Patent Application
20070057180
The technical field relates generally to ion analysis and more particularly to ion analysis in mass spectrometry.
Mass spectrometry methods are very useful for characterizing and/or quantifying chemical, biological and biochemical entities. Tandem mass spectrometry (MS/MS) methods have been shown to be particularly useful for characterizing individual components of a mixture and/or obtaining enhanced structural information from analytes that yield limited fragmentation.
In a typical MS/MS configuration, an ion of a specific mass to charge ratio (m/z) is selected in a first mass analyzer, and transferred to a collision cell where an inert gas is introduced to collide with the selected ion to generate fragmentation. The ions thus formed are then analyzed by a second mass analyzer to obtain a mass spectrum.
In U.S. Pat. No. 5,847,386, Thompson et al. suggest using tapered quadrupole rods to create an axial field in the collision cell of a quadrupole tandem mass spectrometer to facilitate movement of ions through a low energy system.
In U.S. Pat. No. 6,833,544, Campbell et al. disclose use of a linear ion trap component in a mass spectrometer system as a collision cell for collision induced dissociation. Campbell discloses use of segmented rods to form a gradient to move ions through a collision cell.
However, the need remains for improved apparatus and methods for processing ions in mass spectrometry.
A segmented rod multipole apparatus is disclosed for processing ions in a mass spectrometry system. The apparatus comprises a set of elongated-segmented rods having ends and defining an elongated volume therebetween. The elongated volume has a longitudinal axis and each elongated-segmented rod has a plurality of segments. The apparatus further comprises a circuit for applying a Radio Frequency (RF) voltage to the elongated-segmented rods to provide an RF field in the volume, and a circuit for applying DC voltages to the segments. Different DC voltages can be applied to different segments to provide a DC field in the elongated volume wherein the DC field comprises a selectively controllable potential region or regions.
A method of processing ions also is disclosed. The method of processing ions comprises providing the segmented rod apparatus of the invention, providing analyte ions in the elongated volume; applying an RF field to the volume; applying a DC voltage selectively to the segments of the elongated segmented rods; providing analyte ions and reactive reagent in the elongated volume; and processing the analyte ions. Processing the analyte ions with reactive reagent in the elongated volume may comprise a reaction selected from the group consisting of ion-ion reactions, election transfer reactions, proton transfer reactions, electron induced dissociation, energy modulation of the analyte ions, alternating gradient fragmentation, collisional activation, photodissociation, ion-molecule reactions and combinations thereof.
Processed ions may be transferred to a mass analyzer for determination of a mass spectrum. Any mass analyser may be used, however the method is particularly well suited for use with a time-of-flight mass analyser.
The apparatus and method described herein provide for processing ions in mass spectrometry. More particularly the apparatus provides for trapping ions in an electric field in an ion processing cell, processing ions in the ion processing cell, and releasing the ions from the ion processing cell to a mass analyzer. Trapping may include trapping ions in a region of the electric field such as in a potential well. The ion processing cell comprises segmented quadrupole rods and a means for admitting a reactive reagent to the ion processing cell. The ion processing cell receives analyte ions from an analyte ion source and the reactive reagent from a reactive reagent source. The reactive reagent may include reactive reagent ions, reactive reagent molecules, an electron beam, a proton beam or a photon beam. The nature of the reactive reagent selected depends on the chemical nature of the analyte and the information sought in the analysis. Processing of the ions may include trapping and collecting analyte ions and/or subjecting the analyte ions to ion-ion proton transfer reactions, ion-molecule proton transfer reactions, ion-ion electron transfer reactions, ion-ion electron transfer dissociation, alternating gradient fragmentation, charge reduction, collisional activation, photodissociation (such as, for example, infrared multiphoton dissociation), ion-molecule reactions, ion-ion reactions, or a combination thereof. After processing, the processed ions may be transferred to any mass analyzer for determination of a mass spectrum. However, the apparatus is particularly well suited for use with time-of-flight mass analyzers.
Typically, the apparatus is used in a mass spectrometer system comprising an analyte ion source, an ion processing cell and a mass analyzer. Analyte ions are formed in the analyte ion source. For example, analyte ions may be generated in the analyte ion source by electron impact, chemical ionization, MALDI (Matrix Assisted Laser Desorption Ionization), electrospray, fast atom bombardment, and the like. The thus formed ions may be passed directly to the ion processing cell. Optionally, the system may further comprise a mass filter which transmits only selected ions of a specific mass to charge ratio (m/z) to the ion processing cell, and/or ions of a specific mass to charge ratio (m/z) may be selected in the processing cell prior to processing.
Analyte ions are admitted to the ion processing cell which comprises a set of segmented rods. The rods may be configured as a quadrupole, hexapole or other multipole structure. An RF voltage is applied to all rods to provide an RF field in an elongated region between the rods, and DC voltages are selectively and controllably applied to segments of the segmented rods, thus providing for the trapping of ions, control of movement of ions and controllable transfer of ions. Reactive reagent ions may be admitted to the ion processing cell and reacted with at least a portion of the analyte ions.
Optionally, processing may include manipulation of the DC and/or RF voltage in a timed sequence to modify the field or a portion of the field in the ion processing cell to provide conditions which may facilitate the reaction between the analyte and reactive reagent ions and/or provide for one or more additional processing steps. The term processed ion is used herein to refer to an ion that has been subjected to at least one processing step in the ion processing cell.
Ions admitted to the processing cell and trapped in the ion processing cell may be a sample (group) of ions having a mixture of m/z values or ions of a selected m/z value. Optionally, when a sample of ions having a mixture of m/z values is admitted to the ion processing cell, ions of a specific m/z value may be selected in the processing cell by ejecting ions having undesired m/z values from the processing cell. As discussed herein, processing ions in the ion processing cell includes ion-ion reactions, fragmenting ions, manipulation of physical properties of ions, selecting ions, isolating ions, and the like, and combinations thereof. After processing in the ion processing cell, the processed ions are transferred to a mass analyzer for analysis. Mass analyzers may include time-of-flight mass analyzers, quadrupole mass analyzers, momentum mass analyzers, ICR mass analyzers, and the like.
As shown in
In some embodiments, it is advantageous to use segmented rods 20, 22, 24, 28 in which the segments 30 are formed by coating a non-conducting rod with a conducting material at discrete positions interposed between uncoated areas of the non-conducting rod. For example, such a rod may be formed by applying a metalized layer onto a rod formed from an insulating material, such as a ceramic, for example, then removing portions of the metal coating. To remove portions of the metal coating a band may be cut in the metal around the circumference of the rod and the cut band of metal removed from around the circumference. Removal of the metal band forms a nonconducting gap 40. The process is repeated to form multiple gaps 40 interposed between remaining bands of metal. The bands of metal remaining on the rod form multiple segments 30. Segmented rods 20, 22, 24, 28 should be taken to mean either rods comprising discrete individual segments or a rods comprised of a nonconductive material selectively coated with a bands of conducting material to give regions of metal coated rod interposed between uncoated areas of nonconducting material, or a combination thereof.
A gas inlet 89 for admitting inert gas into the ion processing cell 10 and a reagent ion source 82 are also provided.
In some embodiments, individual leads 70 may be attached to additional segments 30 or in other embodiments to all segments 30. The connection of a lead 70 to a specific segment 30 provides for direct control of the DC voltage and/or control of the DC potential field associated with the segment 30 so connected. Increasing the number of segments 30 attached to leads 70 provides for highly selective control of the field in elongated volume 21 including the ability to establish regions with different field conditions, such as for example potential wells, within the elongated volume 21.
As multiple leads 70 are used, the DC voltages can be manipulated to a group of segments 30 and/or individual segments 30 in a timed sequence. The DC voltages to segments 30 may be controlled and changed to create different configurations of the potential regions and/or a plurality of regions of differing field conditions in the elongated volume 21 (e.g., a number of regions and/or the nature of regions may be changed and/or field strengths to particular regions may be manipulated). The DC and RF circuitry provide for making such changes in a timed sequence. The timed sequence may involve changes in DC voltage and/or changes in RF voltages. Thus, ions can be trapped in the elongated volume 21 and/or in a portion of the elongated volume 21 and subjected to a sequence of potential field conditions.
As shown in
As shown in
The ion processing cell 10 provides many experimental options for processing ions. The options for processing ions described herein are exemplary of the many kinds of analyses the ion processing cell 10 facilitates.
Time-of-flight mass analyzers typically receive pulses of ions for analysis, with a considerable portion of the ions generated in an analyte ion source between pulses not captured in packets for analysis. In one exemplary embodiment, interposing the ion processing cell 10 between an analyte ion source and a time-of-flight mass analyzer provides for collecting ions generated between pulses and storing ions for release into the mass analyzer in the next pulse. Collection and storage permits analysis of a larger portion of analyte ions in the time-of-flight system (e.g., by increasing the duty cycle of the time-of-flight analyzer). In some embodiments, it may be possible to collect, store and analyze most of the ions formed in the analyte ion source.
Referring to
Typically, ions are collected in the presence of an inert gas to slow the ions. The gas may be admitted to the ion processing cell 10 through gas inlet 89. The gas pressure should be sufficient to slow the ions but not so high as to induce fragmentation of the ions. Gas pressures of 1 to 20 mTorr are typically sufficient. The optimum pressure depends on the ions to be analyzed and the type of inert gas used. For many applications, a pressure of 5 to 10 mTorr is used and use of a pressure of about 5 mTorr is common. Suitable inert gases include argon, krypton, and nitrogen, for example.
Alternatively, ions produced in the analyte source may be collected and stored in the ion processing cell 10 using ion gates near the entrance orifice 12 and the exit orifice 14. The gates may be the shield electrodes 16, 18 shown in
Referring to
The end and intermediary segments 31, 32, 34; the shields 16, 18; and the ion guide multipoles 62, 64 may perform functions other than acting as ion gates in some embodiments and these structures are not necessarily employed as ion gates when present in a specific embodiment. The segments 31, 32, 34, the shield electrodes 16, 18, or the ion guide multipoles 62, 64, or some combination thereof, may be used as ion gates in a specific embodiment.
Ions collected in the ion processing cell 10 may be simply collected and transferred to the mass analyzer as a group (e.g. packet). As discussed above, such a process can enhance the duty cycle of a time-of-flight mass analyzer. The enhancing of the duty cycle typically increases sensitivity. Alternatively, ions may be collected and reacted and/or otherwise manipulated in the ion processing cell 10 prior to transfer to the mass analyzer.
For example, referring to
Reactive reagent ions may be ions derived from molecules and atoms or alternatively charged entities such as electrons or protons. In the examples described herein, the reactive reagent ions are typically described as derived from molecules and atoms. However, it should be understood that electrons or protons derived from sources such as filaments or radioactive sources, for example, may be reacted with analyte ions to yield similar results in some applications, and accordingly are included within the scope of reactive reagent ions. The reactive reagent ions react with the collected analyte ions. The nature of the analyte ions and the nature of the reactive reagent ions will determine the particular type of reaction that may occur. Accordingly, the ion processing cell 10 has both the advantage of being applicable to a very wide range of species of ions and the advantage, if desired, of being very specialized in the type of ion-ion reaction by careful selection of reactive reagent ions. The reactive reagent ions may react chemically with the analyte ions thereby changing the chemical structure of the analyte ions. The change of structure may include forming adducts and/or interacting to form one or more chemical species that are different from the reactive reagent ions and the analyte ions. The products of the chemical reactions, when analyzed, provide a mass spectrum or other data that facilitates characterization of the analyte ions. The characterization may include structure information and/or information about the physical and/or chemical attributes of the analyte ions and/or provide a species that facilitates quantification.
The ion processing cell 10 may be employed for the analysis of either positive or negative analyte ions. Optionally, circuitry can be provided which permits for changing polarity, including changing the polarity of the ion processing cell 10 and reactive reagent source 82 to accommodate both types of analysis using a single processing cell 10.
Addition of reactive reagent ions radially, as shown in the diagram of
As shown in the potential diagram of
The diagram of
In some embodiments, the analyte ions and reactive reagent ions undergo charge transfer or charge exchange reactions. Charge transfer reactions have many applications. Exemplary applications include charge reduction and/or dissociation. Charge reduction and dissociation applications may be useful in the analysis of many kinds of molecules, particularly analysis of large molecules such as proteins.
For example, a large molecule such as a protein typically bears a number of positive charges when the protein is released from a conventional electrospray or Atmospheric Pressure Chemical Ionization (APCI) analyte ion source into a mass analyzer. Because m/z or mass to charge ratio is the detected entity, an ion specie having multiple charges would yield a series of ions related to the molecular ion specie. For example, an ion with up to four charges and a neutral mass m of 1000 would yield a mass spectrum showing ions of 1001 ((m+1)/1), 501((m+2)/2), 334.3((m+3)/3), and 251((m+4)/4) all related directly to the m/z of the molecular ion species of the analyte ion. In a time-of-flight mass analyser, a plurality of ions generated due to a plurality of charge states can lead to confusion in distinguishing the spectral contribution from the various charge states from the spectral contributions of other components and/or fragments. However, if the multiple-positively charged ion is exposed to a suitable reactive reagent ion of opposite polarity, proton or electron transfer may occur, which reduces the number of charges on the multiply charged ions. For a multiple-positively charged species such as a protein or peptide, protons are transferred from the multiple-positively charged ions to negatively charged reactive reagent ions in charge transfer reactions. The number of positive charges may, in some embodiments, be reduced to one positive charge on the analyte ion and/or fragments from the analyte ion which is particularly desirable. However, partial reduction of charge may be beneficial in some applications as it provides some potential simplification of spectra. Although in conventional analysis it is more common for the analyte ions to have a multiplicity of positive charges, a negativity charged analyte that transfers protons from a positively charged reactive reagent ion is likewise possible.
In another application, electrons can be transferred from the reactive reagent ions to analyte ions in the ion processing cell 10. This transfer of the electrons to positively charged analyte ions can result in charge reduction and/or induce electron transfer dissociation of the analyte ions. For example, use of reactive reagent ions such as fluoranthene provides suitable electrons that can induce fragmentation of a multiple- positively charged peptide ion following electron transfer. Such election transfer dissociations are particularly useful in characterizing peptides and proteins. Typically, such collisions yield at least some fragmentation different than that of conventional collisional activation with an inert gas. For example, in the case of proteins, the fragmentation resulting from election transfer dissociation is particularly useful for obtaining certain key sequence information by identifying c and z ion sequences, for example, and/or characterizing post-translational modifications.
The ability to selectively create regions of different potentials and change the potential permits, not only trapping of ions for processing with reactive reagent ions and transferring them from the ion processing cell to a mass analyzer, but also provides for optionally trapping ions in a specific region within the elongated volume 21 and modulating the energy of ions. For example, moving the trapped ions in an alternating gradient in the ion processing cell can increase the ion energy. An alternating gradient may be produced by providing a field gradient along the segments of the rods and reversing the field gradient in a timed sequence. Exposing ions to the alternating gradient increases ion energy and/or enhances fragmentation generally and/or certain types of fragmentation.
Ions may undergo a single processing step in the ion processing cell 10 or multiple processing steps. The multiple processing steps may comprise repetition of the same type of processing step, one or more different processing steps or a combination thereof. Selection of the processing step or steps to use is determined by the information sought, and the chemical and physical properties of the analyte ions and reactive reagent. Optimization of the experimental design and parameters is typically determined experimentally.
Multiple step processing may include collisional activation of product or fragment ions formed in a previous processing step. To perform collisional activation, ions of the m/z of interest are selected and ions having other m/z values are radially ejected from the elongated volume 21 in the ion processing cell 10. Radial ejection may be done as described by Flory et. al. in U.S. Pat. No. 5,672,870.
Collisional activation is then performed by using an inert gas in the ion processing cell 10 containing the selected ions. The selected ions in the processing cell 10 collide with the inert gas and may undergo collision induced fragmentations (e.g., collisional activation.) Typically an inert gas such as argon or krypton or the like is used as the collision gas. A gas pressure of about 5 m Torr and a collision energy of 20-40 eV is exemplary of typical parameter for collisional activation in the ion processing cell 10. These parameters are exemplary and other parameters may be likewise suitable. The collision gas may be admitted through the reactive reagent ion orifice 80 or via a separate orifice radially positioned.
Upon completion of the collisional activation, the collected ions and fragments may be transferred to the mass analyzer to obtain the mass spectrum or subjected to a further processing step or steps before transfer from the ion processing cell 10 to the mass analyzer to obtain the mass spectrum. Further processing may include selection of specific ions and reaction of the selected ions with reactive reagent ions and/or an additional step or steps of collisional activation (e.g., MSn) of the selected ions, and/or manipulation of the selected ions by modifying the field, and the like, or a combination thereof. The transfer of processed ions to the mass analyzer is accomplished as discussed above by changing the potential of the ion gate 18 at the exit orifice 14. A mass spectrum may be obtained by analysis in the mass analyzer.
The foregoing discussion discloses and describes many exemplary methods and embodiments of the present invention. As will be understood by those familiar with the art, the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims.
