Patent Grant
8119984
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.
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.
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.
In the accompanying drawings:
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
In a variation of the
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
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'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.
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.
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.
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.
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.
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.
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