer tube  170  may be a narrow-bore capillary tube fabricated from a suitable material, which extends between the interior of reagent evaporation chamber  140  and reagent ionization volume  172 . Reagent transfer tube  170 , or a portion thereof, may be heated to prevent condensation of reagent material on the inner surfaces of the tube walls. 
     Referring to  FIG. 2 , the reagent vapor enters reagent ionization volume  172  through an inlet  202  thereof. Reagent ionization volume  172  is located within chamber  130  of mass spectrometer  100 , and functions to ionize (either directly or via a process involving intermediates) at least a portion of the reagent vapor transported thereto in order to produce the desired reagent ions (e.g., fluoranthene anions). For this purpose, reagent ionization volume  172  is provided with electrodes  210  and  215 , across which a potential is applied by a voltage source  205  to establish a controlled discharge, which will preferably take the form of a low-current (e.g., 1-100μamp) discharge such as a Townsend (dark) or glow discharge. As used herein, the term “reagent ionization volume” denotes a structure operable to effect ionization of the reagent vapor, and includes (without limitation) a structure having separated regions in which electrical discharge and ionization take place, per the embodiments depicted in  FIGS. 3 and 7  and described below. Insulative sidewalls  217  extend between electrodes  210  and  215  and form with the electrodes a region that is generally closed to the exterior regions of chamber  130 . Voltage source  205  will preferably include a current limiting circuitry to prevent transition of the low-current (e.g., glow) discharge to a high-current arc discharge. Ionization volume  172  communicates with the interior volume of chamber  130  via a short outlet section or aperture  220 , and is thus maintained at a sub-atmospheric pressure. The actual pressure within reagent ionization volume  172  will be a function of the pressure maintained within chamber  130 , the conductance of outlet section  220 , and the flow rate of carrier gas/reagent vapor into ionization volume  172 . Typically, the reagent ionization volume will be operated to maintain the region at which the electrical discharge occurs at a pressure of between 0.5-10 Torr, although certain implementations may utilize pressures as low as 0.1 Torr or as high as 50 Torr. It has been observed that operation of the controlled discharge at sub-atmospheric pressure promotes stability of the discharge and reduces the temporal variation in the number of reagent ions produced relative to an ionization volume that operates at atmospheric or near-atmospheric pressures. 
     In a variation of the  FIG. 2  design, reagent ionization volume  172  may be adapted with a second inlet for introducing a flow of discharge gas into its interior region. The discharge gas may be of the same composition as the carrier gas (e.g., nitrogen, argon or helium), and the carrier gas and the discharge gas may be supplied from a common source via separately metered lines. This “split-flow” configuration enables independent control of the pressure within ionization volume  172  (which will depend on the combined discharge and carrier gas flow rates) and the flow rate of reagent vapor to ionization volume  172  (which will be governed by the vapor pressure within evaporation chamber evaporation chamber  140  and the carrier gas flow rate). 
     It should be recognized that the position and physical configuration of discharge chamber  172  may be optimized and/or adjusted in view of space constraints, ion flow path considerations, and other operational or design parameters. It is generally desirable to select an electrode gap (the distance between electrodes  210  and  215 ) that places the product of the gap and operating pressure at or close to the minimum of the Paschen breakdown curve in order to minimize the potential required to be applied by voltage source  205 . 
     Reagent ions are produced within ionization volume  172  by the direct or indirect interaction of reagent vapor molecules with electrons produced by the electrical discharge. The reagent ions exit ionization volume  172  through outlet section  220  and flow into chamber  130  under the influence of a pressure and/or electrical field gradient. The reagent ions may then be focused by tube lens  185  before passing into the succeeding chamber of mass spectrometer through an aperture in skimmer lens  180 . It will be recognized that the analyte ions and reagent ions traverse a common path through the various ion transport optics (tube lens  185 , skimmer lens  180 , plate lens  190 , and RF multipole ion guides  192  and  195 ) between chamber  130  and the reaction region, which may take the form of a two-dimensional quadrupole ion trap mass analyzer  197 , as depicted in  FIG. 1 . 
     The analyte and reagent ion sources may be operated to provide a continuous supply of analyte and reagent ions into chamber  130 . For ETD, the analyte and reagent ions are injected sequentially into a reaction region (e.g., ion trap  197 ). Selection of the ions to be delivered to ion trap  197  (i.e., the analyte or reagent ions) may be accomplished by applying DC voltages of suitable magnitude and polarity to the various ion transport optics, such that only the analyte ions are delivered to ion trap  197  at a first set of applied DC voltages, and only the reagent ions are delivered at a second set of DC voltages. Other implementations of the invention may utilize a dedicated switching structure, such as the split-lens switch disclosed in U.S. Pat. No. 7,456,397. by Hartmer et al. In certain implementations, one of the RF multipole ion guides of the ion transport optics (which may be constructed from a set of rod electrodes having square or rectangular cross-sections) may be made mass selective by adding a resolving DC component to the applied RF voltages to filter ions outside of a specified range of mass-to-charge ratios (m/z&#39;s) to prevent the entry of undesirable ion species during the reagent ion injection period. Alternatively, isolation waveforms may be applied to the ion guide electrodes to resonantly eject the undesirable ion species. 
     A notable feature of the foregoing embodiment is that the reagent and analyte ion flows are maintained separate and unmixed until they arrive at reduced-pressure chamber  130 . The undesirable reaction of the analyte ions with background gas molecules and reagent ions within chamber  130  may be alleviated by positioning skimmer lens  180  close to the outlets of the ion transfer tube  115  and reagent ionization volume  172 , such that the number of collisions that the analyte ions undergo within chamber  130  is minimized. 
     In a preferred mode of operation of mass spectrometer  100 , reagent ions are produced intermittently rather than continuously. It will be understood that reagent ions need only be generated during a small fraction of the total analysis cycle time, e.g., when injecting ETD reagent ions into ion trap  197  for subsequent reaction with analyte ions; at other times, the reagent ions are not needed and are diverted from the ion path and destroyed. It may therefore be beneficial to pulse reagent ion production on and off such that the reagent ions are generated on an “as needed basis” in order to reduce wear on components of the reagent ion source (for example, electrodes  210  and  215 ) and to reduce the rate of deposition of material on skimmer lens  180  and other components within chamber  130  (and thereby alleviating cleaning and maintenance requirements). Pulsing reagent ion production may be effected by switching on and off the potential applied to electrodes  210  and  215  to selectively establish the discharge, or by switching on and off (e.g., via a pulse valve) the carrier gas flow to evaporation chamber  140 . 
       FIG. 3  depicts an alternative embodiment of the front-end analyte/reagent ion source, in which reagent ionization volume  310  is divided into a discharge region  320  and an ionization region  330  by apertured electrode  340 . Discharge region  320  is defined by electrodes  340  and  350  and insulative sidewall  360 . A voltage source (not depicted) applies a suitable potential across electrodes  340  and  350  to generate an electrical (e.g., glow) discharge. Carrier gas and entrained reagent vapor enter discharge region  320  via inlet  370 , and flow thereafter through aperture  375  to ionization region  330 , in which ionization of the reagent vapor is believed to primarily occur. Again, ionization may result from a direct or indirect (mediated) interaction with electrons produced in the electrical discharge. While reagent ionization volume  310  is constructed such that the axis defined between electrodes  340  and  350  within discharge region  320  is transverse to the flow axis within ionization region  330 , other implementations of the divided ionization volume design may be implanted in a co-axial geometry, i.e., where the electrode-defined axis within the discharge region is directed co-linear or parallel to the flow axis within the ionization region. The reagent ions then pass from ionization region  330  to chamber  130  via outlet  380 . By placing a conductance-limited aperture  375  between discharge region  320  and ionization region  330 , the pressure within discharge region  320  may be controlled independently of the pressure within chamber  130  without requiring an excessively small outlet  320  that could adversely affect the efficiency of reagent transport. 
       FIG. 7  depicts a variation on the  FIG. 3  reagent ionization volume design, wherein the carrier gas and entrained reagent vapor are introduced into reagent ionization volume  705  via an inlet  710  having a flow axis that is transverse to the primary axis (defined between electrodes  340  and  350 ) of discharge region  320  and parallel to the flow axis within ionization region  330 . Ionization of reagent vapor molecules occurs in ionization region  330  by direct or indirect interaction with electrons, produced within discharge region  320 , and entering ionization region  330  through aperture  375 . The resultant reagent ions are then transported into chamber  130  through outlet  380 . 
     While embodiments of the invention have been described and depicted in connection with a conventional tube lens/skimmer lens structure, these embodiments may be readily adapted for use with other ion optical arrangements.  FIG. 4  depicts one such alternative arrangement, in which the analyte and reagent ions (from reagent ionization volume  705 ) are directed through an S-lens  410  rather than into the tube lens and skimmer shown in  FIGS. 1 and 2 . S-lens  410 , the design and operation of which are discussed in detail in U.S. Patent Application Publication No. US2009/0045062A1. by Senko et al. (incorporated herein by reference), is constructed from a set of aligned ring electrodes having progressively increasing inter-electrode spacing in the direction of ion travel. RF voltages are applied to the ring electrodes to radially confine the ions and focus them to a flow centerline. It has been found that S-lens  410  provides more efficient transport of analyte ions to downstream regions relative to a conventional skimmer structure, thereby improving instrument sensitivity. It has been observed, however, that under certain conditions transport of reagent ions (e.g., fluoranthene ions) through the full length of S-lens  410  may result in the destruction of excessive numbers of the reagent ions. To avoid this undesirable result, reagent ionization volume  172  may be moved such that the reagent ions are introduced in a gap between electrodes of the S-lens or between the final ring electrode and extraction lens  420 , so that the reagent ions do not traverse the entire length of S-lens  410 . 
     In certain types of mass spectrometric analysis, it may be necessary to supply (sequentially or concurrently) two or more distinct reagent ion species to the ion trap or other reaction region of the mass spectrometer. For example, Coon et al. (“Protein Identification Using Sequential Ion/Ion Reactions and Tandem Mass Spectrometry”,  Proc. Nat. Acad. Sci. , Vol. 102, No. 27, pp. 9463-9468(2005)) describes experiments in which ETD, produced by reaction of analyte peptide ions with fluoranthene ions, is followed by proton transfer reaction (PTR) to reduce the charge states of the ETD product ions, which occurs by reaction with deprotonated benzoic acid ions.  FIG. 5  depicts a reagent vapor source  500  adapted to supply two different reagents (e.g., ETD and PTR reagents) to reagent ionization volume  172 . Reagent vapor source  500  includes first and second evaporation chambers  510  and  520  that are separate and divide from each other. First evaporation chamber  510  contains a quantity of a first reagent substance  530  (e.g., fluoranthene) in condensed phase form, and second evaporation chamber similarly contains a second reagent substance  540  (e.g., benzoic acid) in condensed-phase form. First and second evaporation chambers  510  and  520  are provided with independently controllable heaters  550  and  560  to vaporize the corresponding reagents. Separate carrier gas flows are directed into first and second evaporation chambers  510  and  520  through inlets  570  and  580 . The carrier gas and entrained reagent vapor exit first and second evaporation chambers  510  and  520  via outlets  585  and  590 . The gas outlets are coupled to a proximal end of reagent transfer tube  170  by tee  595 . The reagents, or a selected one thereof, are transported through reagent transfer tube  170  to reagent ionization volume  172 . 
     If the reagents are to be supplied to the reaction region in a sequential manner, selection of the desired reagent ion may be effected by operating at least one of the ion transport optics in a mass-selective manner, to selectively transmit the desired ion species while excluding the undesired ion species. As discussed above, this may be accomplished by applying a filtering DC component to an RF ion guide, or by employing an isolation waveform. Alternatively, a flow switch may be provided to allow transport of the selected reagent to ion transfer tube  170  while inhibiting the flow of the non-selected reagent. For example, selection of a reagent may be achieved by turning on the flow of its carrier gas and turning off the flow of the carrier gas corresponding to the non-selected reagent, such that only the selected reagent is delivered to tee  595 . According to another alternative, selection of a reagent may be effected through use of an appropriate valve structure in outlets  585  and  590  or tee  595  to controllably obstruct or divert the flow of carrier gas containing the non-selected reagent to prevent its entry into reagent transfer tube  170 . 
     Although reagent vapor source  150  is configured to provide two reagents to the reagent ionization volume, those skilled in the art will recognize that its design may be easily modified to provide three or more reagents, if required by the mass spectrometric analysis technique to be utilized. 
       FIG. 6  depicts in fragmentary view an alternative embodiment of the invention, wherein a controlled discharge is generated within reagent transfer tube  170  proximate to the outlet end thereof in place of a separate ionization volume. A conductive wire  610  is placed within the interior of reagent transfer tube  170  (which is itself fabricated from a conductive material). An insulator  615 , which may take the form of a fused silica tube, is radially interposed between wire  610  and the inner surface of reagent transfer tube  170 . Application of a suitable potential across wire  610  and reagent transfer tube  170  causes an electrical discharge (e.g., a glow discharge) to be produced at a region near the outlet end that is maintained at a sub-atmospheric pressure close to the pressure within chamber  130  (preferably between 0.5 and 10 Torr). The location and stability of the discharge may be optimized by appropriately tuning design and operational parameters, including (without limitation) the sizes and relative positioning of wire  610 , insulator  615  and reagent transfer tube  170 , the voltage applied to wire  610 , and the geometry (e.g., flared or rolled) of the outlet end of transfer tube  170 . The location and stability of the discharge will also be affected by the gas pressure at the outlet end of reagent transfer tube  170 . 
     It should be further recognized that the specific implementation depicted and described herein, i.e., where the reagent ion source takes the form of an ETD reagent ion source supplying ions to an analytical two-dimensional ion trap, are intended to be illustrative rather than limiting. A reagent ion source constructed in accordance with the invention may be beneficially utilized for supplying reagent ions of any suitable type and character to one or more reaction regions, which will not necessarily include a trapping structure. 
     It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.