Mass spectrometer with high duty cycle

Abstract

A method of mass spectrometry comprising: a) accumulating precursor ions in a first ion accumulator; b) performing a separation cycle including pulsing packets of precursor ions from the first ion accumulator to an ion separator and separating the precursor ions such that precursor ions having different values ​​of a first physicochemical property elute from the ion separator at different times; c) mass filtering the precursor ions eluting from the ion separator to transmit selected precursor ion species; and d) fragmenting or reacting the selected precursor ion species to produce a set of fragment or product ion species from the selected precursor ion species. e) accumulating the set of fragment or product ion species in a second ion accumulator; f) ejecting the set of fragment or product ion species from the second ion accumulator into a TOP mass analyzer, where operation of the TOP mass analyzer is synchronized with the ejection of ions from the second ion accumulator such that a plurality of different species of the set of fragment or product ion species are simultaneously pulsed by a pusher electrode into a time-of-flight region of the TOP mass analyzer; and g) repeating steps c)-f) at least once during a separation cycle, where the selected precursor ion species is different each time steps c)-f) are performed.

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims the priority and benefit of UK Patent Application No. 2204106.5 filed on March 23, 2022. The entire content of this application is incorporated herein by reference.

[0002] (Field of the Invention) The present invention generally relates to a mass spectrometer that pulses ions into a separation region to separate the ions according to their mass - to - charge ratio.

Background Art

[0003] In both data - dependent acquisition (DDA) and tandem (MS / MS) mass spectrometry, precursor ions are filtered such that only selected species of precursor ions pass downstream for analysis at any given time. Therefore, the mass spectrometer selects different species of precursor ions that pass downstream for analysis. This process continues until the desired species of precursor ions are analyzed. Generally, in order to analyze a sample as quickly as possible, it is desirable to sequentially select and transmit as many precursor ions per unit time as possible. However, in conventional instruments such as quadrupole - time - of - flight (QToF) mass spectrometers, simply increasing the speed at which precursor species are selected results in a linear decrease in the sensitivity of the detected ions. For example, if the analyzer settings are changed such that the number of precursor species passing through increases from 25 different precursors per second to 50 different precursors per second, this results in the number of ions in each fragment ion scan being halved.

[0004] It is desirable to increase the overall duty cycle of such instruments.

Summary of the Invention

[0005] A first aspect of this disclosure provides a method for mass spectrometry, the method being: a) A step of accumulating precursor ions in a first ion accumulator, b) A step of performing a separation cycle, which includes pulsing packets of precursor ions from a first ion accumulator to an ion separator, and separating the precursor ions such that precursor ions having different values ​​of the first physicochemical properties elute from the ion separator at different times. c) A step of mass filtering the precursor ions eluted from the ion separator to allow the selected precursor ion species to pass through, d) A step of fragmenting or reacting the selected precursor ion species to generate a set of fragments or product ion species from the selected precursor ion species, e) A step of accumulating a set of fragments or product ion species in a second ion accumulator, f) A step of releasing a set of fragment or product ion species from a second ion accumulator to a TOF mass spectrometer, wherein the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator, and multiple different species of the set of fragment or product ion species are simultaneously pulsed in the time-of-flight domain of the TOF mass spectrometer by pusher electrodes, g) A step in which steps c) to f) are repeated at least once during the separation cycle, wherein the selected precursor ion species is different each time steps c) to f) are performed. Includes.

[0006] The inventors have recognized that the timescale over which a second ion accumulator can accumulate fragment or product ions and release them for analysis in a TOF mass spectrometer is equivalent to the timescale over which any given precursor ion elutes from an upstream separator. Therefore, fragment or product ion species derived from a first precursor ion species eluting from the ion separator during a given separation cycle of the ion separator can be accumulated, captured, and released to the TOF mass spectrometer before the second precursor ion species elutes from the ion separator within the same separation cycle of the ion separator. This method provides a relatively high duty cycle because, while a selected precursor ion species is fragmented, other precursor ion species are not discarded but remain delayed within the ion separator. Furthermore, the synchronization between the second ion accumulator and the TOF mass spectrometer allows for the simultaneous analysis of multiple fragment or product ion species of the selected precursor, thus resulting in a relatively high duty cycle.

[0007] The ion separator may also be an ion mobility separator, and its physicochemical properties may be ion mobility.

[0008] For example, the ion separator may be an ion mobility separator configured to drive ions through a background gas placed therein. The ions may be driven through the background gas by an electric field to separate them according to their mobility. For example, the ions may be driven by applying a static DC potential gradient along the ion separator and / or by repeatedly moving the DC potential along the ion separator. The electric field may be positioned to bias the ions downstream (towards the TOF mass spectrometer) through the ion separator. Alternatively, the background gas may flow downstream, and the electric field may be configured to bias the ions upstream against the gas flow to separate them according to their mobility.

[0009] Other embodiments are conceivable in which the ion separator is a field asymmetric ion mobility separator (FAIMS) device or a differential mobility separator (DMS). Less preferably, the ion separator mass may be a mass-to-charge ratio separator, and its physicochemical properties may be mass-to-charge ratio.

[0010] Each time step c) is performed, only the selected precursor ion species may be allowed to permeate, while other precursor ion species may not.

[0011] Alternatively, each time step c) is performed, the mass filter may allow precursor ions having a limited range of mass-to-charge ratios to pass through, allowing multiple precursor ion species to pass through simultaneously, while precursor ions having a mass-to-charge ratio outside this limited range are filtered out. Step d) may then include simultaneously fragmenting or reacting the multiple permeated precursor ion species to generate a plurality of sets of fragment or product ion species therefrom. Step e) may include simultaneously accumulating the sets of fragment or product ion species in a second ion accumulator. Step f) may include simultaneously releasing the sets of fragment or product ion species from the second ion accumulator to a TOF mass spectrometer, wherein the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator, and a plurality of different species of the sets of fragment or product ion species are simultaneously pulsed in the time-of-flight domain of the TOF mass spectrometer by pusher electrodes. Step g) may include repeating steps c) through f) at least once during the separation cycle, so that the range of mass-to-charge ratio transmitted through the mass filter is different each time steps c) through f) are performed.

[0012] A voltage is applied to the electrodes of the mass filter to allow only a single precursor ion species or a limited range of mass-to-charge ratios, such as multiple precursor ion species, to pass through at any given time. According to step g) above, the voltage changes over time during the separation cycle, and each time steps c) through f) are performed, at least one different precursor ion species passes through the mass filter.

[0013] The mass filter may be a quadrupole mass filter, such as a quadrupole rod set mass filter or a quadrupole rod set mass filter segmented axially. However, it is intended that other types of mass filters may be used in this disclosure.

[0014] The fragment or product ion species may be biased into a second ion accumulator so that (i) fragment or product ion species derived from the same precursor ion species reach the second ion accumulator over substantially the same period of time and are accumulated in the second ion accumulator for at least part of this period, and / or (ii) fragment or product ion species derived from precursor species permeated by the mass filter at different times reach the second ion accumulator over their respective different periods.

[0015] Each time the sequence from step c) to e) is performed, fragments or product ion species may be accumulated in the second ion accumulator for the same or correlated period of time that their precursor ion species permeate through the mass filter.

[0016] For example, fragments or product ion species may accumulate in the second ion accumulator for a duration substantially the same as, or correlated with, the duration between the start time when the voltage applied to the mass filter is switched to initiate the permeation of precursor ion species and the end time when the voltage applied to the mass filter to terminate the permeation of precursor ion species.

[0017] The second ion accumulator may therefore be synchronized with the mass filter so that substantially all fragment or product ions derived from any given precursor ion species are accumulated in the second ion accumulator. This, along with the manner in which the second ion accumulator and the TOF mass spectrometer are synchronized, allows for a relatively high duty cycle of the analyzer.

[0018] Furthermore, it is intended that each time the sequence from step c) to e) is performed, fragment or product ion species may accumulate in the second ion accumulator for a time shorter than the time it takes for their precursor ion species to pass through the mass filter. For example, fragment or product ion species may accumulate in the second ion accumulator for a time shorter than the duration between the start time when the voltage applied to the mass filter is switched to initiate the permeation of the precursor ion species and the end time when the voltage applied to the mass filter to terminate the permeation of the precursor ion species.

[0019] Optionally, each time the sequence from step c) to e) is performed, only the fragment or product ion species derived from the selected precursor ion species, and some ions of the optionally selected precursor ion species itself, are accumulated in the second ion accumulator, while other precursor ion species and the fragment or product ions derived therefrom are not accumulated in the second ion accumulator.

[0020] Therefore, embodiments of the present disclosure do not simply capture fragment or product ions and release them to a TOF mass spectrometer in order to improve the duty cycle. Rather, the timescale on which fragment or product ions accumulate is set to match the timescale on which their precursor ion species permeate through the mass filter. The timescale on which the precursor ion species permeate through the mass filter may then be set to match the timescale on which the precursor ion species elute from the separator. Thus, embodiments of the present disclosure provide a very high duty cycle.

[0021] Therefore, each time the sequence of steps b) and c) is performed, the mass filter may be controlled to allow the symmetric precursor ion species to pass through only for a time that is the same as, correlated with, or shorter than, the time it takes for the precursor ion species to elute from the ion separator.

[0022] For example, the mass filter may be controlled to allow symmetric precursor species to pass through for a duration substantially equal to, correlated with, or shorter than, the duration between the start time when precursor ions begin to elute from the separator and the end time when precursor ions stop eluting from the ion separator.

[0023] Step f) may include releasing fragment or product ion species from the second ion accumulator in reverse order of mass-to-charge ratio, i.e., starting with ions with relatively high mass-to-charge ratios and gradually releasing ions with progressively lower mass-to-charge ratios, so that different fragment or product ion species reach the pusher simultaneously and are simultaneously pulsed into the time-of-flight domain of the TOF mass spectrometer by the pusher electrode.

[0024] A transfer region may be provided between the second ion accumulator and the TOF mass spectrometer, and the pressure in the transfer region and the energy at which ions are released from the second ion accumulator are such that fragments or product ions having a relatively low mass-to-charge ratio can catch up with fragments or product ions having a higher mass-to-charge ratio, so that the ions reach the pusher simultaneously and are pulsed simultaneously by the pusher electrode into the flight time region of the TOF mass spectrometer.

[0025] Optionally, each time step f) is performed, substantially all of the fragment or product ion species released from the second ion accumulator reach the pusher substantially simultaneously and are pulsed simultaneously by the pusher electrode into the flight time region of the TOF mass spectrometer.

[0026] Step f) may include releasing substantially all of the fragment or product ion species from the second ion accumulator substantially simultaneously. A transfer region is provided between the second ion accumulator and the TOF mass spectrometer, and the ions are separated within the transfer region according to the second physicochemical property, such that the fragment or product ion species arrive at the TOF mass spectrometer at a time that depends on their second physicochemical property value, and the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator such that fragment or product ion species having a selected range of values of the second physicochemical property value are pulsed simultaneously by the pusher electrode into the flight time region of the TOF mass spectrometer.

[0027] The method may include, in a mass spectrometer performing the method, receiving an input signal representing a selected range of values of the second physicochemical property, and the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator such that fragment or product ion species having a selected range of values of the second physicochemical property value are pulsed simultaneously by the pusher electrode into the flight time region of the TOF mass spectrometer.

[0028] The second physicochemical property may be a mass-to-charge ratio, which allows ions to separate in the transport region according to that ratio. For example, the operating conditions in the transport region may be such that fragment or product ion species with a relatively low mass-to-charge ratio move faster than fragment or product ion species with a higher mass-to-charge ratio. For example, the product of the pressure P in the transport region and the length L of the ion flight path through the transport region (i.e., P × L) may be such that ions do not substantially collide with background gas molecules in the transport region.

[0029] Alternatively, the second physicochemical property may be ion mobility, thereby separating ions in a transport region according to their ion mobility. For example, the transport region may function as an ion mobility separator configured to drive ions through a background gas placed within it. The ions may be driven through the background gas by an electric field to separate according to their mobility. For example, the ions may be driven by applying a static DC potential gradient along the transport region and / or by repeatedly moving the DC potential along the transport region. The electric field may be configured to bias the ions downstream through the transport region. Alternatively, the background gas may flow downstream, and the electric field may be configured to bias the ions upstream against the gas flow, thereby separating the ions according to their mobility.

[0030] Steps a) through g) may be repeated during a single experimental run. To avoid misunderstanding, the term "during a single experimental run" means during the continuous analysis of the analyte. For example, steps a) through g) may be repeated while ions are generated substantially continuously (e.g., substantially continuous or pulsed) from the analyte and / or during a single liquid chromatography or gas chromatography run.

[0031] This disclosure also provides a mass spectrometer configured to perform the methods described herein.

[0032] Therefore, a first aspect of this disclosure also provides a mass spectrometer, the mass spectrometer being, The first ion accumulator, Ion separator and, Mass filter and, Fragmentation or reaction device, A second ion accumulator, A TOF mass spectrometer having a time-of-flight domain and a pusher electrode, A control circuit that controls a mass spectrometer, a) Precursor ions are accumulated in the first ion accumulator, b) Perform a separation cycle which includes pulsing packets of precursor ions from a first ion accumulator to an ion separator, and separating the precursor ions within the ion separator such that precursor ions having different values ​​of the first physicochemical properties elute from the ion separator at different times. c) Using a mass filter, the precursor ions eluting from the ion separator are mass filtered to allow the selected precursor ion species to pass through. d) The selected precursor ion species is fragmented or reacted within a reaction device to generate a set of fragments or product ion species therefrom. e) A set of fragments or product ion species is accumulated in a second ion accumulator. f) A set of fragment or product ion species is released from a second ion accumulator to a TOF mass spectrometer, and the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator so that multiple different species of the fragment or product ion species set are simultaneously pulsed in the time-of-flight domain of the TOF mass spectrometer by pusher electrodes. g) Repeat steps c) to f) at least once during the separation cycle, and the mass filter comprises a control circuit configured to control the selected precursor ion species so that it is different each time steps c) to f) are performed.

[0033] The control circuit may be configured to control the mass spectrometer such that step f) includes releasing fragment or product ion species from the second ion accumulator in reverse order of mass-to-charge ratio, i.e., starting with ions with relatively high mass-to-charge ratios and gradually releasing ions with progressively lower mass-to-charge ratios, so that different fragment or product ion species reach the pusher simultaneously and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

[0034] A transfer region may be provided between the second ion accumulator and the TOF mass spectrometer, and the mass spectrometer is configured to maintain pressure within the transfer region and to release ions from the second ion accumulator with energy such that fragments or product ions with relatively low mass-to-charge ratios catch up with fragments or product ions with higher mass-to-charge ratios, so that the ions reach the pusher simultaneously and are simultaneously pulsed into the time-of-flight domain of the TOF mass spectrometer by the pusher electrode.

[0035] The mass spectrometer may include a transfer region between a second ion accumulator and a TOF mass spectrometer, and the control circuit is configured to control the mass spectrometer such that step f) includes substantially simultaneously releasing all fragment or product ion species from the second ion accumulator into the transfer region, separating the fragment or product ion species in the transfer region according to a second physicochemical property, and arriving at the TOF mass spectrometer in a time dependent on their second physicochemical property values, the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator so that fragment or product ion species having a selected range of values ​​of the second physicochemical property values ​​are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

[0036] The mass spectrometer may include a user interface configured to input a selected range of values ​​for a second physicochemical property, and the analyzer is configured such that a control circuit controls the operation of the TOF mass spectrometer based on the selected range of values, synchronizing the operation of the TOF mass spectrometer with the release of ions from a second ion accumulator, so that fragment or product ion species having the selected range of values ​​for the second physicochemical property are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

[0037] The mass spectrometer includes an ion source. The mass spectrometer may also include a liquid or gas chromatography separator for separating the analyte upstream of the ion source.

[0038] The mass spectrometer may be configured to repeat steps a) to g) during a single experimental run, for example, while ions are being generated substantially continuously (e.g., substantially continuously or in a pulsed manner) by an ion source, and / or during a single liquid chromatography or gas chromatography separation run.

[0039] It is conceivable that the second ion accumulator can be omitted. In these embodiments, the second ion accumulator is not synchronized with the TOF mass spectrometer to control the fragment or product ions (from any given precursor ion species) that are simultaneously mass-analyzed by the TOF mass spectrometer. Rather, this is done by the operation of the TOF mass spectrometer, which is synchronized with the time in which the fragment or product ions are generated. Thus, the time for which the mass filter permeates the selected precursor ion species into the fragmentation or reaction region is synchronized with the TOF mass spectrometer to control the fragment or product ions from the selected precursor ion species that are simultaneously mass-analyzed.

[0040] Therefore, in a second aspect, the present disclosure provides a method for mass spectrometry, the method being: a) A step of accumulating precursor ions in a first ion accumulator, b) A step of performing a separation cycle, which includes pulsing a packet of precursor ions entering from a first ion accumulator into an ion separator, and separating the precursor ions such that precursor ions having different values ​​of first physicochemical properties elute from the ion separator at different times. c) A step of switching the mass filter that mass filters the precursor ions eluting from the ion separator to start the permeation of the selected precursor ion species, d) A step of fragmenting or reacting the selected precursor ion species to generate a set of fragments or product ion species from the selected precursor ion species, e) A step of transferring a set of fragments or product ion species through a transfer region to a TOF mass spectrometer having a pusher electrode and a time-of-flight region, wherein the ions are separated in the transfer region according to a second physicochemical property, the fragments or product ion species arrive in the TOF mass spectrometer at a time dependent on the second physicochemical property values, and the operation of the TOF mass spectrometer is synchronized with the time at which the mass filter is switched, so that fragments or product ion species having a selected range of values ​​of the second physicochemical property arrive substantially simultaneously at the pusher electrode, and the pusher electrode initiates the permeation of selected precursor ion species so that they are simultaneously pulsed into the time-of-flight region of the TOF mass spectrometer; f) a step which is repeated at least once during the separation cycle, wherein the selected precursor ion species is different each time steps c) to e) are performed.

[0041] A fragment or product ion species of any given selected precursor ion species undergoes a separation cycle in the transport region, which is triggered based on the time it takes for the mass filter to switch over to begin permeating the precursor ion species.

[0042] The method may include, in a mass spectrometer performing the method, the steps of: receiving an input signal representing a selected range of values ​​for a second physicochemical property; and then controlling the operation of the TOF mass spectrometer so that fragments or product ion species having a selected range of values ​​for the second physicochemical property reach the pusher electrode substantially simultaneously and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

[0043] Fragments or product ions of any given precursor ion species may reach the TOF mass spectrometer at a time correlated with the time when the mass filter begins to permeate those precursor ion species. For example, selected precursor ion species permeated in step c) do not need to be axially captured before fragmentation or reaction in step d). Fragments or product ions generated in step d) do not need to be axially captured before reaching the TOF mass spectrometer.

[0044] A method according to a second aspect of the present disclosure may have any of the features described in relation to the first aspect of the present disclosure, except that it does not require the use of a second ion accumulator.

[0045] For example, each time step c) is performed, only the selected precursor ion species may be allowed to pass through, while other precursor ion species are not. Alternatively, each time step c) is performed, the mass filter may allow precursor ions having a limited range of mass-to-charge ratios to pass through, thereby allowing multiple precursor ion species to pass through simultaneously, while precursor ions having a mass-to-charge ratio outside this limited range are filtered out. Step d) may then include the step of simultaneously fragmenting or reacting the multiple permeated precursor ion species to produce a plurality of sets of fragment or product ion species therefrom. Step e) may include the step of transferring the plurality of sets of fragment or product ion species through a transfer region to a TOF mass spectrometer, the operation of which is synchronized with the time at which the mass filter is switched to begin allowing precursor ion species to pass through so that fragment or product ion species having a selected range of values ​​for a second physicochemical property reach the pusher electrode substantially simultaneously and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer. Step g) may include repeating steps c) to f) at least once during the separation cycle, so that the range of mass-to-charge ratios transmitted through the mass filter is different each time steps c) to f) are performed.

[0046] The Disclosure also provides a mass spectrometer configured to perform the methods described in relation to the second aspect of the Disclosure.

[0047] Therefore, this disclosure provides a mass spectrometer, which is a mass spectrometer, The first ion accumulator, Ion separator and, Mass filter and, Fragmentation or reaction device, A TOF mass spectrometer having a time-of-flight domain and a pusher electrode, A transfer region between the fragmentation or reaction device and the TOF mass spectrometer, A control circuit that controls a mass spectrometer, a) Precursor ions are accumulated in the first ion accumulator, b) Perform a separation cycle which includes pulsing packets of precursor ions from a first ion accumulator to an ion separator, and separating the precursor ions within the ion separator such that precursor ions having different values ​​of the first physicochemical properties elute from the ion separator at different times. c) Switch the mass filter that mass filters the precursor ions eluting from the ion separator, and start the permeation of the selected precursor ion species. d) The selected precursor ion species is fragmented or reacted within a reaction device to generate a set of fragments or product ion species therefrom. e) A set of fragment or product ion species is transferred to a TOF mass spectrometer through a transfer region, where the ions are separated in the transfer region according to a second physicochemical property, the fragment or product ion species arrive in the TOF mass spectrometer at a time dependent on their second physicochemical property values, and the operation of the TOF mass spectrometer, synchronized with the time the mass filter is switched, initiates the transmission of selected precursor ion species so that fragment or product ion species having a selected range of values ​​of the second physicochemical property arrive substantially simultaneously at the pusher electrode, and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer. f) a control circuit is configured to repeat steps c) to e) at least once during the separation cycle, such that the selected precursor ion species is different each time steps c) to e) are performed. [Brief explanation of the drawing]

[0048] Hereafter, various embodiments of the present invention will be described only as examples, with reference to the accompanying drawings. [Figure 1] A schematic diagram of an embodiment of the mass spectrometer according to this disclosure is shown. [Figure 2]This illustrates an example of how a mass filter can be controlled as a function of time to selectively transmit different precursor ion species over different time periods. [Figure 3] The plot is the same as in Figure 2, except that the fragment or product ion species are superimposed. [Figure 4] This shows an example of how fragment or product ions can be mass-analyzed using a time-of-flight (TOF) mass spectrometer. [Figure 5] Figure 1 shows how the mass spectrometer may operate according to one embodiment of the present disclosure. [Figure 6] Figure 5 illustrates how the process described in relation to it can be repeated multiple times for different precursor ion species eluting from the ion separator during the same separation cycle. [Figure 7] Figure 1 shows how the mass spectrometer may operate according to another embodiment of this disclosure. [Figure 8] Figure 7 illustrates how the process described in relation to it can be repeated multiple times for different precursor ion species eluting from the ion separator during the same separation cycle. [Modes for carrying out the invention]

[0049] Figure 1 shows a schematic diagram of an embodiment of the mass spectrometer according to the present disclosure. The analyzer comprises an ion source 1, a first ion accumulator 2, an ion separator 3, a mass filter 4, a fragmentation or reaction device 5, a second ion accumulator 6, a transfer region 7, and a time-of-flight (TOF) mass spectrometer 8. It should be understood that other ion optics devices, such as one or more RF ion guides, electrostatic lenses, or impact-cooled RF ion guides, are also provided within the analyzer and are configured to bias ions through them by using an axial electric field or by moving a potential barrier along them.

[0050] During use, ions are supplied from ion source 1, which may be an ion source that generates ions from the analyte sample, or an ion optical device that supplies ions. Ions from ion source 1 may be accumulated in a first ion accumulator 2 and then released into separator 3 as ion packets. Ion accumulator 2 may accumulate ions and periodically pulse them into separator 3. Accumulator 2 may be configured to allow ions to enter accumulator 2 continuously, or it may prevent ions from entering accumulator 2 when ion packets are being pulsed into separator 3. In any case, accumulator 2 accumulates ions from ion source 1 in between the times when ion packets are being pulsed into separator 3.

[0051] The separator 3 is controlled to separate each packet of ions received from the accumulator 2 according to one or more physicochemical properties. For example, the separator 3 may separate ions according to ion mobility, mass-to-charge ratio, or a combination of both of these physicochemical properties. The separated ions then pass from the separator 3 to the mass filter 4. The mass filter 4 is controlled to allow only a single mass-to-charge ratio (or a limited range of mass-to-charge ratios) to pass through at any given time, and as ions separated from the ion packets elute from the separator 3, the single mass-to-charge ratio (or limited range) changes over time.

[0052] Figure 2 illustrates an example of how the mass filter 4 can be controlled as a function of time as ions separated from the ion packet elute from the separator 3. More specifically, Figure 2 shows a plot of the mass-to-charge ratios transmitted by the mass filter 4 as a function of time since the ion packet was pulsed into the separator 3. In this example, the mass filter 4 is controlled to transmit only ions with a first, relatively low mass-to-charge ratio at a first time. The mass filter is then controlled to transmit only ions with a second, higher mass-to-charge ratio at a second, later time. The mass filter is then controlled to transmit only ions with a third, higher mass-to-charge ratio at a third, later time. The mass filter is then controlled to transmit only ions with a fourth, lower mass-to-charge ratio at a fourth, later time. The mass filter is then controlled to transmit only ions with a fifth, higher mass-to-charge ratio at a fifth, later time.

[0053] The mass filter 4 may be, for example, a resolved quadrupole mass filter, but it is intended that other types of mass filters may be used. It will also be understood that the mass filter 4 may be controlled to transmit different mass-to-charge ratios at different times in ways other than those shown in the example in Figure 2.

[0054] After a delay time, another packet of ions may be pulsed from the ion accumulator 2 into the separator 3, and the above process may be repeated to separate and filter the ions in this new packet. The delay time may be the time it takes for all of the target ions in one ion packet (i.e., the one set to pass through the mass filter 4 at a certain point) to pass through the separator 3 and reach the filter 4, but may be set to a shorter or longer delay time if desired. Any desired number of ion packets may be separated sequentially and then filtered in the manner described above.

[0055] The separated ions that have been passed through filter 4 (i.e., not removed by the filter) are transferred to a fragmentation or reaction device 5. The fragmentation or reaction device 5 may be a collision-induced dissociation (CID) device, an electron capture dissociation (ECD) device, an electron transfer dissociation (ETD) device, a surface-induced dissociation (SID) device, or a device that fragments ions according to any other technique. For example, the device may fragment the ions by reacting them with a reagent ion or molecule, or the device may be used to react ions with a reagent ion or molecule to form other types of product ions, such as adduct ions.

[0056] Figure 3 shows the same plot as Figure 2, except that the fragment or product ion species generated from the precursor ions are superimposed. In the illustrated example, the first precursor species, transmitted through mass filter 4, is shown as the first dark ellipse, and fragments or reacts to produce two types of fragments or product ions, which are shown by lighter colored ellipses above and below the first dark ellipse. In this example, one of the fragment or product ion species has a higher mass-to-charge ratio than the first precursor species, and the other has a lower mass-to-charge ratio. In this example, each of the second to fifth precursor species (represented by dark ellipses) fragments or reacts to produce three types of fragments or product ions (represented by light ellipses).

[0057] It should be understood that different precursor ions arrive at the fragmentation or reaction device 5 at different times due to their separation in separator 3, and therefore different precursor ions fragment or react at different times, producing their fragments or product ions at different times. Figure 3 shows that fragment or product ion species are produced over a time that precisely matches the time their precursor ion species are permeated through filter 4, however this may not be the case, and it should be understood that the time at which any given fragment or product ion species is produced may differ from the time that the precursor ion species was permeated through filter 4, due to, for example, the time it takes for the precursor ion species to permeate to (or through) the fragmentation or reaction device 5, and / or the time it takes for the precursor species to undergo fragmentation or reaction within device 5.

[0058] The separation of precursor ion species induced by separator 3 may be preserved among the fragments or product ion species derived from these precursor ion species. In particular, different precursor ion species arrive at the fragmentation or reaction device 5 at different times, and therefore, their respective fragments or product ion species are produced at corresponding different times. The fragments or product ion species may be biased outward through the fragmentation or reaction device 5 so that fragments or product ion species derived from different precursor species arrive at the second ion accumulator 6 at different different times. Thus, fragments or product ion species derived from the same precursor species arrive at the second ion accumulator 6 over substantially the same period of time and are accumulated in the second ion accumulator.

[0059] The second ion accumulator 6 accumulates ions and periodically pulses them into the transport region 7. The second ion accumulator 6 may be configured to allow ions to enter it continuously, or it may prevent ions from entering the accumulator 6 when packets of ions are being pulsed into the transport region 7. In any case, the accumulator accumulates ions from the fragmentation or reaction device 5 during the periods between the times when packets of ions are pulsed from the accumulator 6 into the transport region 6. However, the second ion accumulator 6 may be omitted, and the fragmentation or reaction device 5 may instead pulse ions into the transport region 7 by, for example, providing an ion gate between the fragmentation or reaction device 5 and the transport region 7. Alternatively, the second ion accumulator 6 and the fragmentation or reaction device 5 may be two parts of the same device.

[0060] Fragment or product ions are transferred through a transfer region 7 to a TOF mass spectrometer 8. As is well known in the art, the TOF mass spectrometer 8 has a pusher that pulses ion packets into the time-of-flight domain (e.g., a field-free region) and toward an ion detector. As the ions pass through the time-of-flight domain, they separate according to their mass-to-charge ratio and then collide with the ion detector. Thus, the separated ions reach the ion detector at different times, and the time it takes for an ion to reach the detector is related to its mass-to-charge ratio. The mass-to-charge ratio of any given ion can be determined from the duration between the time the ion was pulsed into the time-of-flight domain and the time it was detected by the ion detector. Thus, the TOF mass spectrometer 8 can acquire data from the signals detected by the detector, determine the mass-to-charge ratio of the ions pulsed into the mass spectrometer 8 and their intensities, and form a mass spectrum.

[0061] Ions may be reflected by one or more ion mirrors between the pusher and the ion detector to provide a relatively long flight path through the time-of-flight domain. This allows ions with different mass-to-charge ratios to be separated to a greater extent as they travel through the time-of-flight domain, and thus provides the TOF mass spectrometer 8 with higher mass resolution. Therefore, the TOF mass spectrometer 8 may be a multi-reflecting TOF (MRTOF) mass spectrometer that uses ion mirrors to reflect the ions multiple times as they drift from the pusher towards the ion detector.

[0062] Fragments or product ions derived from any given species of precursor ions reach the TOF mass spectrometer 8 within a limited period of time related to the time it takes for those precursor species to leave the separator 3.

[0063] Figure 4 shows an example of how fragment or product ions are mass-analyzed by a TOF mass spectrometer 8 according to a conventional (uncoded high-frequency pulsing) approach. In this example, three fragment or product ions with three different mass-to-charge ratios are generated from a single precursor species in the fragmentation or reaction device 5 and then, over the same period, are sent from a second ion accumulator 6 through the transfer region 7 to the TOF mass spectrometer 8. The three fragment or product ions reach the pusher of the TOF mass spectrometer 8 over a time window 9 shown in Figure 4. The TOF mass spectrometer 8 periodically pulses the pusher, and therefore the pusher repeatedly samples the fragment or product ions through the time window 9 and mass-analyzes them. The time during which the TOF pusher is pulsed is indicated by a vertical line 10 on the x-axis. These multiple pulses 10 of the TOF pusher during duration 9 cause multiple TOF mass spectra to be generated for the fragment or product ions derived from each precursor ion.

[0064] Figure 4 shows only fragment or product ions from one of the precursor species that permeate through filter 4, but it should be understood that fragment or product ions derived from other precursor ion species that permeate through filter 4 (at later or earlier times for each of the above species) will also be mass-analyzed in the TOF mass spectrometer 8 in the corresponding manner (at later or earlier times). This conventional data acquisition method has proven useful because, for example, it allows for accurate measurements in the separation time domain of separator 3, and thus ultimately enables the determination of physicochemical properties such as ion mobility and collision cross-section. This approach also allows processing software to group together fragment or product ions that are common to the same precursor species.

[0065] However, the approach shown in Figure 4 will provide the TOF mass spectrometer 8 with a relatively low sampling duty cycle. This is because a relatively long duration is provided between any given pusher pulse 10 and the next pusher pulse, which allows time for the ion with the heaviest mass-to-charge ratio pushed by the given pusher pulse to arrive at the ion detector of the TOF spectrometer 8, so that another packet of ions is pushed toward the ion detector by the next pusher pulse. This means that the duration between pusher pulses 10 is greater than or equal to the time of flight from the pusher to the detector of the largest mass-to-charge ratio to be detected. Thus, the sampling duty cycle decreases with respect to the square root of the largest mass-to-charge ratio to be detected. The TOF sampling duty cycle with such a technique is typically in the range of 0.1% to 50%. These values ​​are typical maximum duty cycles.

[0066] To improve the overall experimental duty cycle of the analyzer, ion transfer between the various components of the analyzer is synchronized.

[0067] Figure 5 shows how the mass spectrometer of Figure 1 may operate according to embodiments of the present disclosure. The upper part of Figure 5 schematically shows a portion of the analyzer of Figure 1, and the lower part of Figure 5 shows plots illustrating how precursor species of ions are analyzed in the analyzer. The plots show examples of mass-to-charge ratios (y-axis) of ions present in different regions shown in the analyzer, i.e., at different times (x-axis).

[0068] As described above in relation to Figure 1, the separator 3 performs a separation cycle in which different ion species are pulsed from the first accumulator 2 to the separator 3, where they are separated according to their physicochemical properties, and then eluted at different times according to their physicochemical property values. The ions eluted from the separator 3 are received by the mass filter 4. At any given time during the separation cycle of the ion separator 3, the mass filter 4 may be configured to allow only a selected mass-to-charge ratio, or a selected range of mass-to-charge ratios, to pass through, for example, only a single precursor ion species. This is shown in Figure 5 by a dark ellipse below the mass filter 4, representing a first precursor ion species having a specific mass-to-charge ratio that is passed through the mass filter over a period of time. The precursor species is then passed through to the fragmentation or reaction device 5, where it fragments or reacts to form fragments or product ions. This process is shown in Figure 5 by six ellipses arranged in a row below the fragmentation or reaction device 5. The dark ellipses represent the precursor species, and the five lighter ellipses represent different types of fragments or product ions produced from the precursor species. It should be understood that fewer or more different types of fragments or product ions may be produced. Also, some of the precursor ions may or may not be present after fragmentation or the reaction.

[0069] Next, the fragment or product ions are transferred to a second ion accumulator 6 during the ion accumulation period. After this ion accumulation period, different types of fragment or product ions are released from the second ion accumulator 6 into the transfer region 7 at different times. More specifically, the fragment or product ions are released from the ion accumulator 6 in reverse order of mass-to-charge ratio, i.e., the fragment or product ion species with the highest mass-to-charge ratio are released first, followed by fragment or product ion species with progressively lower mass-to-charge ratios in sequence. This is illustrated in Figure 5 by the five fragment or product species shown below the transfer region 7. The spacing of the five fragment or product ion species along the time axis indicates that the fragment or product ion species with the highest mass-to-charge ratio is released first, followed by fragment or product ion species with progressively lower mass-to-charge ratios, each released at progressively later times.

[0070] The method by which fragment or product ion species are released from the second ion accumulator 6 and the method by which the transfer region 7 is configured are selected so that at least some of the different fragment or product ion species arrive at the TOF mass spectrometer 8 substantially simultaneously. This is illustrated in Figure 5 by the row of five fragment or product ion species shown below the TOF mass spectrometer 8. For example, different fragment or product species with substantially the same energy may be released from the ion accumulator 6 so that there are relatively few collisions between the ions and the background gas as the ions move to the TOF mass spectrometer 8, and the transfer region 7 may be maintained at a relatively low pressure. Under these conditions, it should be understood that the transfer region 7 operates as a time-of-flight domain where ions with a relatively low mass-to-charge ratio move faster than ions with a higher mass-to-charge ratio. Thus, fragment or product ion species with a relatively low mass-to-charge ratio, released from the ion accumulator 6 after heavier (i.e., higher mass-to-charge ratio) fragment or product ion species, catch up with the heavier fragment or product ion species and arrive at the TOF mass spectrometer 8 substantially simultaneously with them. The pusher of the TOF mass spectrometer 8 is synchronized with the release of ions from the ion accumulator 6 and is designed to pulse the pusher when multiple different types of fragment or product ions reach the pusher of the TOF mass spectrometer 8 simultaneously. Thus, the TOF mass spectrometer is provided with a relatively high duty cycle.

[0071] The second ion accumulator 6 may be a mass-selective ion trap. The ion trap may be a 3D quadrupole field ion trap or a linear ion trap. Ions may be emitted from the ion trap into the transfer region 7 and toward the TOF mass spectrometer 8 in reverse order of mass-to-charge ratio. This can be achieved in various ways, as is known in the art. For example, the RF voltage and / or DC voltage applied to the electrodes of the ion trap may be changed over time (e.g., scanned) to sequentially destabilize ions in the ion trap as their mass-to-charge ratio decreases, and thus emit them into the transfer region 7 and toward the TOF mass spectrometer 8. Alternatively or additionally, an AC voltage may be applied to the electrodes of the ion trap to resonantly excite ions of a specific mass-to-charge ratio and emit them from the ion trap into the transfer region 7 and toward the TOF mass spectrometer 8. The frequency of this AC voltage may be changed over time (e.g., scanned) to sequentially emit ions with decreasing mass-to-charge ratios. For example, the accumulator 6 may mass-selectively discharge ions in order of decreasing mass-to-charge ratio, as described in U.S. Patent No. 6,770,872, which is incorporated herein by reference.

[0072] The inventors have recognized that the timescale over which the second ion accumulator 6 can accumulate fragment or product ions and release them in the reverse order of mass-to-charge ratio is equivalent to the timescale over which any given precursor ion elutes from the upstream separator 3. For example, in several embodiments, the second ion accumulator 6 accumulates and releases fragment or product ion species from any given precursor ion species over a period of about 1 ms, and this period of about 1 ms is approximately the same as the period over which the precursor ion species elutes from the separator 3. Thus, fragment or product ion species derived from a first precursor ion species that elutes from the ion separator 3 during a given separation cycle of the separator 3 can be accumulated, captured, and released (in the reverse order of mass-to-charge ratio) before the second precursor ion species elutes from the ion separator 3 within the same separation cycle of the separator 3. This will be explained in more detail with reference to Figure 6. Therefore, the mass spectrometer has a relatively high duty cycle because precursor ions are fragmented and analyzed, while other precursor ions remain delayed in the separator rather than being discarded.

[0073] Figure 6 illustrates how the process described in relation to Figure 5 can be repeated multiple times for different precursor ion species eluting from separator 3 during the same separation cycle.

[0074] The lower plot in Figure 6 corresponds to the process described in relation to Figure 5, in which the first precursor ion species eluting from separator 3 is fragmented or reacted, and the resulting fragments or product ions accumulate in accumulator 6 and are then released to TOF mass spectrometer 8 in the reverse order of mass-to-charge ratio.

[0075] The central plot in Figure 6 shows the same process being performed on a second precursor ion species, which elutes from separator 3 earlier, during the same separator separation cycle as the first precursor ion species. More specifically, according to the central plot in Figure 6, the mass filter 4 is set to allow the second precursor ion species, represented by the dark ellipse in the central plot, to pass through. The second precursor ion species then passes through to the fragmentation or reaction device 5, where it fragments or reacts to form fragment or product ions. This is indicated by five lighter ellipses arranged in a column with the dark ellipse, but it should be understood that fewer or more different types of fragment or product ions may be produced. The fragment or product ions derived from the second precursor ion species are then transferred to the second ion accumulator 6, where they are released in reverse order of mass-to-charge ratio, so that at least some of the different fragment or product ion species reach the TOF mass spectrometer 8 substantially simultaneously.

[0076] The upper plot of Figure 6 shows the same process performed on a third precursor ion species, which elutes from separator 3 earlier, during the same separator separation cycle as the first and second precursor ion species. More specifically, according to the upper plot of Figure 6, the mass filter 4 is set to allow the third precursor ion species, represented by the dark ellipse, to pass through. The third precursor ion species then passes through to the fragmentation or reaction device 5, where it fragments or reacts to form fragment or product ions. This is indicated by five lighter ellipses arranged in a column with the dark ellipse, but it should be understood that fewer or more than five different types of fragment or product ions may be produced. The fragment or product ions are then transferred to the second ion accumulator 6, from which they are released in reverse order of mass-to-charge ratio, so that at least some of the different fragment or product ion species reach the TOF mass spectrometer 8 substantially simultaneously.

[0077] The mass spectrometer may be configured to allow only fragments or product ions derived from any given precursor ion species to enter the accumulator 6 after all fragments or product ions derived from previously analyzed precursor ion species have been discharged from the accumulator. This can be achieved, for example, by controlling the time at which the mass filter 4 is switched to allow the given precursor ion species to pass through, to be at or after the time when the accumulator 6 has released the lightest mass-to-charge ratio fragment or product ion derived from the preceding precursor ion species. Alternatively, an ion gate controlled by the mass spectrometer can be provided upstream of the accumulator 6 to prevent ions from entering the accumulator until the accumulator 6 is controlled to release all fragments or precursor ions in the reverse order of mass-to-charge ratio.

[0078] Although it is shown that three precursor ion species are analyzed during a single separation cycle of separator 3, it is intended that only two precursor ion species, or more than three precursor ion species, may be analyzed sequentially in the manner described during a single separation cycle.

[0079] Furthermore, although this method has so far been described only in the context of separator 3 performing a single separation cycle, it should be understood that multiple separation cycles may be performed during the same experiment, and the above method may be repeated for each of these separation cycles during the experiment.

[0080] The above method of synchronizing ion transfer between various components of the analyzer improves the overall experimental duty cycle of the analyzer. For example, the mass filter 4 is synchronized with the ion separator 3 so that during a single separation cycle of the separator 3, the mass filter 4 switches the permeation of different precursor ion species at different times. The precursor ions permeated by the mass filter 4 are fragmented or reacted in the fragmentation or reaction device 5 and then passed to the second ion accumulator 6. The second ion accumulator 6 is synchronized with the mass filter 4 (and therefore with the ion separator 3), and during a single separation cycle of the separator 3, (i) a first set of fragments or product ions derived from a first precursor ion species, which elutes from the separator 3 and permeates through the mass filter 4, accumulates in the second ion accumulator 6 and is then released into the transport region 7, and (ii) a second set of fragments or product ions derived from a second precursor ion species, which elutes from the separator 3 and permeates through the mass filter 4, accumulates in the second ion accumulator 6 and is then released into the transport region 7. Thus, the mass spectrometer has a relatively high duty cycle because precursor ions are fragmented and analyzed while other precursor ions remain delayed in the separator 3 rather than being discarded. Furthermore, the TOF mass spectrometer 8 may be configured such that its pusher is pulsed in synchronization with the release of ions from the second ion accumulator 6, so that multiple fragments or product ion species from the same precursor arrive at the pusher simultaneously. Thus, the TOF mass spectrometer is provided with a relatively high duty cycle.

[0081] Figure 7 illustrates another method according to this disclosure, which is the same as described above in relation to Figures 5 and 6, up to the point when the fragment or product ions are released from the second ion accumulator 6. In the embodiment shown in Figure 7, different fragment or product ion species are released substantially simultaneously from the second ion accumulator 6 into the transport region 7. This is illustrated in Figure 7 by the five fragment or product species shown below the transport region 7.

[0082] The transfer region 7 is configured to separate fragment or product ion species according to their physicochemical properties. For example, ions may be separated in the transfer region 7 according to their ion mobility, mass-to-charge ratio, or a combination of both of these physicochemical properties. For example, the transfer region 7 may be maintained at a relatively low pressure so that collisions between ions and the background gas within it are relatively small while the ions are moving to the TOF mass spectrometer 8. Under these conditions, it should be understood that the transfer region 7 operates as a time-of-flight domain where ions are separated according to their mass-to-charge ratio. Therefore, fragment or product ion species with relatively low mass-to-charge ratios reach the TOF mass spectrometer 8 before ions with higher mass-to-charge ratios. This is illustrated in Figure 7 by the five fragment or product ion species shown below the TOF mass spectrometer 8. These species are shown spaced apart on the time axis, indicating that the fragment or product ion species with the smallest mass-to-charge ratio arrives first at the TOF mass spectrometer 8, followed by fragment or product ion species with progressively higher mass-to-charge ratios, arriving at their respective progressively higher times.

[0083] Alternatively, fragment or product ion species may be pulsed within the transport region 7 and separated according to their mobility through the background gas placed in the transport region 7. The ions may be driven through the background gas by an electric field to separate according to their mobility. For example, the ions may be driven by applying a static DC potential gradient along the transport region 7 and / or by repeatedly moving the DC potential along the transport region 7. The electric field may be positioned to bias the ions toward the TOF mass spectrometer 8 through the transport region 7. Alternatively, the background gas may be flowed in a direction that biases the ions toward the TOF mass spectrometer 8, while the electric field may be positioned to bias the ions in the opposite direction to the gas flow to separate the ions according to their mobility. In these mobility-based separation embodiments, the pulsed ions within the transport region 7 may reach the TOF mass spectrometer 8 in order of increasing or decreasing mobility.

[0084] In the embodiment described in relation to Figure 7, the transport region 7 acts as an ion separator, in which different ion species are pulsed into the transport region 7, separated there according to their physicochemical properties, and then eluted to the mass spectrometer 8 at different times according to their physicochemical property values, thereby performing a separation cycle. This separation cycle may be shorter than the separation cycle of the ion separator 3.

[0085] After separating ions within the transfer region 7 according to their physicochemical properties, the ions may be transferred to an ion guide that guides the separated ions to the TOF mass spectrometer 8 while maintaining their separation. For example, a voltage may be applied to the ion guide to generate a series of potential wells (and thus potential barriers) spaced apart axially along the ion guide. The voltage may be a DC voltage, but alternatively, it is intended that pseudopotential wells may be formed by using an AC voltage. The voltage applied to the electrodes may vary over time so that the series of potential wells move from the upstream end of the ion guide along the ion guide to the TOF mass spectrometer 8. As ions with different values ​​of physicochemical properties reach the upstream end of the ion guide at different times, ions with different ranges of physicochemical properties will be trapped in different potential wells, and thus move within the wells to reach the TOF mass spectrometer 8 at different times. The ion guide may operate in the same manner as the ion guide described, for example, in U.S. Patent No. 7,829,841 (incorporated herein by reference).

[0086] In the embodiment shown in Figure 7, fragments or product ions having a limited range of physicochemical properties (in which the transport region 7 separates the ions) arrive simultaneously at the pusher of the TOF mass spectrometer 8, which may be synchronized with the release of ions from the second ion accumulator 6, thereby pulsing the ions having the selected range of physicochemical properties into the time-of-flight domain of the TOF mass spectrometer 8.

[0087] Figure 8 illustrates how the process described in relation to Figure 7 can be repeated multiple times for different precursor ion species eluting from the first ion separator 3 during the same separation cycle.

[0088] The lower plot in Figure 8 corresponds to the process described in relation to Figure 7, in which the first precursor ion species eluting from separator 3 is fragmented or reacted, and the resulting fragments or product ions accumulate and are then released all at once into TOF mass spectrometer 8.

[0089] The central plot in Figure 8 shows the same process being performed on a second precursor ion species, which elutes from separator 3 earlier, during the same separation cycle of separator 3 as the first precursor ion species. More specifically, according to the central plot in Figure 8, the mass filter 4 is set to allow the second precursor ion species, represented by the dark ellipse in the central plot, to pass through. The second precursor ion species then passes into the fragmentation or reaction device 5, where it fragments or reacts to form fragment or product ions. This is indicated by five lighter ellipses arranged in a column with the dark ellipse, but it should be understood that fewer or more different types of fragment or product ions may be produced. The fragment or product ions derived from the second precursor ion species are then transferred to the second ion accumulator 6, from which they are released simultaneously, so that different fragment or product ion species reach the TOF mass spectrometer 8 at different times.

[0090] The upper plot of Figure 8 shows the same process performed on a third precursor ion species, which elutes from separator 3 earlier, during the same separator separation cycle as the first and second precursor ion species. More specifically, according to the upper plot of Figure 8, the mass filter 4 is set to allow the third precursor ion species, represented by the dark ellipse, to pass through. The third precursor ion species then passes through to the fragmentation or reaction device 5, where it fragments or reacts to form fragment or product ions. This is shown by five lighter ellipses arranged in a column with the dark ellipse, but it should be understood that fewer or more different types of fragment or product ions may be produced. The fragment or product ions are then transferred to the second ion accumulator 6, from which they are released simultaneously, so that different fragment or product ion species reach the TOF mass spectrometer 8 at different times.

[0091] The mass spectrometer may be configured to allow only fragment or product ions derived from any given precursor ion species to enter the second accumulator 6 after all fragment or product ions derived from a previously analyzed precursor ion species have been discharged from the accumulator 6. This can be achieved, for example, by controlling the time at which the mass filter 4 is switched to allow the given precursor ion species to pass through, to coincide with or after the time at which the accumulator 6 has released the fragment or product ions derived from the preceding precursor ion species. Alternatively, an ion gate controlled by the mass spectrometer can be provided upstream of the accumulator 6 to prevent ions from entering the accumulator 6 until the accumulator 6 is controlled to release all fragment or precursor ions.

[0092] Although it is shown that three precursor ion species are analyzed during a single separation cycle of separator 3, it is intended that only two precursor ion species, or more than three precursor ion species, may be analyzed sequentially in the manner described during a single separation cycle.

[0093] Furthermore, although this method has so far been described only in the context of separator 3 performing a single separation cycle, it should be understood that multiple separation cycles may be performed during the same experiment, and the above method may be repeated for each of these separation cycles during the experiment.

[0094] While the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made without departing from the scope of the invention as described in the appended claims.

[0095] For example, embodiments in which a second ion accumulator 6 is present have been described in relation to Figures 7 and 8, but it is intended that the second ion accumulator 6 may be omitted. In such embodiments, each precursor ion species may be fragmented or reacted within the fragmentation or reaction device 5 without capturing the ion, and the resulting fragment or product ion is transferred to the transfer region 7 without being captured. In such embodiments, the fragment or product ion of any given precursor ion species enters the transfer region 7 at a time correlated with the time when the mass filter 4 begins to permeate the precursor ion species. Thus, the start of the separation cycle in the transfer region 7 for the fragment or product ion of any given precursor ion species is triggered by the time when the mass filter 4 switches to begin permeating that precursor ion species. As described above, fragment or product ions having a limited range of physicochemical properties reach the pusher of the TOF mass spectrometer 8 at different times. The pusher may pulse ions having a selected range of physicochemical properties into the time-of-flight domain of the TOF mass spectrometer 8, in synchronization with the time it takes for the mass filter 4 to switch to allow new precursor ion species to pass through.

[0096] This specification has described embodiments in which the analyzer includes a first ion accumulator 2 and / or a second ion accumulator 6. An ion attenuation device may be provided upstream of the first and / or second ion accumulators to attenuate ions moving toward them. This may be used to reduce the number of ions entering the ion accumulators 2, 6, for example, to reduce the space charge effect in the ion accumulators and / or to prevent saturation of the detector in the TOF mass spectrometer 8. Additionally or alternatively, an ion attenuation device may be provided downstream of the first and / or second ion accumulators 2, 6 to attenuate ions moving downstream from there. This may be used, for example, to prevent saturation of the detector in the TOF mass spectrometer 8.

[0097] Although the mass filter 4 is described as stepping between different, separate mass permeation windows as ions elute from the separator 3, it is intended that the mass filter 4 may instead have a progressively scanned mass permeation window. For example, the mass permeation window may increase by one mass-to-charge ratio unit at a time. Alternatively, the mass filter 4 may have a mass permeation window configured to allow a range of mass-to-charge ratios (e.g., a range consisting of multiple Da values) to pass through simultaneously. This mass permeation window may be progressively scanned, and each time the mass permeation window moves to allow a new mass range to pass through, the new mass range may still partially overlap with the previous mass range that the filter 4 was configured to allow to pass through.

[0098] Alternatively or additionally, although filter 4 is described as a mass-to-charge ratio filter, it is intended that the filter may filter ions according to another physicochemical property such as ion mobility or FAIMS.

[0099] Alternatively or additionally, although separator 3 is described as an ion-mobility separator, it is intended that the separator may separate ions according to other physicochemical properties such as the mass-to-charge ratio.

[0100] In less desirable embodiments, the first ion accumulator 2 may be omitted, and the ion source 1 may instead pulse the ions to the separator 3, for example, by being a pulsed ion source, or by providing an ion gate between the ion source 1 and the separator 3.

Claims

1. A method of mass spectrometry, a) A step of accumulating precursor ions in a first ion accumulator, b) A step of performing a separation cycle, which includes pulsing packets of precursor ions from the first ion accumulator to the ion separator, and separating the precursor ions such that precursor ions having different values ​​of the first physicochemical properties elute from the ion separator at different times. c) A step of mass filtering the precursor ions eluted from the ion separator to allow the selected precursor ion species to pass through, d) A step of fragmenting or reacting the selected precursor ion species to generate a set of fragments or product ion species from the selected precursor ion species, e) The step of accumulating the set of fragments or product ion species in a second ion accumulator, f) A step of releasing a set of fragment or product ion species from a second ion accumulator to a TOF mass spectrometer, wherein the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator, and multiple different species of the set of fragment or product ion species are simultaneously pulsed in the time-of-flight domain of the TOF mass spectrometer by pusher electrodes, g) a step in which steps c) to f) are repeated at least once during the separation cycle, wherein the selected precursor ion species is different each time steps c) to f) are performed. A method that includes this.

2. The method according to claim 1, wherein the ion separator is an ion mobility separator and the physicochemical property is ion mobility.

3. The aforementioned fragment or product ion species is biased by the second ion accumulator, (i) Fragments or product ion species derived from the same precursor ion species reach the second ion accumulator over the same period of time and / or accumulate in the second ion accumulator during at least part of this period (ii) The method according to claim 1, wherein fragments or product ion species derived from precursor species transmitted through a mass filter at different times reach the second ion accumulator over different periods.

4. The method according to claim 1, wherein each time the sequence of steps c) to e) is performed, fragments or product ion species are accumulated in the second ion accumulator for the same or correlated period of time as the period during which their precursor ion species are permeated by the mass filter.

5. The method according to claim 1, wherein each time the sequence of steps c) to e) is performed, only the fragment or product ion species derived from the selected precursor ion species is accumulated in the second ion accumulator, while other precursor ion species and the fragment or product ions derived therefrom are not accumulated in the second ion accumulator.

6. The method according to claim 1, wherein each time the sequence of steps b) and c) is performed, the mass filter is controlled to allow the target precursor ion species to pass through only for a period of time equal to, correlated with, or shorter than, the period over which the precursor ion species elutes from the ion separator.

7. The method according to claim 1, wherein step f) includes releasing the fragment or product ion species from the second ion accumulator in reverse order of mass-to-charge ratio, that is, starting with ions with relatively high mass-to-charge ratios and gradually releasing ions with progressively lower mass-to-charge ratios, so that different fragment or product ion species reach the pusher simultaneously and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

8. The method according to claim 7, wherein a transfer region is provided between the second ion accumulator and the TOF mass spectrometer, and the pressure within the transfer region and the energy of ions released from the second ion accumulator are such that fragments or product ions having a relatively low mass-to-charge ratio catch up with fragments or product ions having a higher mass-to-charge ratio, so that the ions reach the pusher simultaneously and are simultaneously pulsed into the time-of-flight domain of the TOF mass spectrometer by the pusher electrode.

9. The method according to claim 7, wherein each time step f) is performed, all of the fragment or product ion species emitted from the second ion accumulator simultaneously reach the pusher and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

10. The method according to claim 1, wherein step f) includes simultaneously releasing all of the fragment or product ion species from the second ion accumulator, a transfer region is provided between the second ion accumulator and the TOF mass spectrometer, the ions are separated in the transfer region according to a second physicochemical property, the fragment or product ion species arrive at the TOF mass spectrometer in a time dependent on their second physicochemical property values, and the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator so that the fragment or product ion species having a value within a selected range of the second physicochemical property values ​​are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

11. The method according to claim 10, wherein a mass spectrometer performing the method includes the step of receiving an input signal representing the selected range of values ​​of the second physicochemical property, the operation of the TOF mass spectrometer is synchronized with the release of the ions from the second ion accumulator so that fragment or product ion species having the selected range of values ​​of the second physicochemical property are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

12. The method according to claim 10, wherein the second physicochemical property is a mass-to-charge ratio, and ions are separated in the transport region according to the mass-to-charge ratio.

13. The method according to claim 10, wherein the second physicochemical property is ion mobility, and ions are separated in the transport region according to their ion mobility.

14. The method according to any one of claims 1 to 13, wherein steps a) to g) are repeated during a single experimental run.

15. A mass spectrometer, The first ion accumulator, Ion separator and, Mass filter and, Fragmentation or reaction device, The second ion accumulator, A TOF mass spectrometer having a time-of-flight domain and a pusher electrode, A control circuit, wherein the mass spectrometer a) Precursor ions are accumulated in the first ion accumulator, b) Performing a separation cycle which includes pulsing packets of precursor ions from the first ion accumulator to the ion separator, and separating the precursor ions within the ion separator such that precursor ions having different values ​​of the first physicochemical properties elute from the ion separator at different times. c) Using the mass filter, the precursor ions eluting from the ion separator are mass filtered to allow the selected precursor ion species to pass through. d) The selected precursor ion species is fragmented or reacted within the fragmentation or reaction device to generate a set of fragments or product ion species therefrom. e) The set of fragments or product ion species is accumulated in the second ion accumulator, f) The set of fragment or product ion species is released from the second ion accumulator to the TOF mass spectrometer, and the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator so that multiple different species of the set of fragment or product ion species are simultaneously pulsed in the time-of-flight domain of the TOF mass spectrometer by the pusher electrode. g) a mass spectrometer comprising a control circuit configured to control the mass filter such that the selected precursor ion species is different each time steps c) to f) are performed, wherein steps c) to f) are repeated at least once during the separation cycle.

16. The mass spectrometer according to claim 15, wherein the control circuit is configured to control the mass spectrometer such that step f) includes releasing the fragment or product ion species from the second ion accumulator in reverse order of mass-to-charge ratio, that is, starting with ions with relatively high mass-to-charge ratios and gradually releasing ions with progressively lower mass-to-charge ratios, so that different fragment or product ion species reach the pusher simultaneously and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

17. The mass spectrometer according to claim 16, wherein a transfer region is provided between the second ion accumulator and the TOF mass spectrometer, and the mass spectrometer is configured to maintain pressure within the transfer region and to release ions from the second ion accumulator with energy such that fragments or product ions having a relatively low mass-to-charge ratio catch up with fragments or product ions having a higher mass-to-charge ratio, so that the ions reach the pusher simultaneously and are simultaneously pulsed into the time-of-flight domain of the TOF mass spectrometer by the pusher electrode.

18. A mass spectrometer according to claim 15, comprising a transfer region between the second ion accumulator and the TOF mass spectrometer, wherein the control circuit is configured to control the mass spectrometer such that step f) simultaneously releases all of the fragment or product ion species from the second ion accumulator into the transfer region, the fragment or product ion species separate in the transfer region according to a second physicochemical property, and arrives at the TOF mass spectrometer in a time dependent on the second physicochemical property values, the operation of the TOF mass spectrometer is synchronized with the release of ions from the second ion accumulator so that fragment or product ion species having a selected range of values ​​of the second physicochemical property values ​​are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

19. The mass spectrometer according to claim 18, comprising a user interface configured to input a selected range of values ​​for the second physicochemical property, wherein the control circuit controls the operation of the TOF mass spectrometer based on the selected range of values, synchronizing the operation of the TOF mass spectrometer with the release of the ions from the second ion accumulator, so that fragment or product ion species having the selected range of values ​​for the second physicochemical property are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

20. A method of mass spectrometry, a) A step of accumulating precursor ions in a first ion accumulator, b) A step of performing a separation cycle, which includes pulsing packets of precursor ions from the first ion accumulator to the ion separator, and separating the precursor ions such that precursor ions having different values ​​of the first physicochemical properties elute from the ion separator at different times. c) A step of switching the mass filter that mass filters the precursor ions eluted from the ion separator to start the permeation of the selected precursor ion species, d) A step of fragmenting or reacting the selected precursor ion species to generate a set of fragments or product ion species from the selected precursor ion species, e) A step of transferring the set of fragments or product ion species through a transfer region to a TOF mass spectrometer having a pusher electrode and a time-of-flight region, wherein the ions are separated in the transfer region according to a second physicochemical property, the fragments or product ion species arrive in the TOF mass spectrometer at a time dependent on the second physicochemical property values, and the operation of the TOF mass spectrometer is synchronized with the time the mass filter is switched, so that the selected precursor ion species begin to permeate the pusher electrode so that the fragments or product ion species having a selected range of values ​​of the second physicochemical property that arrive simultaneously at the pusher electrode are simultaneously pulsed into the time-of-flight region of the TOF mass spectrometer; f) A step in which steps c) to e) are repeated at least once during the separation cycle, wherein the selected precursor ion species is different each time steps c) to e) are performed. A method that includes this.

21. The method according to claim 20, comprising the steps of: receiving an input signal representing the selected range of values ​​of the second physicochemical property in a mass spectrometer performing the method described above; and then controlling the operation of the TOF mass spectrometer so that fragments or product ion species having the selected range of values ​​of the second physicochemical property simultaneously reach the pusher electrode and are simultaneously pulsed by the pusher electrode into the time-of-flight domain of the TOF mass spectrometer.

22. A mass spectrometer, The first ion accumulator, Ion separator and, Mass filter and, Fragmentation or reaction device, A TOF mass spectrometer having a time-of-flight domain and a pusher electrode, The transfer region between the fragmentation or reaction device and the TOF mass spectrometer, A control circuit that controls the mass spectrometer, a) Precursor ions are accumulated in the first ion accumulator, b) Performing a separation cycle which includes pulsing packets of precursor ions from the first ion accumulator to the ion separator, and separating the precursor ions within the ion separator such that precursor ions having different values ​​of the first physicochemical properties elute from the ion separator at different times. c) Switch the mass filter that mass filters the precursor ions eluting from the ion separator and start the permeation of the selected precursor ion species. d) The selected precursor ion species is fragmented or reacted within the fragmentation or reaction device to generate a set of fragments or product ion species therefrom. e) The set of fragments or product ion species is transferred through a transfer region to a TOF mass spectrometer, where the ions are separated in the transfer region according to a second physicochemical property, and the fragments or product ion species arrive in the TOF mass spectrometer at a time dependent on their second physicochemical property values, and the operation of the TOF mass spectrometer is synchronized with the time the mass filter is switched so that the selected precursor ion species are transmitted through the pusher electrode so that the fragments or product ion species having a selected range of values ​​of the second physicochemical property that arrive simultaneously at the pusher electrode are simultaneously pulsed into the time-of-flight domain of the TOF mass spectrometer. f) Repeat steps c) to e) at least once during the separation cycle, such that the selected precursor ion species is different each time steps c) to e) are performed. A control circuit configured as follows, A mass spectrometer equipped with the following features.

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