Methods and apparatus for improved tandem mass spectrometry duty cycle

A method for parallel accumulation and serial fragmentation of ions, wherein ions are injected into a device capable of serial ejection using a pseudopotential barrier created by an RF voltage. In all instances, the ions may be filtered prior to accumulation in the device capable of serial ejection. In some cases this filtering may take the form of discrete isolation windows using isolation waveforms with multiple notches. In some cases these waveforms may be applied to a quadrupole mass filter. Following accumulation of the precursor ions, the initial population may be serially ejected using a pseudopotential barrier created by an RF voltage. Following serial ejection, the individual precursor ion populations are analyzed. In some cases, this analysis might involve additional rounds of ion isolation and manipulation (e.g., MSn, CID, ETD, etc.).

FIELD OF THE INVENTION

This invention relates generally to mass spectrometry and mass spectrometers and, more particularly, to methods and apparatus for any of ion fragmentation, ion reaction or tandem mass spectrometry, including multistage tandem mass spectrometry.

BACKGROUND OF THE INVENTION

Modern mass spectrometers are capable of highly sophisticated ion manipulations. Tandem mass spectrometry, including multistage tandem mass spectrometry or MSn, synchronous precursor selection, ion/ion reactions, and fast spectral acquisition rates are all part of the standard mass spectrometry toolbox. Due, in large part, to the development of these modern capabilities, mass spectrometer users are routinely performing experiments that would have been impossible only a few years prior to this writing. For example, the types of experiments that are now routinely performed include analyzing a yeast proteome in less than one hour, accurate relative quantitation across ten channels using synchronous precursor selection-based MS3analysis of Tandem Mass Tag (TMT) labeled samples, and previously-unachievable glycopeptide sequence coverage using electron transfer dissociation (see Hebert, A. S. et al. The one hour yeast proteome.Molecular and Cellular. Proteomics2014, 3, 339-347; Erickson, B. K. et al. Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometer. Analytical Chemistry 2015, 2, 1241-1249; Saba, J. et al. Increasing the Productivity of Glycopeptides Analysis by Using Higher-Energy Collision Dissociation-Accurate Mass-Product-Dependent Electron Transfer Dissociation.International Journal of Proteomics2012). In the above, and in the remainder of this document, the symbolism MSn, or related symbolism in which “n” is replaced by a specific number, refers to multistage tandem mass spectrometry. In this document, the term “tandem mass spectrometry” is used in a broad sense to include such multistage techniques, in addition to traditional MS/MS (i.e., MS2) mass spectrometry. During an MS2mass spectrometer analysis, a precursor is isolated and then fragmented to yield a first generation of product ions. During high order MSnexperiments, in which n is greater than 2, after a first sequence of precursor ion isolation and fragmentation, to yield a first generation of fragment ions, one or more species of the first generation of fragment ions are further isolated and fragmented to form a second-generation of fragment ions, where this sequence of events (fragmentation of an earlier generation of fragment ions) may be reiterated any number of times.

FIG. 1Adepicts the components of a general conventional mass spectrometer system1that may be employed for tandem mass spectrometry. An ion source, which may take the form of an electrospray ion source5, generates ions from an analyte material supplied from a sample inlet. For example, the sample inlet may be an outlet end of a chromatographic column, such as liquid or gas chromatograph (not depicted), from which an eluate is supplied to the ion source. The ions are transported from ion source chamber10that, for an electrospray source, will typically be held at or near atmospheric pressure, through several intermediate-vacuum chambers20,25and30of successively lower pressure, to a high-vacuum chamber35. The high-vacuum chamber35houses a quadrupole mass filter (QMF)51, an ion reaction cell52(such as, a collision cell, fragmentation cell, or ion routing multipole), and a mass analyzer40. Efficient transport of ions from ion source5to the high-vacuum chamber35is facilitated by a number of ion optic components, including quadrupole radio-frequency (RF) ion guides45and50, octopole RF ion guide55, skimmer60, and electrostatic lenses65and70. Ions may be transported between the ion source chamber10and first intermediate-vacuum chamber20through an ion transfer tube75that is heated to evaporate residual solvent and break up solvent-analyte clusters. Intermediate-vacuum chambers20,25and30and high-vacuum chamber35are evacuated by a suitable arrangement of pumps to maintain the pressures therein at the desired values. In one example, intermediate-vacuum chamber20communicates with a port of a mechanical pump (not depicted), and intermediate-vacuum chambers25and30, and high-vacuum chamber35, communicate with corresponding ports of a multistage, multiport turbomolecular pump (also not depicted).

Electrodes80and85(which may take the form of conventional plate lenses) positioned axially outward from the mass analyzer40may be used in the generation of a potential well for axial confinement of ions, and also to effect controlled gating of ions into the interior volume of the mass analyzer40. The mass analyzer40, which may comprise a quadrupole ion trap, a quadrupole mass filter, a time-of-flight analyzer, a magnetic sector mass analyzer, an electrostatic trap, or any other form of mass analyzer, is provided with at least one detector49that generates a signal or signals representative of the abundance of ions of each m/z. If the mass analyzer40is provided as a quadrupole mass filter, then a detector at the position shown inFIG. 1Awill generally be employed so as to receive and detect those ions which selectively pass through the mass analyzer40from an entrance end to an exit end. If, alternatively, the mass analyzer40is provided as a linear electrodynamic ion trap or other form of mass analyzer, then one or more detectors at alternative detector positions may be employed. Various alternative analyzer methods and detector geometries are also envisaged.

Ions enter an inlet end of the mass analyzer40as a continuous or quasi-continuous beam after first passing, in the illustrated conventional apparatus, through a quadrupole mass filter (QMF)51and an ion reaction cell52. The QMF51may take the form of a conventional multipole structure operable to selectively transmit ions within an m/z range determined by the applied RF and DC voltages. The reaction cell52may also be constructed as a conventional multipole structure to which an RF voltage is applied to provide radial confinement. The reaction cell may be employed, in conventional fashion, as a collision cell for fragmentation of ions. In such operation, the interior of the cell52is pressurized with a suitable collision gas, and the kinetic energies of ions entering the collision cell52may be regulated by adjusting the DC offset voltages applied to QMF51, collision cell52and lenses53and80.

The mass spectrometer system1shown inFIG. 1Amay operate as a conventional triple quadrupole mass spectrometer, wherein ions are selectively transmitted by QMF51, fragmented in the ion reaction cell52(employed as a collision cell), and wherein the resultant product ions are mass analyzed so as to generate a product-ion mass spectrum by mass analyzer40and detector49. Samples may be analyzed using standard techniques employed in triple quadrupole mass spectrometry, such as precursor ion scanning, product ion scanning, single- or multiple reaction monitoring, and neutral loss monitoring, by applying (either in a fixed or temporally scanned manner) appropriately tuned RF and DC voltages to QMF51and mass analyzer40. The operation of the various components of the mass spectrometer systems may be directed by a controller or a control and data system15, which will typically consist of a combination of general-purpose and specialized processors, application-specific circuitry, and software and firmware instructions. The control and data system15may also provide data acquisition and post-acquisition data processing services. As is well known, the mass spectrometer system comprises one or more power supply units41,42,43to provide the appropriate RF and DC voltages for containing the ions with various multipole ion guides, ion filters and collision cells. The power supply units also provide the appropriate DC voltages and drag fields to the various lenses, ion guides, multipole rod electrodes and/or other ion optics components for the purpose of urging the ions along a general pathway from the ion source to the detector.

FIG. 1Bis a schematic depiction of an exemplary mass spectrometer system150that may be employed for more complex mass spectrometry experiments and measurements, such as MSnexperiments and measurements. The mass spectrometer illustrated inFIG. 1Bis a hybrid mass spectrometer, comprising more than one type of mass analyzer. Specifically, the mass spectrometer system150includes a quadrupole ion trap mass analyzer116as well as an ORBITRAP™ analyzer112, which is a type of electrostatic trap mass analyzer. Since, as will be described below, and in accordance with the present teachings, various analysis methods employ multiple mass analyzers, and as such, a hybrid mass spectrometer system can be advantageously employed to improve duty cycles by using two or more analyzers simultaneously. The ORBITRAP™ mass analyzer112employs image charge detection, in which ions are detected indirectly by the image current they induce on a set of outer electrodes of the analyzer by the motion of ions within an ion trap.

In operation of the mass spectrometer system150, an electrospray ion source101provides ions of a sample to be analyzed to an aperture of a heated ion transfer tube102, at which point the ions enter into a first vacuum chamber. After entry, the ions are captured and focused into a tight beam by a stacked-ring ion guide104or, alternatively, an ion funnel. A first ion optical transfer component103atransfers the beam into downstream intermediate-vacuum regions of the mass spectrometer. Most remaining neutral molecules and undesirable ion clusters, such as solvated ions, are separated from the ion beam by a curved beam guide106. Neutral molecules and ion clusters follow a straight-line path whereas the paths of ions of interest are bent around the ninety-degree turn of the curved beam guide, thereby producing the separation.

A quadrupole mass filter108of the mass spectrometer system150is used in its conventional sense as a tunable mass filter so as to pass ions only within a selected m/z range. A subsequent ion optical transfer component103bdelivers the filtered ions to a curved ion trap (“C-trap”) component110. The C-trap110is able to transfer ions along a pathway between the quadrupole mass filter108and the ion trap mass analyzer116. The C-trap110also has the capability to temporarily collect and store a population of ions and then deliver the ions, as a pulse or packet, into the ORBITRAP™ mass analyzer112. The transfer of packets of ions is controlled by the application of electrical potential differences between the C-trap110and a set of injection electrodes111disposed between the C-trap110and the ORBITRAP™ mass analyzer112. The curvature of the C-trap is designed such that the population of ions is spatially focused so as to match the angular acceptance of an entrance aperture of the ORBITRAP™ mass analyzer112.

Multipole ion guide114and optical transfer component103cserve to guide ions between the C-trap110and the ion trap mass analyzer116. The multipole ion guide114provides temporary ion storage capability such that ions produced in a first processing step of an analysis method can be later retrieved for processing in a subsequent step. The multipole ion guide114can also serve as a fragmentation cell and ion trap, which, in the illustrated apparatus (FIG. 1B), is often referred to as an “ion routing multipole”. Various ion optics along the pathway between the C-trap110and the ion trap mass analyzer116are controllable such that ions may be transferred in either direction, depending upon the sequence of ion processing steps required in a particular analysis method.

The ion trap mass analyzer116is a dual-pressure linear ion trap (i.e., a two-dimensional trap) comprising a high-pressure linear trap cell117aand a low-pressure linear trap cell117b, the two cells being positioned adjacent to one another and separated by a plate lens having a small aperture that permits ion transfer between the two cells and that also acts as a pumping restriction that allows different pressures to be maintained in the two traps. The environment of the high-pressure cell117afavors ion trapping, ion cooling, ion fragmentation by either collision-induced dissociation or pulsed-q dissociation, ion/ion reactions by either electron transfer dissociation or proton-transfer reactions, and some types of photon activation, such as ultraviolet photo dissociation (UVPD). The environment of the low-pressure cell117bfavors analytical scanning with high resolving power and mass accuracy. The low-pressure cell includes a dual-dynode ion detector115.

The use of either electron transfer dissociation or a proton transfer reaction, within a mass analysis method, requires the capability of performing controlled ion-ion reactions within a mass spectrometer. Ion-ion reactions, in turn, require the capabilities of generating reagent ions, and of causing the reagent ions to mix with sample ions. The mass spectrometer system150, as depicted inFIG. 1B, illustrates two alternative reagent-ion sources, a first reagent-ion source199adisposed between the stacked-ring ion guide104and the curved beam guide106and a second (alternative) reagent-ion source199bdisposed at the opposite end of the instrument, adjacent to the low-pressure cell117bof the linear ion trap mass analyzer116. Generally, any particular system will only include one reagent ion source at most. Nonetheless, both reagent ion sources could be included so as to facilitate the capability of performing different types of ion-ion reaction within a single instrument. In other embodiments, a single reagent ion source may be capable of generating multiple distinct ion/ion reagents. Although the following discussion is directed to reagent ion sources for PTR, similar discussion may apply to ETD reagent ion sources or other alternative forms of ion/ion reactions.

A first possible reagent ion source199a, may be located between the stacked ring ion guide104and the curved beam guide106. As illustrated, the reagent ion source199acomprises a glow discharge cell comprising a pair of electrodes (anode and cathode) that are exposed to a reagent gas conduit198athat delivers the reagent gas from a reagent liquid (or solid) reservoir197ahaving a heater that volatilizes the reagent compound. When a high voltage is applied across the electrodes, glow discharge is initiated, which ionizes the reagent molecules flowing between the electrodes. Reagent anions from the glow discharge source are introduced into the ion optics path ahead of the quadrupole mass filter108within which they may be m/z selected. The reagent ions may then be accumulated in the multipole ion guide114, and subsequently transferred into the high-pressure cell117aof the dual-pressure linear ion trap116within which they are made available for the ion-ion reaction. The reaction products may be directly transferred to the low-pressure cell117bor to the ORBITRAP™ mass analyzer112for m/z analysis.

A possible alternative reagent ion source199bmay be located adjacent to the low-pressure linear trap cell117b, where it may comprise an additional high-vacuum chamber192, from which reagent ions may be directed into the high-pressure cell117athrough an aperture in between chamber192and the high-pressure cell. In operation, gaseous reagent compound is supplied from a reagent liquid (or solid) reservoir197bhaving a heater that volatilizes the reagent compound and is directed through a reagent gas conduit198bthat delivers the reagent gas into a partially confined ion generation volume196. In operation, thermionic electrons supplied from an electrically heated filament194are directed into the ion generation volume196with a certain pre-determined energy by application of an electrical potential between the filament194and an accelerator electrode (not shown). The supplied energetic electrons cause ionization of the reagent gas so as to generate reagent ions. The reagent ions may then be guided into the high-pressure cell117aby ion optical transfer component103dunder the operation of gate electrodes (not shown).

FIG. 2is a more-detailed depiction of a general multipole device352which may be employed as an ion guide or as an ion storage device. The multipole device352includes an entrance electrode353a(e.g., an entrance lens) disposed at an entrance end358aof the device and an exit electrode353b(e.g., an exit lens) disposed at an exit end358b. The multipole device352may comprise four elongated, and substantially parallel, rod electrodes arranged as a pair of first rod electrodes361and a pair of second rod electrodes362. The leftmost diagram ofFIG. 2provides a longitudinal view and the adjacent right-hand diagram provides a transverse cross-sectional view, of the ion storage device352. Note that only one of the rod electrodes362is shown in the left-hand depiction, since the view of the second rod electrode362is blocked in the depicted view. The four rod electrodes define an axis59of the device that is parallel to the rod electrodes362,361and that is centrally located between the rod electrodes; in other words, the four rod electrodes362,361are equidistantly radially disposed about the axis59. The rod electrodes are maintained in the proper configuration, relative to one another, by means of one or more support structures357made of an insulating material.

Although the ion storage device352shown inFIG. 2is illustrated with straight, parallel rod electrodes, in some embodiments, the electrodes may be curved. Instead of being limited to just four rod electrodes so as to generate an RF electric field, the ion storage device may alternatively comprise six (6) rods, eight (8) rods, or even more rods so as to increase the contribution of higher-order electric fields (e.g., hexapolar and octopolar). For example, the cross-sectional view within inset370ofFIG. 2illustrates a configuration having a total of eight rods, organized as four rod pairs, specifically rod pairs371,372,373and374, which together define a central axis59. As is well known, during operation, each rod pair is energized with a different respective phase of an applied RF confining voltage.

One common complication with all of the tandem mass spectrometry, and general MSnmethods (e.g., see Ibrahim, Y. et al. Improving the Sensitivity of Mass Spectrometer using a High-Pressure Electrodynamic Ion Funnel Interface.Journal of the American Society of Mass Spectrometry2006, 9, 1299-1305; Scheltema, R. A. et al. The Q Exactive H F, a Benchtop Mass Spectrometer with a Pre-filter, High-performance Quadrupole and an Ultra-high-field Orbitrap Analyzer.Molecular and Cellular Proteomics□2014, 12, 3698-3708), is that successful analysis requires a large quantity of initial precursor ions so as to produce product ion mass spectra having sufficiently strong product-ion signals. For example, the experimental types described above often require more than one hundred thousand precursor ions for good results. Previous efforts to satiate these ion requirements have focused on increasing the permissiveness of the ion pathway (e.g., ion funnels and high-capacity transfer tubes), and a tendency towards analyzing larger amounts of sample (e.g., loading more sample and increasing the chromatography peak capacity). Unfortunately there are physical limitations to these approaches. For example, modern designs of ionization sources are rapidly approaching the theoretical ionization efficiency limit.

As an alternative to increasing the brightness of the ion beam or increasing ion transmission throughput, mass spectrometry sensitivity can be improved by utilizing a larger portion of the ion population generated at the ion source. In the field, this strategy is known as improving the instrument duty cycle. Most efforts to improve mass spectrometer duty cycle have focused on speeding up ion manipulations (e.g., fragmentation or ion-ion reaction) and analysis. In this disclosure, however, the inventors focus on another approach to improving instrument duty cycle during tandem mass spectrometry or higher-order MSnexperiments. The novel approaches taught herein are based upon the concept of injecting and accumulating multiple precursor ions in parallel. In the novel approaches taught herein, the total analysis time spent injecting ions is reduced by accumulating multiple precursors in parallel, which results in shorter average spectral acquisition times and an improved overall duty cycle.

In some of the earliest implementations of this parallel ion accumulation method, all the accumulated precursor ions were manipulated and analyzed in parallel (e.g., see Gillet, L. C. et al. Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis.Molecular and Cellular Proteomics□2012, 6; Egertson, J. D. et al. Multiplexed peptide analysis using data-independent acquisition and Skyline.Nature Protocols.2015, 10, 887-903). These methods are quite fast, because multiple precursor ions are processed in parallel during every MS step. However, these methods suffer from increased spectral complexity and limited dynamic range.

As an alternative, a recently implemented version of this method describes individual analysis of each of the parallel-accumulated precursor ion species. These parallel-accumulated precursor ions are sequentially ejected from an ion trap by trapped ion mobility (TIMS). Following TIMS-based ion ejection, the individual precursor ions are subjected to MS2analysis (Meier, F. et al. Parallel Accumulation-Serial Fragmentation (PASEF): Multiplying Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device.Journal of Proteome Research2015, 12, 5378-5387). As implemented, there are two limitations to this earlier approach. Firstly, all the ions formed at the source are accumulated in parallel in the TIMS device. This step will limit the dynamic range of the method. Secondly, the ions accumulated in parallel are sequentially ejected based upon their mobility, which can be difficult to predict and, most often, must be experimentally measured. This fact limits the applicability of the Meier et al. method because it makes it difficult to apply the method to a sample comprised of previously uncharacterized molecules. Accordingly, there is the need in the art for improved methods of injecting and accumulating multiple precursor ions in parallel with subsequent sequential ion manipulation and analysis.

SUMMARY OF THE INVENTION

To address the above-identified needs in the art, the inventors herein propose an alternative to the parallel accumulation based methods described above. According to the present teachings, ions are injected into a device that is capable of serial ejection, where the serial ejection is effected using a pseudopotential barrier that is generated by an RF voltage. The ions formed at an ion source are filtered prior to accumulation in the device capable of serial ejection. Once the ions have finished accumulating, they are ejected in an m/z dependent order using an offset voltage that progressively overcomes, for each m/z window, a pseudopotential barrier that corresponds to the depth of a pseudopotential barrier. Following ejection, the ions in each serially ejected window are mass analyzed individually. In embodiments, this analysis may be performed in a quadrupole ion trap, an electrostatic trap, such as an ORBITRAP™ mass analyzer or a Cassini trap, or a time-of-flight mass analyzer. In various embodiments, the analysis of the ions within a window or within a plurality of windows might include additional rounds of ion isolation and manipulation (e.g., MSn, fragmentation by collision-induced dissociation, electron capture dissociation, electron transfer dissociation, proton transfer reaction, etc.).

As noted above, many of the earlier methods that utilized parallel accumulation of multiple precursors have a limited dynamic range. As described herein, methods in accordance with the present teachings avoid this pitfall by filtering ions upstream of the pseudopotential-based ion accumulation and separation device. By including this filter, the instrument is not required to accumulate the entire breadth of ions formed at the source. As such, the instrument can accumulate more ions of interest before reaching the space-charge limit of the pseudopotential-based ion accumulation and separation device. In some cases, this up-stream filtering may take the form of discrete isolation windows using isolation waveforms with multiple notches. In some cases these waveforms may be applied to a quadrupole mass filter (e.g., Song, Q. et al. Demonstration of using Isolation Waveforms for Beam Type Selected-Reaction-Monitoring on a QqLIT Mass Spectrometer.Proceedings of the64thConference of the American Society for Mass Spectrometry2016). After the precursor population is accumulated, the precursor ions are ejected in a serial order based upon their individual m/z ratios, as described above.

The other limitation that was discussed above relates to the use of ion mobility to sequentially eject ions that were accumulated in parallel. Ion ejection by mobility can be difficult to perform because most often ion mobilities must be experimentally measured and cannot be accurately predicted based upon the chemical formula or precursor m/z value. As an alternative, we propose sequentially ejecting ions using a pseudopotential barrier generated by an RF voltage.

According to a first aspect of the present teachings, a method for mass spectrometric analysis of ions of a plurality of ion species generated by ionization of a sample is provided, the method comprising: (a) isolating a plurality of portions of the ions, each portion consisting of a subset of the ion species within a respective range of mass-to-charge (m/z) values; (b) simultaneously retaining the isolated plurality of portions of the ions in an ion storage apparatus, wherein the retaining is at least partially facilitated by applying an auxiliary radio-frequency (RF) voltage waveform to a one of two electrode members of the ion storage apparatus, thereby generating a pseudopotential between the two electrode members, each electrode member either consisting of a single electrode or comprising a group of electrodes; (c) releasing the retained isolated portions of the ion species one at a time from the ion storage apparatus, the releasing comprising one or more of: varying a DC potential applied to a one of the electrode members, varying DC potentials applied to both of the electrode members, or by reducing an amplitude of the applied auxiliary RF voltage waveform; and (d) fragmenting or reacting each released portion of the ion species to thereby generate a respective set of product ion species and mass analyzing the product ion species.

In some embodiments, the step (a) may comprise generating each portion, one at a time, by passing a continuous beam of a plurality of ions that includes all of the ion species through a mass filter while operating the mass filter so as to eject all ion species other than ion species within the respective range of mass-to-charge (m/z) values corresponding to the portion, while the step (b) may comprise receiving and trapping each of the generated portions, one at a time, from the mass filter as they are generated. In some alternative embodiments, the step (a) may comprise generating the plurality of portions, simultaneously, by passing a continuous beam of a plurality of ions that includes all of the ion species through a mass filter while operating the mass filter so as to eject all ion species other than ion species within any one of the respective ranges of mass-to-charge (m/z) values corresponding to the plurality of portions while the step (b) may comprise receiving the plurality of portions simultaneously and trapping the plurality of portions as they are received. In some embodiments, the step (b) may comprise simultaneously retaining the isolated plurality of portions of the ions in a multipole apparatus comprising an entrance lens, an exit lens, and a set of parallel rod electrodes disposed between the entrance and exit lenses, the set of rod electrodes being the first electrode member and the exit lens being the second electrode member, wherein the auxiliary RF voltage waveform is applied to the exit lens. However, in some alternative embodiments, the auxiliary RF voltage waveform is instead applied to all of the rod electrodes, wherein the waveform applied to each rod electrode comprises a same phase, amplitude, and frequency as does a voltage waveform applied to each other rod electrode. In accordance with some still further alternative embodiments, the step (b) may comprise simultaneously retaining the isolated plurality of portions of the ions within a multipole apparatus comprising an entrance lens, an exit lens, and a sequence of sections defined along an axis of the ion storage apparatus, wherein a first subset of the plurality of portions of the ions is retained in a first section and a second subset of the plurality of portions of the ions is retained in a second section downstream from the first section, wherein a first one of the electrode members comprises electrodes of the first section and the other one of the electrode members comprises electrodes of the second section. Each section may comprise a respective plurality of rod electrode segments disposed about the axis of the ion storage device or, alternatively, a respective plurality of stacked plate electrodes, each plate electrode having an aperture and disposed such that the axis passes through the aperture.

According to some embodiments, a second plurality of portions of the ions may be isolated and retained in the ion storage apparatus simultaneously with the fragmenting or reacting and mass analyzing of an earlier plurality of portions of the ions. According to some embodiments, a second plurality of portions of the ions may be isolated and retained in the ion storage apparatus simultaneously with the releasing, from the ion storage apparatus, of an earlier plurality of portions of the ions.

According to a second aspect of the present teachings, a mass spectrometer system is provided, the mass spectrometer system comprising: (i) an ionization source; (ii) a mass filter apparatus configured to receive ions from the ionization source; (iii) a fragmentation or reaction cell configured to receive ions filtered according to mass-to-charge ratio (m/z) by the mass filter and to fragment or react the received ions so as to thereby generate product ions; (iv) a mass analyzer configured to receive, mass analyze and detect the product ions; (v) an ion guide having an axis and comprising (a) an entrance lens configured to receive the filtered ions from the mass filter; (b) an exit lens disposed downstream from the entrance lens and configured and to transmit the filtered ions to the fragmentation or reaction cell; and (c) a plurality of electrodes disposed between the entrance and exit lenses; and (vi) one or more power supplies electrically coupled to the ion guide, fragmentation or reaction cell and mass analyzer, wherein the one or more power supplies are configured to: supply an oscillatory radio-frequency (RF) voltage to the plurality of electrodes that confines ions within the ion guide to a vicinity of the axis; supply an auxiliary radio-frequency (RF) voltage waveform either to the exit lens or, with phase synchronicity, to all electrodes of the ion guide; and supply a variable DC potential difference between the plurality of electrodes and the exit lens.

According to some embodiments, the plurality of electrodes may comprise a set of mutually parallel rod electrodes that are parallel to and symmetrically disposed about an axis. According to some alternative embodiments, the plurality of electrodes may comprise a set of stacked plate electrodes, each plate electrode comprising an aperture, the plurality of apertures defining an ion channel through the ion guide between the entrance and exit lenses. In some embodiments, the mass spectrometer system may further comprise: (vii) an electronic controller or computer processor comprising machine-readable program instructions operable to cause the one or more power supplies to vary one or both of an amplitude of the auxiliary RF voltage waveform and the variable DC potential difference such that ions are prevented from exiting the ion guide. The electronic controller or computer processor may comprise further machine-readable instructions that are operable to cause the one or more power supplies to vary one or both of the amplitude of the auxiliary RF voltage waveform and the variable DC potential difference such that ion species are released from the ion guide in accordance with their respective m/z values. In some embodiments, the electronic controller or computer processor may comprise machine-readable instructions that are operable to cause the one or more power supplies to cause the fragmentation or reaction cell to either fragment or react each released ion species as it is received from the ion guide.

According to a third aspect of the present teachings, a mass spectrometer system is provided, the mass spectrometer system comprising: (i) an ionization source; (ii) a mass filter apparatus configured to receive ions from the ionization source; (iii) a fragmentation or reaction cell configured to receive ions filtered according to mass-to-charge ratio (m/z) by the mass filter and to trap and/or fragment or react the received ions so as to thereby generate product ions; (iv) a mass analyzer configured to receive, mass analyze and detect the product ions; (v) an ion guide configured to receive the filtered ions from the mass filter and to transmit the filtered ions to the fragmentation or reaction cell, the ion guide comprising: an entrance end and an ion exit end; an axis extending between the ion entrance and exit ends; and a sequence of sections disposed along the axis from the entrance lens to the exit lens; and (vi) one or more power supplies electrically coupled to the ion guide, the fragmentation or reaction cell, and the mass analyzer, the one or more power supplies configured to: supply a radio-frequency (RF) confining voltage to electrodes of all sections of the ion guide; supply an auxiliary RF voltage waveform to electrodes of a segment, each of a phase, amplitude and frequency of the provided auxiliary RF voltage being identical among all electrodes of the segment; and supply a DC potential difference between the segment to which the auxiliary RF voltage is provided and a second segment that is adjacent thereto.

In some embodiments, the electrodes of each section may comprise a stack of two or more plate electrodes, each plate electrode comprising an aperture, wherein the plurality of apertures of all plate electrodes define an ion channel through the ion guide. In alternative embodiments, each section may comprise a plurality of rod electrode segments that are symmetrically disposed about the axis.

DETAILED DESCRIPTION

The following description is presented to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the described embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiments and examples shown but is to be accorded the widest possible scope in accordance with the features and principles shown and described.

The particular features and advantages of the invention will become more apparent with reference to the appendedFIGS. 2-8, taken in conjunction with the following description. Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. It will be appreciated that there is an implied “about” prior to the quantitative terms mentioned in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting.

As used herein, “a” or “an” also may refer to “at least one” or “one or more.” Also, the use of “or” is inclusive, such that the phrase “A or B” is true when “A” is true, “B” is true, or both “A” and “B” are true. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, and as commonly used in the art of mass spectrometry, the term “DC” does not specifically refer to or necessarily imply the flow of an electric current but, instead, refers to a non-oscillatory voltage which may be either constant or variable. Likewise, as used herein, and as commonly used in the art of mass spectrometry, the term “AC” does not specifically refer to or necessarily imply the existence of an alternating current but, instead, refers to an oscillatory voltage or oscillatory voltage waveform. The term “RF” refers to an oscillatory voltage or oscillatory voltage waveform for which the frequency of oscillation is in the radio-frequency range.

The reader should be aware that, throughout this document, the term “DC” is used in accordance with its general usage in the art so as to mean “non-oscillatory” without necessary implication of the existence of an associated electrical current. Thus, the usage of the terms “DC voltage”, “DC voltage source”, “DC power supply”, “DC potential” etc. in this document are not, unless otherwise noted, intended to necessarily imply the generation or existence of an electrical current in response to the “DC voltage” or “DC potential” or to imply the provision of an electrical current by a “DC voltage source” or a “DC power supply”. As used in the art, and as used herein, unless otherwise noted, the term “DC” is made in reference to electrical potentials (and not electrical current) so as to distinguish from radio-frequency (RF) potentials. A “DC” electrical potential, as commonly used in the art and as used herein, may be static but is not necessarily so; in other words, the DC potential could be variable. In this document, the terms “upstream” and “downstream” are used, in a relative sense, to convey a relative position of a component or entity along an ion pathway through various components from an ion source to an ion destination, where “upstream” represents components or positions along the pathway that are nearer to the ion source and “downstream” represents components or positions along the pathway that are nearer to the ion destination.

Pseudopotential-based ion ejection has been well studied, and is best summarized in the work by Gerlich (Gerlich, D. Inhomogenous RF Fields: A Versatile Tool for the Study of Processes with Slow Ions.State-selected and State-to-State Ion-Molecule Reaction Dynamics. Part1. 1992, 1-177). Briefly, the application of an auxiliary, inhomogeneous RF field creates a pseudopotential barrier of the form:

Upseudopotential=C⁢⁢UA⁢⁢C2ω2⁡(mz)Eq.⁢1
Where UACand ω are the amplitude and angular frequency of the RF, m and z are the mass and charge of the ion of interest, and C is a geometry dependent parameter. The pseudopotential barrier may be offset or overcome by a DC potential, UDC:

UD⁢⁢C=C⁢⁢UA⁢⁢C2ω2⁡(mz)=UpseudopotentialEq.⁢2
Note that the algebraic sign (positive or negative) of the m/z term in the denominator transfers to both the pseudopotential, Upseudopotential, and the DC potential, UDC, in either Eq. 1 or Eq. 2. For positively-charged ions, Upseudopotentialand UDCare both positive; for negatively-charged ions, Upseudopotentialand UDCare both negative. Regardless of the sign of z, in the absence of a DC potential that is able to overcome the pseudopotential barrier ions are motivated to migrate away from the region of space in which the ions oscillate in response to auxiliary field.

An ion will leave the pseudopotential-based ion separator when the “height” of the pseudopotential barrier (in the case of positively-charged ions) or “depth” of the barrier (in the case or negatively-charged ions) is just offset by the DC field created by the application of the DC potential. The rising portion of the pseudopotential barrier (in the case of positive ions) is sometimes loosely referred to as a “pseudopotential well” because of its resemblance to the rising pseudopotential barriers that maintain ion confinement within a restricted spatial region within a conventional RF ion trap, such as a Paul trap (three-dimensional trap) or a linear ion trap (two-dimensional trap). In the remainder of this document, it is assumed, for convenience, that ions are positively charged. Accordingly, ions are assumed to move down-potential and pseudopotentials are illustrated as “peaks” in the drawings. If negatively-charged ions are to be considered, then all potentials and pseudopotentials should be reversed in sign relative to those that are drawn and described herein.

Operationally, by application of an oscillatory RF voltage to at least one electrode of a pair of adjacent electrodes, it is possible to cause ions to physically oscillate about or around a region of space near or between the electrode or electrodes. In these areas of higher oscillation the ions will acquire more energy; as such, they will tend to move away from these higher energy regions towards lower energy regions. This bias or restriction of the ions to a particular region of space somewhat resembles the situation that would hypothetically occur if it were possible to create a static DC potential local maximum at the center of the region of oscillation. Since such a free-space electrostatic extremum is not possible, this fictitious potential that generates this real ion confinement is referred to as a pseudopotential.

When the multipole device352(FIG. 2) is employed as an ion guide, movement of ions in one direction along the axis59is facilitated by application of DC lens potentials to entrance and exit electrodes. Such DC potential offsets are schematically depicted in box310ofFIG. 2, where the graph portions322,324and326are a schematic depiction of the hypothetical variation of electrical potential along axis59of device352. In box310, as in elsewhere in this document, it is assumed that all ions are positively charged. However, as one of ordinary skill in the art will readily understand, the concepts described herein are not limited to positively charged ion species. The ion manipulations described herein are equally valid with regard to the manipulation of negatively charged species, provided that the algebraic signs of DC potentials are reversed. Graph portion324represents the DC electrical potential along the axis59where it is surrounded by the multipole rods and graph portions322and324represent DC potential applied to the entrance and exit electrodes (lenses)353a,353bthat keep the ions moving in the direction of the arrows.

Conventionally, trapping of ions within the multipole device352may be achieved by raising the DC potential of the exit electrode353bso that the DC potential(s) of both entrance and exit lenses are greater than the DC potential along axis59within the multipole. However, such conventional ion trapping does not discriminate among different m/z values. In order to release stored ions in order of their m/z values in accordance with methods of the present teachings, the inventors have recognized that a pseudopotential may be created between the multipole rods and one or both of the entrance and exit lenses by application of an auxiliary RF voltage.

FIG. 3illustrates a known ion storage apparatus452in which the rods362and the rods361(as shown in and previously described in reference toFIG. 2) are replaced by series of rod segments. Specifically, in the illustrative depiction ofFIG. 3, each individual rod361of the apparatus352is replaced by six rod segments461a-461fand each individual rod362of the apparatus352is replaced by the six rod segments462a-462f. Each collection of four rod segments comprises a section of the apparatus452. For example, six such sections,465a-465f, are illustrated inFIG. 3as well as inFIGS. 6A and 6B. Although each section (465a-465f) of the apparatus452, as described and illustrated herein, is comprised of four rod segments, each such section could, alternatively, be configured as a general multipole device comprising a larger number of rod segments. In conventional operation, all of the segments461a-461fare supplied with the same RF voltage and phase from a power supply via a set of isolating capacitors (not illustrated). Likewise, all of the segments462a-462fare supplied with the same RF voltage that is phase-shifted relative the RF phase supplied to rod segments461a-461f.

In conventional operation, variable DC voltages are applied to the different sections of the apparatus452, such that each collection of four segments that make up a section is set at a particular respective DC voltage. As illustrated in box410ofFIG. 3, the set of such applied voltages comprise a series of voltage steps424a-424fthat decrease in a direction from the entrance lens453ato the exit lens453b. The various voltage steps424a-424fthat are applied to the sections465a-465f, and the voltages422,426applied to the entrance and exit lenses, can create an internal field along the axis59and within the device452that assists in urging ions in the direction of the arrows within the device.

In accordance with some embodiments of the present teachings, the operation of the multipole device352(previously described with reference toFIG. 2) may be modified using pseudopotentials so that the device functions as an ion selector. For example, box312ofFIG. 4schematically illustrates the creation of pseudopotential barriers along the axis59between: (a) the multipole rods361,362in the four rod multipole configuration, or, in the eight-rod configuration, between the rods371,372,373,374and (b) the exit electrode353b. In the example operation procedure corresponding to box312, the pseudopotential barriers are generated by application of an auxiliary RF voltage to the exit electrode. Three different pseudopotential-modified electrical potential profiles325a,325b,325care schematically illustrated, corresponding to ion species of three different ink values, in accordance with Eq. 1 above. More specifically, the example profiles325a,325band325cpertain to ion species of (m/z)a, (m/z)band (m/z)c, respectively, where (m/z)a>(m/z)b>(m/z)c. With reference to the profile325a, it may be noted that the DC potential difference between the multipole rods and the exit electrode353b(i.e., between graph portions324and326) is sufficiently great to overcome the pseudopotential barrier that would otherwise be formed in the vicinity of the gap363b. As a result, the ions associated with the profile325aare able to pass through the gap363band to exit the apparatus352through the exit electrode353b.

Still with reference to box312ofFIG. 4, it may be noted that the profiles325band325ccomprise maxima as a result of the superimposition of the pseudopotential on top of the regular DC potential gradient. Thus, the profiles325band325care pseudopotential barriers to the passage of ion species of corresponding respective m/z values. Pseudopotential barriers325band325cprevent ions of the corresponding respective ions species from exiting the apparatus352along the axis (the ions still being confined transverse to the axis by the trapping RF potentials applied to the multipole rods, assuming that they have previously been completely thermalized within the multipole device). Therefore, the trapped ion species, for example, the species corresponding to the profiles325band325c, will be prevented from exiting the quadrupole device352through exit electrode353b. Note that the three illustrated pseudopotential-modified electrical potential profiles325a,325b,325cofFIG. 4are merely examples of a hypothetical infinite number of such profiles, one for each respective m/z value in accordance with Eq. 1.

Still with reference to box312ofFIG. 4, it should be noted that it is possible to progressively release the ions corresponding to potential profiles325band325cfrom apparatus352by either progressively lowering the DC potential corresponding to graph portion326or progressively raising the DC potential on the multipole rods corresponding to graph portion324, or both. Alternatively, the amplitude of the applied auxiliary RF voltage applied to exit lens353bmay be progressively ramped downwards so as to progressively decrease the magnitude of the imposed pseudopotential, in accordance with Eq. 1. Alternatively, any two or all three options for releasing ions stored in the ion separator device may be employed at the same time. In this fashion, ions that are stored in the multipole apparatus352may be progressively released in accordance with their m/z values, specifically in the reverse order of their m/z values, with ions having greater m/z values being released prior to the release of ions with lesser m/z values. Thus, when operated in accordance with the present teachings, the ion storage device352, as well as other devices employed in accordance with the present teachings, may be regarded as an ion separator.

Another method for generating the pseudopotential-modified electrical potential profiles325a,325b,325c, and others, for different m/z values in the vicinity of gap363b, is by applying the auxiliary RF voltage to the multipole rods (e.g. rods361,362or371,372,373,374) instead of to an exit lens353b. In such experimental setups, the auxiliary RF voltage must be applied with synchronous phase on all such rods (Kaiser N. K. et al. Controlled ion ejection from an external trap for extended m/z range in FT-ICR mass spectrometry. J Am Soc Mass Spectrom. 2014 June; 25(6):943-9). This auxiliary RF voltage is superimposed on-top of the main RF voltage that confines the ions transverse to the axis59. When applied to the multipole rods in this fashion, the auxiliary RF voltage creates further pseudopotential-modified electrical potential profiles in the vicinity of the electrode gap363abetween the entrance lens353aand the multipole rods, as illustrated in box314ofFIG. 4. As is illustrated in this specific example, the offset DC potential at the entrance lens (that is, the potential difference between potential322and potential324) is identical to the offset potential at the exit lens (i.e., the difference between potential324and potential326). As a result, the illustrated pseudopotential-modified electrical potential profiles323a,323band323ccorrespond to the same respective m/z values for which the corresponding profiles at the opposite side of the apparatus352are, respectively, profiles325a,325band325c. If, at a certain time, t0, the two offset DC potentials are not identical, then the m/z values of ions that are selectively admitted into the apparatus352at time, t0may differ from the m/z values of ions that are selectively released from the opposite end of the apparatus at the same time, t0.

Pseudopotential-based sequential ion ejection is technically simpler than the mobility based approaches described in the background section, because pseudopotential-based ion separation ejects ions based upon their m/z ratios. As such, it is possible to accurately predict when un-characterized ions will leave the pseudopotential-based ion separator using the m/z information collected in an initial MS1survey mass spectrum. Using the methods of the present teachings, it is not necessary to experimentally measure the mobility of each precursor ion species, or indeed any other specific property of each ion, other than its m/z ratio, prior to performing the separation.FIG. 5is a set of graphs of remaining trapped ions (values normalized to 100%) of different m/z values as the ions are selectively released from an apparatus of the type shown inFIG. 2that is operated as described above. Specifically, graphs302,304,306,308and310pertain to ion species whose m/z values are 1022, 1122, 1322, 1522 and 1721, respectively (all values in thomsons, Th). The curves shown inFIG. 5are plotted as functions of progressively decreasing lens offset voltage, UDC(Eq. 2), applied to an exit lens, while the auxiliary RF voltage applied to the same lens was held constant. Accordingly, the data points depicted inFIG. 5were generated reading from right to left across the diagram, thus confirming that ion species are released from the apparatus in the reverse order of their m/z values. When selecting sets of ions comprising a plurality of m/z values that are to be isolated and temporarily contemporaneously trapped in an ion separator in accordance with the present teachings, it is preferable to select the ion m/z values such that none of the steeply rising portions of the transmission curves (curves such as those shown inFIG. 5) overlap one another. By selecting the contemporaneously trapped ions in this fashion, it may be assured that there will not be appreciable subsequent co-release of ions of different ink values from the ion separator.

In accordance with the present teachings, the apparatus452may also be operated as an ion selector.FIGS. 6A and 6Billustrate two examples of such operation in accordance with the present teachings. Specifically, the operation may be achieved by configuring one or more power supplies (not shown) to provide one or more additional auxiliary RF voltages to chosen elements of the apparatus. The auxiliary RF voltages may be applied so as to create one or more pseudopotential barriers, each such pseudopotential barrier being at either: (a) the gap463abetween the entrance lens453aand the first section465aof the apparatus452, (b) one of the gaps463b-463fbetween sections or (c) the gap463gbetween the last section465fand the exit lens453b. For example, box414ofFIG. 6Aschematically illustrates pseudopotential-modified electrical potential profiles425a,425band425ccreated in the vicinity of the gap463gby application of an auxiliary RF voltage to the exit lens453b. The example profiles425a,425band425cpertain to ion species of (m/z)d, (m/z)eand (m/z)f, respectively, where (m/z)d>(m/z)e>(m/z)f. As noted above, the three illustrated pseudopotential-modified electrical potential profiles425a-425care merely examples of a hypothetical infinite number of such pseudopotential-modified profiles that may be generated at the gap463g, one such profile for each respective m/z value in accordance with Eq. 1. In particular, the profile425amonotonically decreases in the direction of the exit lens453bwithin the gap463gand therefore allows the egress of ions having the m/z value, (m/z)d. In contrast, the profiles425band425c, which are applicable to the ion species (m/z)eand (m/z)f, respectively, both comprise maxima within the gap463g, since the illustrated potential difference between the DC voltage424fapplied to the last section465fand the DC voltage426applied to the exit lens453b, is insufficient to overcome the pure pseudopotential barrier generated by the auxiliary RF voltage. Thus, with the illustrated example pseudopotential-modified electrical potential profiles425a,425b, the ion species (m/z)eand (m/z)fwill be selectively trapped within the apparatus452while, at the same time, the (m/z)eions will be pass out of the apparatus. The trapped ions may be preferentially allowed to exit, in reverse order of their respective m/z values, by varying either the amplitude of the applied auxiliary RF voltage or by varying the DC voltage difference between the DC voltage424fapplied to the last apparatus section465fand the DC voltage426applied to the lens453b.

In other embodiments in accordance with the present teachings, auxiliary RF voltages could be applied to one or more of the sections465a-465fby applying the auxiliary RF voltage with synchronous RF phase and with equal amplitude and frequency to all rod segments comprising the particular section. In such cases, pseudopotential-modified electrical potential profiles will be created in gaps at both ends of the section to which the auxiliary RF voltage is applied. By controlling either the amplitude of the auxiliary RF voltage applied to the section in question or the DC voltage difference between the section in question and the components to either side of the section in question, then the m/z values of ions both entering and exiting the section may be selectively controlled.

In accordance with the present teachings, the ability to apply pseudopotential-generating auxiliary RF voltages to selected sections of the apparatus452provides the capability to partition the apparatus so that different ion species may be independently accumulated in different regions of the apparatus. As one example, multiple ion species having relatively low m/z values may be accumulated in different respective regions while, simultaneously, different ion species having greater m/z value(s) are allowed to pass through with minimal or no accumulation. Such operation may be advantageous in situations in which the ion species that are allowed to pass through are present in relatively high abundance so that little or no accumulation is needed.FIG. 6Bschematically illustrates one example of such ion partitioning within the apparatus452. In the example ofFIG. 6B, it is assumed that auxiliary RF voltages are applied to sections465band465d, as indicated by shading of the rod segments to which such auxiliary RF fields are applied. As described above, within each section, the auxiliary RF voltage is applied with identical amplitude, frequency and phase to all rod segments (e.g., six rod segments, 8 rod segments, 12 rod segments, etc.) of the section. The application of an auxiliary RF voltage to the section465bcreates a first pseudopotential at the gap463band a second pseudopotential at the gap463c. Similarly, the application of an auxiliary RF voltage to the section465dcreates a third pseudopotential at the gap463dand a fourth pseudopotential at the gap463e. Because a separate pseudopotential is created at each end of any section to which an auxiliary RF voltage is applied, there will generally be at least one intervening section to which no auxiliary RF voltage is applied disposed between each consecutive pair of sections that receive such auxiliary RF voltage waveforms. For example, inFIG. 6B, the section465cis such an intervening section that does not receive an auxiliary RF voltage. AlthoughFIG. 6Bonly depicts two sections (sections465band465d) that receive an auxiliary RF voltage, and only depicts six total sections, it is to be understood that additional sections could receive an auxiliary RF voltage, that the apparatus could comprise either greater or fewer total sections, and that an auxiliary RF voltage could be applied to either or both of the sections adjacent to the entrance lens453aor the exit lens453b.

Box700ofFIG. 6Bis a schematic depiction of four hypothetical profiles701,702,703,704of “effective DC potential” across the length of the apparatus452with relation to four different ion species having mass-to-charge ratios of (m/z)1, (m/z)2, (m/z)3, and (m/z)4, respectively, where (m/z)1<(m/z)2<(m/z)3<(m/z)4. All four effective DC potentials701-704are identical except for the regions at the section gaps463b,463c,463d, and463eat which pseudopotentials are superimposed upon the applied actual DC potentials. Note that the applied DC potentials consist of the horizontal portions of the profiles. Similarly to the conventional operation of the apparatus (FIG. 3), the applied DC potentials comprise a series of downward voltage steps across the apparatus from the entrance to the exit in order to ultimately urge ions completely through the apparatus. For example, voltage steps outlined by open-ended boxes723aand723finFIG. 6Bare analogous to various voltage steps depicted in the profile shown in box410ofFIG. 3. The switchable voltage step outlined by open-ended box723gis also analogous to the voltage step between applied potential424fand applied potential426depicted inFIG. 3except that, inFIG. 6B, this step is shown in a configuration that allows the temporary accumulation of trapped ions within the apparatus.

Still with reference toFIG. 6B, it is to be noted that the voltage steps outlined by open-ended boxes725b-725einFIG. 6Bare different in magnitude from the conventional voltage steps (e.g., the voltage steps outlined at723aand723f) and comprise a series of voltage steps that decrease in magnitude in sequence from box725bto box725e. The voltage steps at725b,725c,725dand725ecorrespond, respectively, to the section gaps463b,463c,463dand463eat which the applied DC potentials are superimposed upon the (m/z)-dependent pseudopotentials that result from application of auxiliary RF voltages to the sections465band465das described above. Accordingly, pseudopotential-modified potential profiles occur within the boxes725b-725e. The modified potentials710,720,730and740at box725bcorrespond to the section gap463b. Similarly, the modified potentials711,721,731and741at box725ccorrespond to the section gap463c. Similarly, the modified potentials712,722,732and742at box725dcorrespond to the section gap463d. Similarly, the modified potentials713,723,733and743at box725ecorrespond to the section gap463e.

Each modified potential depicted in box700ofFIG. 6Bexhibits the effect of the superimposition of an (m/z)-dependent pseudopotential upon an applied DC voltage step. At the position of open-ended box725b, the applied DC voltage step is of sufficiently great magnitude to overcome the blocking effect of the pseudopotentials corresponding to all the referenced ion species, i.e., each of the ion species having mass-to-charge ratios of (m/z)1, (m/z)2, (m/z)3, and (m/z)4. Accordingly, any of the plurality of these ions that enter the apparatus452through the entrance lens453awill proceed at least through gaps463aand463band into the section465b.

At the position of box725c, the (m/z)1species will encounter pseudopotential barrier711. This species will therefore be obstructed form proceeding further and will be trapped in section465b, since the pseudopotential is the greatest for this ion species. However, the pseudopotentials for the (m/z)2species, (m/z)3species, and (m/z)4species are insufficiently great to overcome the applied DC potential drop at725c. Thus, these latter three ion species will continue their forward progress through the gap463cand into the section465c.

At the position of box725d, corresponding to the section gap463d, the magnitude of the applied DC potential drop is less than the applied DC potential drop at box725c. Accordingly, at725d, the (m/z)2ion species will encounter pseudopotential barrier722. Since the pseudopotential corresponding to this ion species is greater than the pseudopotentials corresponding to the (m/z)3species and the (m/z)4species, the (m/z)2ion species will thus be trapped in section465c. At the same position, the pseudopotentials for the ion species (m/z)3and (m/z)4are insufficiently great to overcome the applied DC potential drop at725d. Thus, these latter two ion species will continue their forward progress through the gap463dand into the section465d.

A similar separation of the (m/z)3species from the (m/z)4species occurs at the position of box725e, at which the (m/z)3species encounters the pseudopotential barrier733but the (m/z)4species does not encounter such a barrier. Thus, the (m/z)3species will be trapped in section465dwhile the (m/z)4species may proceed forward through the apparatus452to the minimum applied DC potential adjacent to the exit lens453b. Alternatively, the applied potential on the exit lens453bmay be configured to allow the (m/z)4species to exit the apparatus.

By the above-described process, it is possible to independently control the accumulation of ions species of different m/z values through the apparatus452. Following accumulation, the ion species may then be released from the apparatus to a downstream component of a mass spectrometer system in the order (m/z)4followed by (m/z)3followed by (m/z)2followed, finally, by (m/z)1. In the illustrated example ofFIG. 6B, the (m/z)4species may be released by re-configuring the applied DC potential at the exit lens453b. The accumulated (m/z)3species then may be released by either lowering the amplitude of the auxiliary RF voltage applied to section465dby an appropriate amount, by raising the applied DC potential on section465dby an appropriate amount, by lowering the DC potential applied to section465e, or by some combination of the above. The appropriate amount of any such voltage or potential lowering or raising is chosen such that the potential barrier733disappears while, at the same time, the potential barriers722and711remain. As the same time that the (m/z)3species is being released from the apparatus, the same amplitude or potential adjustments may cause the (m/z)2species to migrate forward to position725e. Following the release of the (m/z)3species from the apparatus, a similar procedure may be employed to release just the (m/z)2species while maintaining the trapping of the (m/z)1species. Finally, the (m/z)1species is released.

In the above-described fashion, the accumulation of each one of different ion species comprising different respective m/z values may be independently controlled, even though the introduction of, the accumulation of, and/or the release of different species may occur at least partially contemporaneously. In view of the above teachings, one of ordinary skill in the art would be able to readily envisage various different modes of operation of a segmented ion separator apparatus, as exemplified by the separator apparatus452, said various different modes of operation comprising sequences or orders of ion species introduction, accumulation, and release that are different than those explicitly described above. Such different sequences and/or orders of events may possibly include different sequences of applied auxiliary RF and DC voltages to the components of the apparatus, as would be readily understood by one of ordinary skill in the art.

It should be appreciated that, in various alternative embodiments of apparatuses in accordance with the present teachings, any instance of a set of rod electrodes as described in this document may be replaced by a stacked ring ion guide. Further, it should be appreciated that any instance of an entrance lens or exit lens as described herein may likewise be replaced by a stacked ring ion guide. Accordingly,FIG. 6Cillustrates a longitudinal cross section of another ion storage apparatus852in accordance with the present teachings in which both the rod electrode sets and the entrance and exit lenses are replaced by a single continuous stack of ring electrodes, each such ring electrode comprising an electrode plate867, a representative one of which is illustrated in face-on view inFIG. 6D.

In the ion storage apparatus852, a plurality of electrode plates867comprise a generally evenly-spaced-apart stack or series of electrodes progressing from an entrance end801to an exit end802of the apparatus. The electrodes may all be formed similarly to the single plate electrode867illustrated inFIG. 6D, each such electrode comprising an aperture868. When arranged as a stack, as schematically depicted inFIG. 6C, the set of aligned apertures868together form an ion channel869that extends from the entrance end801to the exit end802of the apparatus852. It should be kept in mind that, although the plates867are depicted, inFIG. 6D, as being rectangular in shape and having circular apertures868, neither the shapes of the plates nor the shapes of the apertures are limited to any particular shape or shapes. For example, the apertures may be oval or polygonal in shape. As another example the plates may comprise essentially circular rings. Further, the plates may comprise various mounting structures, such as tabs or grooves, for the purpose of mounting within an alignment structure (not shown) and may also comprise electrical contact points or leads (not shown) for purposes of supplying electrical AC and DC voltages to the various plates.

As is known in the art, an RF confining voltage may be applied to the stacked electrode plates867of the apparatus852so as to confine ions to a restricted region about an axis859that is centrally located within the ion channel869. The RF confining voltage is applied such that all electrode plates within the stack receive the same RF amplitude but such that the RF phase applied to adjacent plates is 180-degrees out of phase. In other words, if the plates are consecutively numbered, commencing with plate “number1” at the entrance end801of the apparatus, then the RF applied to all odd numbered plates is in phase and the RF applied to all even numbered plates is likewise in phase but there is an RF phase difference of 180-degrees between the even- and odd-numbered plates. The plate-to-plate alternating RF phase serves to maintain ions in the vicinity of the central axis859within the ion channel869of the apparatus852. In the schematic depiction illustrated inFIG. 8C, the various electrode plates867are illustrated as being mutually aligned such that the ion channel869and the axis859are essentially straight. Nonetheless, it should be kept in mind that the plates may, in some embodiments be offset relative to one another (either offset vertically within the plane of the drawing ofFIG. 6Cor offset out of the plane of the drawing) such that portions of or the entirety of the channel869is curved.

The novel aspects of the operation of the stacked ring ion guide apparatus852in accordance with the present teachings are that, in addition to the RF confining voltage, an further auxiliary RF voltage may be applied to certain selected subsets of the plate electrodes and adjustable DC offset voltages may be applied to the same selected subsets. The auxiliary RF voltage applied to each such selected subset, which is applied in addition to the RF confining voltage, is applied such that all electrodes of the selected subset receive the same RF amplitude and same synchronous frequency and phase. The selective application of the auxiliary RF voltage thus logically divides the stacked ring ion guide into segments, even though the physical structure of the plate electrodes need not differ between different segments. For example, in the schematic illustration ofFIG. 6C, the apparatus852includes seven such segments,865a-865g, which are formed through the selective application of the auxiliary RF voltage to the plate electrodes (shaded) of segments865b,865dand865f. In this example, the plate electrodes of the other segments865a,865c,865eand865gdo not receive the auxiliary RF voltage.

The selective application of an auxiliary RF voltage to certain subsets of the plate electrodes of the stacked ring ion guide apparatus852creates a pseudopotential barrier at each end of each segment that receives an auxiliary RF voltage, in a similar fashion as described above with regard to the apparatus452(FIGS. 6A-6B). Accordingly, with the application of auxiliary RF voltages as depicted inFIG. 6C(i.e., to the shaded electrodes of segments865b,865dand865f), a respective pseudopotential barrier is generated between each pair of adjacent segments. Thus, application of the auxiliary RF voltages to selected segments taken together with coordinated application of DC offset voltages between segments permits the apparatus852ofFIG. 6Cto be operated as a selective ion accumulation apparatus similar to the previously described operation of the rod-electrode-based apparatus452(FIGS. 6A-6B). Voltage profiles similar to those illustrated in the lower half ofFIG. 6Bmay be applied likewise to and between the segments of the apparatus852to achieve similar ion accumulation/selection/transmission results as described previously.

An additional (but not necessarily essential) feature of the apparatus852(FIG. 6C) is that the entrance and exit lenses are incorporated as part of the same electrode stack that is utilized for ion accumulation, storage, selection, and transmission. InFIG. 6C, the entrance and exit segments853a,853bof the apparatus852(FIG. 6C) are analogous to the entrance lens453aand exit lens453b, respectively, of the apparatus452(FIGS. 6A-6B). In general, no auxiliary RF voltages are applied to electrodes of the entrance and exit segments853a,853b. However, the RF confining voltage is generally applied, and DC offset voltages may be applied, to the electrodes of the entrance and exit segments853a,853b. The stacked ring ion guide device852(FIG. 6C) provides an optional operational feature, relative to the apparatus452(FIGS. 6A-6B), in that an axial field or “drag field” may be applied within one or more of the segments, including segments,865a-865gand entrance and exit segments853a,853b. The axial, or drag field, may be applied to assist ion movement in the direction of the arrows depicted on axis859within any such segment by applying varying DC offset voltages between individual plate electrodes867of the segment. It may also be noted that axial/drag fields may be created within any of the rod-based apparatuses352,452,552described herein using any one of a variety of methods, such as the methods taught in U.S. Pat. No. 7,675,031 in the names of inventors Konicek et al.; U.S. Pat. No. 5,847,386 in the names of inventors Thomson et al; and U.S. Pat. No. 6,163,032 in the name of inventor Rockwood, among others.

According to another implementation of the present teachings, as exemplified by the schematically illustrated apparatus552shown inFIG. 7, it is possible to create a series of pseudopotential barriers by dividing a linear ion guide into a series of discrete sections, e.g., sections565a-565c, using a series of lenses (e.g., lenses553a-553d) that are disposed between each set of rod electrodes. According to the exemplary embodiment shown inFIG. 7, the multipole apparatus is comprised of four rods. As illustrated, section565acomprises rod electrodes561aand562a, section565bcomprises rod electrodes561band562b, and section565ccomprises rod electrodes561cand562c. Although the sections are shown with four rods, various embodiments of the apparatus may comprise multipole sections that include more than four rods, such as six, eight, ten, twelve rods, etc. Alternatively, the rod electrodes of one, some, or all of the sections could be replaced by a respective stacked ring ion guide that comprises a plurality of plate electrodes as previously noted. Each of the lenses553a-553dis provided with a respective DC voltage that is controlled so as to either: (a) permit all ions to pass through the lens, in the general direction from the apparatus entrance end558ato the exit end558b, without discrimination according to the ions' m/z values; (b) prevent all ions from passing through the lens (i.e., trap all ions) or (c) to selectively permit ions to pass through the lens in accordance with their m/z values. The first two listed operations are conventional; the last operation is performed with application of an auxiliary RF voltage to the lens so as to create a pseudopotential profile, as described above. Each lens may be operated independently of the others and the same operation may be performed by more than one of the apparatus sections565a-565c, such that ions of different m/z values may be temporarily partitioned into different sections and caused to exit from the apparatus552at different times.

According to other modes of operation of the apparatus552, an auxiliary RF voltage may be applied with synchronous phase to all rod electrodes of a section, while the DC voltages applied to the neighboring lenses are simultaneously adjusted so as to selectively admit ions into the section in accordance with their m/z values and, simultaneously, selectively release ions from the section in accordance with their m/z values. The m/z values of the ions that are admitted into the section may differ from the m/z values of ions that are being released from the section. More than one section of the apparatus may be selectively populated in this fashion.

FIG. 8is a flow chart of a generalized method (method600) for operating a mass spectrometer in accordance with the present teachings. In Step601of the method600, a survey mass spectrum may be measured in order to characterize the ions that are being delivered to the mass filtering and mass analysis stages of a mass spectrometer from an ionization source, possibly as modified by in-source fragmentation. The measurement of this mass spectrum, which is sometimes referred to as an “MS1” spectrum or “survey scan”, or “survey mass spectrum”, may be performed in order to select precursor ion species of certain m/z values for subsequent MSnanalyses. The Step601may be skipped in some circumstances such as, for example, when a sample is well-characterized, if the precursor ions have been previously characterized, or if the method is comprised of expected “targeted” precursors. In Step602, a sample portion of ions or, otherwise, a continuous stream of ions is filtered, such as by a quadrupole mass filter, so as to eliminate ions within all mass-to-charge (m/z) regions except for ions within a plurality of certain pre-selected, distinct, separated ranges of m/z (i.e., m/z ranges). In some cases, these pre-selected regions are determined based upon the survey scan collected in step601. Typically, each m/z range will encompass a respective, pre-determined, m/z value of a precursor ion species, which is to be further manipulated after the elimination of other ions species. In some embodiments, the execution of Step602may comprise sequential isolations of each of the various m/z ranges, in sequential order, in a fashion similar to conventional mass filtering. In such embodiments, the execution of Step602may comprise repeatedly eliminating all ions except for ions within a specific respective one of the pre-selected m/z ranges, where each such isolation step operates on a different portion of a continuous ion stream. In alternative embodiments, the execution of Step602may comprise a multi-notch isolation, whereby the plurality of pre-selected m/z ranges are co-isolated. The principles of multi-notch isolation are described, for example, in U.S. Pat. No. 9,048,074 as well as in Soni, M H and Cooks R G, Selective Injection and Isolation of Ions in Quadrupole Ion Trap Mass Spectrometry Using Notched Waveforms Created Using the Inverse Fourier Transform, Anal. Chem., 1994, 66 (15), pp 2488-2496, both of which are hereby incorporated by reference in their entirety.

In Step603of the method600(FIG. 8), the various ion species within the plurality of pre-selected, distinct, separated, m/z ranges, as filtered in Step602, are collected and accumulated within an ion separation device that is provided with the capability of generating an auxiliary oscillatory voltage that can generate one or more pseudopotential barriers for at least some ion species. The application of the auxiliary oscillatory voltage may be active at the time that ions are accumulated in the ion separation device. In such cases, the pseudopotential barriers may be employed to temporarily trap ions. Alternatively, the initial ion trapping may be effected by conventional means (e.g., DC lens voltages), after which the auxiliary oscillatory voltage is applied. The ion separation device is, preferably, a multipole device comprised of sets of rods (e.g., 4 rods, 6 rods, 8 rods, etc.). In some embodiments, the ion separation device may be a multipole device that is otherwise employed as an ion guide at times when the pseudopotential barrier is not applied, or as an ion trap or ion activation cell, or when methods in accordance with the present teachings are not executed. The accumulation of ions within the ion separation device will generally, but not necessarily, occur simultaneously with the ion filtering step602, as ion species within the isolated m/z ranges may pass through the mass filter device unimpeded directly to the ion separation device. Otherwise, ion storage within the ion separation device, to which the pseudopotential barrier is applied, may not occur simultaneously with ion filtering if a different device operates as an intermediate ion separation device or ion storage device. The ion separation device associated with the pseudopotential barrier may comprise any one of the exemplary ion separation devices described in this document. However, other forms of ion separation devices that employ one or more pseudopotential barriers, possibly within segmented or partitioned ion traps, or possibly within sequentially arranged multipole traps, are also contemplated even if not explicitly described herein.

In Step604of the method600, ions within a single one of the m/z ranges are selectively released from the ion separation device by lowering of the pseudopotential barrier as described previously. In other embodiments, the ions may be given enough energy to overcome the pseudopotential barrier. The released ions will generally comprise precursor ions within a single one of the m/z ranges. Following release of these ions from the pseudopotential-based ion separation device, the individual precursor ion populations can undergo further ion manipulations and m/z analysis or analyses in Step606. In various alternative experimental situations, the analysis or analyses may occur in a multipole ion trap, a linear quadrupole mass analyzer, an electrostatic trap mass analyzer (such as an ORBITRAP™ mass analyzer or a Cassini trap mass analyzer), or a time-of-flight mass analyzer. In some cases, the ion manipulations might involve additional rounds of ion isolation, and still further manipulation. In some cases, the further ion manipulations and m/z analysis or analyses may employ additional ion traps, ion filters, or mass analyzers included within the same mass spectrometer system within which the preceding method steps are executed.

The exact form of the ion manipulations and analyses performed on the released ions in Step606will vary depending upon the type of application or experiment. For example, in a common form of ion manipulation, the released precursor ions are transmitted from the ion separation device to an ion fragmentation or reaction cell. These precursor ions may then be manipulated in the fragmentation or reaction cell in accordance with the general techniques of tandem mass spectrometry. For example, the released precursor ions may be fragmented or otherwise manipulated by controlled ion-ion reactions so as to generate product ions. Electron transfer dissociation is one type of ion/ion reaction. Proton transfer is another ion-ion reaction that could take place in such a reaction cell. The so-generated product ions are then mass analyzed in mass analyzer components of a mass spectrometer (Step606).

The fragmentation or reaction cell may have one of many known types that receive precursor ions and that generate product ions by fragmentation or reaction of the precursor ions. For example, in various embodiments, the cell may be of a type in which precursor ions are caused to collide with neutral gas molecules such that internal vibrational energy is imparted to the ions, ultimately leading to breakage of certain chemical bonds. Such cell types include fragmentation cells that operate according to the method of collision induced dissociation (CID) or higher-energy collisional dissociation (HCD). Alternatively, the ions may be caused to fragment in the cell by the process of surface-induced dissociation (SID). Alternatively, the cell may be a cell that causes fragmentation by electron capture dissociation (ECD), in which precursor ions are bombarded with electrons. Alternatively, the cell may be coupled to a light source, such as an ultraviolet (UV)-emitting or infrared (IR)-emitting laser that imparts photonic energy to the precursor ions that causes them to dissociate. All such examples of fragmentation/reaction cells, as well as others, are contemplated for use in conjunction with methods, apparatuses, and systems in accordance with the present teachings.

The fragmentation or reaction and mass analysis operations of Step606may optionally be accompanied by simultaneous execution of Step603aand, possibly, also Step602a, as indicated by dotted lines inFIG. 8. In the optional Step603a, the ion separation device may be replenished or augmented with one or more filtered sets of ions (each such set comprising ions within a one of the pre-determined m/z ranges) to replace or augment the ions released in the prior execution of Step606. Alternatively, Step603amay comprise the introduction into the ion separation device of ions of one or more m/z ranges that were not previously introduced into the ion separation device during an experiment in question. Such replenishment or introduction of a new set of ions will generally occur once the ion separation device has been emptied of all sets of ions and will generally be accompanied by ion filtering in Step602a.

After execution of the fragmentation or reaction and product-ion mass analyses of Step606, if there are additional trapped m/z ranges in the ion separation device (Step608), then execution of the method600returns to Step604at which point trapped ions within a different m/z range (with respect to the m/z range released just prior) are released into the ion fragmentation or reaction cell. The progression of selective releasing of different sets of ions, where each set corresponds to a different respective m/z range, may be better understood with reference toFIG. 5. With reference to bothFIG. 8andFIG. 5, assume that the selective filtering in Step602of the method600has caused sets of ions corresponding to just those ions corresponding to curves302,306and310to be accumulated in an ion separation device (Step603of the method600). Following the accumulation, the lens offset voltage (which is used to overcome an applied pseudopotential barrier) may be ramped downwards according to the values from listed right to left across the horizontal axis ofFIG. 5. The graph200shows that initial release of the ions corresponding to curve310will begin at an offset voltage of about −5.8 V and, further, that such ions will be essentially emptied from the ion separation device at an offset voltage of about −8.0 V. The graph further indicates that initial release of the ions corresponding to curve306will begin at an offset voltage of about −8.5 V and that such ions will be essentially fully emptied from the ion separation device at an offset voltage of about −10.0 V. Finally, the ions corresponding to curve302will begin to be released at about an offset voltage −10.5 V, and that these latter ions will be essentially fully emptied from the ion separation device at an offset voltage of about −13.0 V. The release of each such set of ions corresponds to a separate iteration or re-iteration of Step604ofFIG. 8.

Once the ion separation device has been emptied of all previously trapped sets of ions, it is determined, in Step610of the method600, if there are additional sample portions which are to be analyzed. Such different sample portions will generally correspond to different samples of a continuous stream of ions that is generated by an ion source in response to a continuous stream of fluid sample that is provided to the ion source. If a subsequent sample portion is to be analyzed (Step610), then execution of the method600returns to either Step601or Step602. A subsequent sample portion could include the same sets of ions that were generated in a previous sample portion or, otherwise, could include different sets of ions. If it is known or can be assumed that the subsequent sample portion merely includes the same sets of ions that were generated in a previous sample portion, the Step601might be bypassed. However, the ions could differ between iterations of Step602because of changing sample composition caused by fractionation in a chromatographic column. Even in the event that a subsequent sample portion includes exactly the same sets of ions as a prior sample portion (for example, if the composition of the sample stream has not changed), the analysis of the subsequent portion might be directed to different sets of ions than were analyzed in the analysis of the prior portion. For example, once again with reference toFIG. 5, if the sets of ions corresponding to curves302,306and310are accumulated in the prior iteration of Step602(and subsequently fragmented after accumulation in the following Step606) then the subsequent iteration of Step602may comprise accumulation of the sets of ions corresponding to curves304and308. Inspection of graph200inFIG. 5shows that choosing, in such fashion, which sets of ions are to be accumulated and analyzed in each iteration of the Steps602-610allows maximum discrimination of ion species.

The discussion included in this application is intended to serve as a basic description. The present invention is not intended to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Any patents, patent applications, patent application publications, or other literature mentioned herein are hereby incorporated by reference herein in their respective entirety as if fully set forth herein, except that, in the event of any conflict between the incorporated reference and the present specification, the language of the present specification will control.