Declustering ion guide

Abstract

A method of mass spectrometry and / or ion mobility spectrometry, comprising: providing an ion director including a plurality of electrodes and having a background gas in the ion director; applying an RF voltage to the electrodes of the ion director to radially confine ions within the ion director; transporting a cluster of analyte ions and adduct substances into the ion director; in a first mode, applying one or more AC voltages to the ion director to oscillate the clusters such that the clusters collide with molecules of the background gas and cause adduct substances in the clusters to separate from the analyte ions, wherein the one or more AC voltages have an amplitude and / or frequency different from that of the RF voltage; and (i) changing the velocity at which the clusters are propelled along the ion director during the first mode; and / or (ii) changing the amplitude and / or frequency of the one or more AC voltages as the clusters travel along the ion director.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to UK patent application 2004961.5, filed on April 3, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention generally relates to a declustering device for removing adducts from analyte ions, and to a mass spectrometer or migration spectrometer including such a device. Background Technology

[0004] There is a growing interest in the study of relatively large molecules in biomolecules, such as those in biopharmaceuticals interested in intact antibodies or structural biology fields interested in protein complexes and membrane proteins. Such analytes are ionized by an ion source before being analyzed in a mass spectrometer. However, when leaving the ion source, such as when leaving an electrospray ionization source, the resulting analyte ions tend to cluster with other substances. These other substances are called adducts and may, for example, contain substances such as salts or solvent molecules. Because clusters of analyte ions with adducts will have different physicochemical properties than individual analyte ions (e.g., different mass-to-charge ratios and / or ion mobilities), it may be necessary to remove adducts from the analyte ions before analysis or selection. For example, it may be necessary to remove adduct molecules before mass filtering the analyte ions, for example, because it may be necessary to transfer analyte ions with a specific mass-to-charge ratio, while clusters will have different, possibly unknown, mass-to-charge ratios.

[0005] Cluster removal techniques are known for removing adducts from analyte ions. For example, ions generated by an ion source can be accelerated downstream through a potential difference in the instrument, causing the analyte-adduct clusters to collide with background gas molecules and detach the adducts. It is also known to radially confine ion clusters within an RF ion director and push the clusters radially outward toward the RF electrode of the ion director, thereby causing RF heating of the clusters and separating the analyte ions from the adducts.

[0006] However, there is a need for an alternative technique for removing cluster adducts from analyte ions. Summary of the Invention

[0007] This invention provides a method for mass spectrometry and / or ion mobility spectrometry, comprising: providing an ion director including a plurality of electrodes and having a background gas in the ion director; applying an RF voltage to the electrodes of the ion director to radially confine ions in the ion director; transporting clusters of analyte ions and adduct substances into the ion director; in a first mode, applying one or more AC voltages to the ion director to oscillate the clusters such that the clusters collide with molecules of the background gas and cause adduct substances in the clusters to separate from the analyte ions, wherein the one or more AC voltages have an amplitude and / or frequency different from that of the RF voltage; and (i) changing the velocity at which the clusters are propelled along the ion director during the first mode; and / or (ii) changing the amplitude and / or frequency of the one or more AC voltages as the clusters travel along the ion director.

[0008] The speed at which the cluster is propelled along the ion guide axis in the first mode can be varied to change the passage time of the cluster through the de-cluster ion guide, and thus to change the time the cluster is oscillated by the AC voltage. This alters the amount of de-clustering performed in the first mode. Similarly, the amplitude and / or frequency of one or more AC voltages can be varied to change the amount of de-clustering performed (for ions with a given mass-to-charge ratio), and / or to change (e.g., optimize) the amount of de-clustering for ions with different mass-to-charge ratios at different times.

[0009] Embodiments of the present invention alter the conditions under which clusters are subjected to one or more AC voltages in order to change or control the amount of cluster removal. For example, this may be advantageous if the presence of some adduct is relevant to the analysis of the analyte. For instance, it may be important to remove solvadducts while retaining non-covalently bound drug molecules.

[0010] Embodiments of the present invention alter the conditions under which clusters are subjected to one or more AC voltages in order to change or control the amount of declustering or conformational change of analyte ions (e.g., proteins). For example, it may be necessary to provide a relatively small amount of conformational rearrangement or unfolding of analyte ions.

[0011] The implementation separates the RF voltage requirements for radial confinement of ions from the AC voltage requirements for declustering, thereby allowing more degrees of freedom to optimize radial confinement and / or declustering.

[0012] The amplitude and / or frequency of the one or more AC voltages may vary over time.

[0013] The method may include transferring ions from the ion director to a mass filter and mass filtering the ions in the mass filter. The mass-to-charge ratio or range of mass-to-charge ratio selectively transferred by the mass filter may vary over time in sync with the amplitude and / or frequency of the one or more AC voltages, optionally so as to transfer substantially only ions that have already been declustered in the ion director.

[0014] Alternatively, the method may include transferring ions from the ion director to a mobility filter and performing mobility filtering on the ions in the mobility filter; wherein the mobility or mobility range selectively transferred by the mobility filter varies over time in sync with the amplitude and / or frequency of the one or more AC voltages over time, optionally so as to transfer substantially only ions that have already been declustered in the ion director.

[0015] The method may include separating the clusters by mass-to-charge ratio or ion mobility before transferring the clusters to the ion director, and changing the amplitude and / or frequency of the one or more AC voltages over time based on the mass-to-charge ratio or ion mobility of the clusters transferred to the ion director.

[0016] For example, clusters can be separated using a scanning / stepping mass filter (e.g., a quadrupole mass filter), a mass-selective ion trap, or other separator devices to transport ions with different mass-to-charge ratios to the ion director at different times. The separator can be scanned / stepped in this manner over a period of time, and changes in one or more AC voltages can be synchronized with this time, causing clusters with different mass-to-charge ratios to experience different AC amplitudes and / or frequencies in the ion director. Similarly, clusters can be separated using an ion mobility separator to transport ions with different mobilities to the ion director at different times. The separator can separate ions over a period of time, and changes in one or more AC voltages can be synchronized with this time, causing clusters with different mobilities to experience different AC amplitudes and / or frequencies in the ion director.

[0017] The method may include performing mass analysis on ions from the ion director in a mass analyzer; wherein the operation of the mass analyzer varies over time to change the mass-to-charge ratio or mass-to-charge ratio range that the mass analyzer can analyze or is optimized to analyze; and wherein this mass-to-charge ratio or mass-to-charge ratio range varies over time in sync with changes in the amplitude and / or the frequency of the one or more AC voltages, optionally so that mass analysis is performed substantially only on ions that have already been declustered in the ion director.

[0018] For example, a mass analyzer may be a quadrupole mass analyzer that includes a quadrupole mass filter (synchronized with one or more AC voltages) that scans or steps over time to transmit different mass-to-charge ratios to the detector at different times.

[0019] Alternatively, the mass analyzer may be a time-of-flight mass analyzer, which includes pusher and / or puller electrodes that are repeatedly and intermittently activated to pulse ions entering the time-of-flight region to the detector. The operation of the time-of-flight mass analyzer may vary over time to perform mass analysis on different mass-to-charge ratio ranges at different times, wherein said range varies over time in sync with changes in the amplitude and / or frequency of one or more AC voltages.

[0020] The time-of-flight mass analyzer can operate in enhanced duty cycle (EDC) mode. In this mode, ions pulsate toward the time-of-flight mass analyzer and the pusher and / or puller electrodes, and the activation time of the pusher and / or puller electrodes is synchronized with the ion pulsation toward the time-of-flight mass analyzer, such that ions with one or more predetermined mass-to-charge ratios pulsate toward the detector via the pusher and / or puller electrodes. The time delay between the ion pulsation toward the time-of-flight mass analyzer and the activation of the pusher and / or puller electrodes can be varied for different pulses toward the time-of-flight mass analyzer, for example, to optimize the mass analysis of ions with different mass-to-charge ratios in different pulses. This time delay can vary over time in sync with changes in the amplitude and / or frequency of one or more AC voltages.

[0021] Alternatively, the mass analyzer could record mass spectrometry data as a function of time (i.e., as a function of the frequency and / or amplitude of one or more AC voltages). The recorded data could then be post-processed to obtain mass spectrometry data at the desired AC voltage characteristics. For example, the mass spectrometry data could be filtered to retain only the data for declustered ions.

[0022] The one or more AC voltages can be multiple different AC voltages with different amplitudes and / or frequencies, and the different AC voltages can be applied at different axial positions along the length of the ion guide.

[0023] The AC voltage that can be applied at progressively downstream axial positions of the ion guide has progressively lower amplitudes.

[0024] This is beneficial because the mobility of the clusters tends to increase as they move along the ion guide and cause adducted ions to detach, so reducing the amplitude of the AC voltage along the ion guide helps prevent these ions from oscillating with a large amplitude, which would cause the ions to be lost to the electrodes of the ion guide. For the same purpose, different AC voltages can (alternatively or otherwise) have different frequencies.

[0025] The electrodes of the ion guide can define a conduit through which the cluster is guided, wherein in the first mode, the RF voltage applied to the electrodes radially confines the ions and pushes the ions toward a central axis through the conduit, and wherein the AC voltage causes the cluster to oscillate around the central axis.

[0026] This allows the clusters to oscillate with relatively large amplitudes, and the risk of the clusters impacting the electrodes of the ion guide and being lost into the system is relatively low. This contrasts with some conventional techniques in which ions are driven radially toward the RF electrodes to oscillate and heat them.

[0027] The AC voltage causes the ions to oscillate around the central axis, such that the cluster has substantially the same average oscillation amplitude on either side of the axis.

[0028] At least in the first mode, there is essentially no airflow through the ion director. Alternatively, an airflow through the ion director can be provided in a downstream or upstream direction, and a voltage can be applied to the ion director to counteract the force generated on the ions due to the airflow, wherein the airflow and / or voltage are selected or changed to slow down or change the passage time of the cluster through the ion director.

[0029] The step of changing the speed at which the cluster is propelled along the ion guide may include repeatedly traveling a transient DC voltage along the ion guide to propel the cluster along the ion guide; and the amplitude of the transient DC voltage and / or the speed and / or frequency at which the transient DC voltage moves along the ion guide may vary over time, thereby changing the speed at which the cluster is propelled along the ion guide in the first mode.

[0030] An ion director may include multiple electrodes spaced apart along its longitudinal axis, and whenever a transient DC voltage travels along the ion director, the transient DC voltage may be continuously applied along the ion director to different electrodes, or to multiple electrodes in different groups, so that the transient DC voltage moves along the ion director.

[0031] The step of changing the speed at which the cluster is propelled along the ion guide may include generating an axial electric field along the ion guide by simultaneously applying different DC voltages to different electrodes of the ion guide, and changing the different voltages to change the magnitude of the electric field, and thereby changing the speed at which the cluster is propelled along the ion guide in the first mode.

[0032] Alternatively or additionally, the step of changing the speed at which the cluster is propelled along the ion guide may include providing an airflow through the ion guide to propel the cluster along the ion guide, and changing the speed of the airflow.

[0033] During the first mode, the background gas can be maintained at a pressure between 0.01 mbar and 10 mbar.

[0034] The method may include, for example, changing the pressure and / or composition of the background gas over time during a first mode.

[0035] During the first mode, the background gas can be maintained at the first pressure or vary within the first pressure range, while during the second mode, when no AC voltage is applied to the ion guide, the background gas can be maintained at a pressure lower than the first pressure or the first pressure range.

[0036] The method may include operating the ion director in a second mode in which the one or more AC voltages are not applied to the ion director.

[0037] Ideally, the RF voltage should not cause the cluster to oscillate to the point of separating the analyte ions from the adducts in the cluster.

[0038] In this second mode, clusters may essentially not be declustered. The speed at which clusters are propelled along the ion guide can be varied or unchanged in this mode.

[0039] In the second mode, the ion director can operate at a higher transport level than in the first mode, meaning that a larger proportion of analyte ions can be transported in the second mode.

[0040] The method may include switching between a first mode and a second mode when the cluster is passing through the ion guide.

[0041] The method may include repeatedly switching between the first mode and the second mode as the cluster is passing through the ion guide.

[0042] The method may include ionizing an analyte solution to generate the cluster, wherein the analyte solution contains a membrane protein dissolved in a solvent using a detergent, and wherein the analyte ions in the cluster are membrane protein ions, and the adduct substances in the cluster are detergent molecules.

[0043] The method then includes the step of transferring clusters of analyte ions and adduct substances into an ion guide.

[0044] The method may include the step of forming an analyte solution by dissolving membrane proteins in a solvent using a detergent.

[0045] By way of example only, the membrane protein and / or solvent and / or detergent can be any of these substances used in Lengqvist et al.'s JBC, Vol. 279, No. 14, pp. 13311-13316, published April 2, 2004. For example, the membrane protein can be microsomal glutathione transferase-1 (MGST1). The detergent can be Triton X-100. The solvent can be an aqueous solution of ammonium acetate.

[0046] Alternatively, it is envisioned that the membrane protein and / or solvent and / or detergent can be any of the substances used in Science, 2008, 321, 243, by Barrera et al. For example, the membrane protein could be a heteromeric (ATP) binding cassette transporter complex (BtuC2D2) and / or the detergent could be n-dodecyl-β-D-maltose glycoside (DDM).

[0047] Alternatively, it is envisioned that the membrane protein and / or solvent and / or detergent can be any of the substances used in US 2015 / 0346214. For example, the membrane protein can be one of the following: an ammonium channel C-terminally fused to green fluorescent protein (AmtB-GFP), an aquaporin Z membrane protein complex (AQPZ), an ammonium channel membrane protein complex (AmtB), a high-conductivity mechanosensitive channel (MscL), acridine flavin resistance protein B (AcrB), a G protein-coupled receptor (GPCR), a multidrug transporter (EmrE), an integrated membrane protein (LmrP), a multidrug resistance protein (MexB), an inner membrane protein (MacB), a transmembrane P-glycoprotein 1 (P-gp), a lipid A export ATP-binding / permeabilization protein (MsbA), a possible multidrug resistance protein (NorM), or an inward rectifier potassium channel (Kirbac3.1). The detergent can be n-decyl-β-D-maltose glycoside (DM), n-undecyl-β-D-maltose glycoside (UDM), n-dodecyl-β-D-thiomaltopyranoside (DDTM), Cymal-5, Cymal-6, octylglucose neopentyl glycol (OGNG), n-octyl-β-D-glucanoside (OG), tetraethylene glycol monooctyl ether (C8E4), pentaethylene glycol monooctyl ether (C8E5), octaethylene glycol monododecyl ether (C12E8), or anapoe-58 (Brij-58, C16E20). The solvent system can be an aqueous solution of the following: ammonium acetate; ammonium bicarbonate; sodium chloride, TRIS, and β-mercaptoethanol; sodium chloride, glycerol, TRIS, and β-mercaptoethanol; sodium chloride, imidazole, TRIS, and β-mercaptoethanol; or sodium chloride, imidazole, glycerol, TRIS, and β-mercaptoethanol.

[0048] Although the clusters are described as clusters of membrane protein ions and detergents, it is envisioned that the analyte ions and / or adducts could be other types of substances. For example, the adducts in the clusters could be salts or molecules of solvents.

[0049] The method may include mass analysis and / or ion mobility analysis of the analyte ions and any remaining clusters downstream of the ion director to obtain mass peaks and / or mobility peaks of the analyte ions and the remaining clusters, respectively.

[0050] The method may include determining the width and / or signal-to-noise ratio of one or more of the peaks, and changing the frequency and / or amplitude of the one or more AC voltages during the first mode to change the width and / or signal-to-noise ratio of the peaks of the analyte ions and clusters subsequently analyzed.

[0051] The peak width can be the FWHM width.

[0052] The method can be performed on a mass spectrometer or a migration spectrometer, which includes: a first vacuum chamber having an inlet orifice; a second vacuum chamber adjacent to the first vacuum chamber; and a differential pumping orifice separating the first vacuum chamber and the second vacuum chamber; wherein the ion guide is arranged in the first vacuum chamber.

[0053] The first vacuum chamber may include an ion guiding device having a first portion that guides ions along a first axial path, a second portion that guides ions along a second different axial path, and a transition portion that pushes ions from the first axial path onto the second axial path.

[0054] The first axial path can be substantially parallel to the second axial path, but radially offset from the second axial path. This ion guiding device can be arranged to guide ions from the inlet orifice to and through the differential pumping orifice.

[0055] The ion guide may be part of the ion guiding device or downstream of the ion guiding device.

[0056] For example, an ion guide can form part or all of the second part of an ion guiding device.

[0057] The first vacuum chamber includes a gas pumping port for evacuating the gas from the first vacuum chamber, and at least a portion of the second part of the ion guide can be shielded from the gas pumping port by a barrier. Alternatively or additionally, the ion guiding device can be arranged such that the central axis of the first axial path is coaxial with the central axis of the gas pumping port.

[0058] The mass and / or ion mobility analyzer can be located in the second vacuum chamber or in another vacuum chamber downstream of the second vacuum chamber.

[0059] The quality analyzer can be a time-of-flight quality analyzer.

[0060] The present invention also provides a mass spectrometer or migration spectrometer configured to perform any of the methods described herein.

[0061] Therefore, the present invention provides a mass spectrometer or migration spectrometer comprising: an ion director including a plurality of electrodes and a background gas in the ion director; an RF voltage source for applying an RF voltage to the electrodes of the ion director to radially confine ions within the ion director; one or more AC voltage sources for applying one or more AC voltages to the ion director in a first mode to oscillate clusters of analyte ions and adduct substances, such that the clusters collide with molecules of the background gas and cause adduct substances in the clusters to separate from the analyte ions, wherein the AC voltages have an amplitude and / or frequency different from the amplitude and / or frequency of the RF voltages; and a control circuit configured to control the spectrometer to: (i) change the velocity at which the clusters are propelled along the ion director during the first mode; and / or (ii) change the amplitude and / or frequency of the one or more AC voltages as the clusters travel along the ion director.

[0062] The spectrometer may include a DC voltage source connected to an electrode of the ion director, wherein the control circuit controls the DC voltage source to continuously apply a DC voltage to different electrodes in the electrode, thereby repeatedly traveling a transient DC voltage along the ion director; wherein the amplitude of the transient DC voltage and / or the speed and / or frequency of the transient DC voltage traveling along the ion director varies with time to change the speed at which the cluster is propelled along the ion director.

[0063] The spectrometer may include a DC voltage source connected to an electrode of the ion guide, wherein the control circuit controls the DC voltage source to simultaneously apply different DC voltages to different electrodes of the electrode to generate an axial electric field along the ion guide, and changes the different voltages over time to change the magnitude of the electric field to change the speed at which the cluster is propelled along the ion guide. Attached Figure Description

[0064] Various implementation schemes will now be described by way of example and with reference to the accompanying drawings, wherein:

[0065] Figure 1A and Figure 1B A schematic diagram of a declustering apparatus according to an embodiment of the present invention is shown;

[0066] Figure 2 The upstream end of the mass spectrometer, including the ion source, is shown schematically.

[0067] Figures 3A to 3C A view of an ion director that can be used in embodiments of the present invention is shown;

[0068] Figure 4A It shows Figures 3A to 3C A schematic side view of the ion guide shown, and Figure 4B A schematic diagram of a declustering device arranged within an ion guide according to an embodiment is shown;

[0069] Figure 5 A to Figure 5 C shows mass spectrometry data for both the known declustering technique and the declustering technique according to an embodiment of the present invention;

[0070] Figures 6A to 6C Mass spectrometry data for both known declustering techniques and declustering techniques according to embodiments of the present invention are also shown;

[0071] Figures 7A to 7D Mass spectrometry data from a declustering technique according to an embodiment of the invention are shown, wherein the declustering AC voltages have different frequencies;

[0072] Figures 7A to 7D Mass spectrometry data from a declustering technique according to an embodiment of the invention are shown, wherein the declustering AC voltages have different frequencies;

[0073] Figures 8A to 8E Mass spectrometry data from a declustering technique according to an embodiment of the present invention are shown, wherein the declustering AC voltage has a first frequency and different amplitudes;

[0074] Figures 9A to 9E Mass spectrometry data from a declustering technique according to an embodiment of the present invention are shown, wherein the declustering AC voltage has a second frequency and a different amplitude;

[0075] Figure 10 The graph shows how the FWHM of the m / z peak varies as a function of the amplitude of the declustered AC voltage for four different frequencies of declustered AC voltage.

[0076] Figure 11 The graph shows how the peak area over a specific quality range varies as a function of the declustering AC voltage amplitude for declustering AC voltages of different frequencies; and

[0077] Figures 12A to 12D Mass spectrometry data according to the embodiment are shown, wherein the mass filter is scanned synchronously with the amplitude of the declusivation AC voltage. Detailed Implementation

[0078] Embodiments of the present invention provide continuous activation of ionic substances (such as relatively large analyte ions) to remove adduct substances, for example by mass filtration and / or mass analysis of the analyte ions, prior to selection and / or analysis of the analyte ions.

[0079] Figure 1A and Figure 1B A schematic diagram of a declustering apparatus according to an embodiment of the present invention is shown.

[0080] Reference Figure 1A The de-clustering device includes two planar upper and lower electrodes 1, which are spaced apart from each other in the y-axis and have main faces facing each other. In use, a voltage source 9 applies a DC voltage to these electrodes to generate an electric field that confines ions between ion guides. Alternatively, the voltage source 9 may provide an RF voltage to these electrodes 1 to confine ions between the ion guides. The planar electrodes 1 are shown as parallel to each other, but it is contemplated that they may be angled relative to each other, for example, such that when DC and / or AC voltages are applied to these electrodes, ions are driven through the means between the planar electrodes 1. The de-clustering device also includes side electrodes 3, which are spaced apart from each other in the x-axis and are arranged such that the side electrodes 3, together with the upper and lower electrodes 1, define a conduit between them through which ions 5 can be guided. Figure 1A In the depicted embodiment, each sidewall of the declustering device includes a plurality of electrodes 3 extending in the z-axis (i.e., in the direction in which ions 5 travel through the device) and spaced apart along the direction between the upper and lower electrodes 1 (i.e., in the x-axis). These electrodes 3 may be planar and may have a main surface facing the upper and lower electrodes 1. These electrodes 3 may be arranged parallel to each other. In use, a voltage source 11 may apply an RF voltage to these side electrodes 3 to generate an AC electric field and a pseudo-barrier that radially confines ions between ion guides (in the x-axis). For example, opposite phases of the RF voltage may be applied to electrodes 3 adjacent to each other within each sidewall. Side electrodes 3 adjacent and opposite to each other in the x-axis may remain in the same RF phase.

[0081] Although each sidewall has been described as comprising multiple RF electrodes 3, it is contemplated that the sidewall may alternatively comprise only a single RF electrode. Less preferred is that instead of applying an RF voltage to the electrode 3 in each sidewall, it is contemplated that a DC voltage be applied to the electrode 3 in each sidewall in order to confine ions in the x-dimensional space.

[0082] As described above, in use, the RF and / or DC voltages applied to the upper electrode, lower electrode, and side electrode cause the ion cluster 5 entering the ion transport conduit along the axis arranged in the z-dimensional direction to be radially confined relative to the axis (i.e., radially confined in the x and y dimensions).

[0083] In the first declustering mode, one or both of voltage sources 9 and 11 apply an AC voltage to one or more of electrodes 1 and 3 to cause clusters of analyte ions and adduct substances to oscillate in the radial direction. For example, an AC voltage can be applied between the upper electrode and the lower electrode 1 to cause the ion clusters to oscillate in the y-axis. Since a background gas is present in the ion transport conduit, the oscillation of the ion clusters causes them to collide with molecules in the background gas, thereby increasing their internal energy and causing declustering, i.e., separation of the adduct substance from the analyte ions. It is envisioned that the background gas pressure can be maintained below atmospheric pressure or at atmospheric pressure during this process. The energy input to the clusters is related to their size, the electric field strength generated by the AC voltage, and the gas pressure.

[0084] The amplitude and frequency of the AC voltage are chosen such that at least some of the analyte ions remain radially confined by electrodes 1 and 3, and ideally, the analyte ions are not fragmented by collision-induced dissociation (CID). For example, the AC voltage can be applied around a DC offset potential of the RF voltage used for radial confinement of the ions. This ensures that the ion declination caused by the declustering AC voltage is symmetrical about the central axis, rather than extending relatively far from said axis and impacting electrodes 1 and 3. The frequency of the declustering AC voltage can also be chosen such that the declination of the ions of interest from the central axis prevents the ions from being lost to electrodes 1 and 3. The most efficient form of the declustering AC voltage is a square wave, as the ions essentially undergo a declustering field throughout. After declustering, the analyte ions can then be transported forward in a downstream direction through the conduit for further analysis.

[0085] Clusters can be driven by a declustering device to control and vary the amount of cluster removal. This can be achieved by pushing the clusters through the declustering device and changing the force applied to the clusters, thereby altering the speed at which the clusters pass through the device. For example, if the clusters are pushed through the declustering device relatively slowly, in a first declustering operating mode, the clusters will experience oscillations caused by the declustering AC voltage for a relatively long period, resulting in a relatively large amount of cluster removal (e.g., a relatively large proportion of the analyte will be declustered and / or a relatively large amount of adducts will be declustered from each cluster). Conversely, if the clusters are pushed through the declustering device faster, in the first declustering operating mode, the clusters will experience oscillations for a shorter period, resulting in a smaller amount of cluster removal (e.g., a smaller proportion of the analyte will be declustered and / or a smaller amount of adducts will be declustered from each cluster).

[0086] Clusters can be propelled through a de-clustering device by one or more of a number of devices (including one or more of the following). A transient DC voltage can be repeatedly traveled along the de-clustering device to propel clusters through it. The transient DC voltage is continuously applied to electrodes spaced apart along the axial direction of the de-clustering device to create a DC potential barrier moving along the de-clustering device, thereby propelling ions radially confined within the de-clustering device through it. These axially spaced electrodes are not... Figure 1A As shown in the text, but Figure 1B Electrode 3 in the process can be used for this purpose. The step of causing a transient DC voltage to travel along the de-clustering device may include controlling voltage source 13 to continuously apply the transient DC voltage to each of the axially spaced electrodes 3. Alternatively, the transient DC voltage may be continuously applied only to every nth electrode downstream of the electrode to which the transient DC voltage was last applied, where n is an integer greater than 1 (e.g., only to alternating axially spaced electrodes). It is also envisioned that the transient DC voltage may be applied simultaneously to a group of multiple electrodes at any given time, and that the transient DC voltage may be applied sequentially to different groups of multiple electrodes at different corresponding times, such that the transient DC voltage moves along the de-clustering device. Each group of electrodes may consist of axially continuous electrodes of the de-clustering device. Characteristics of the transient DC voltage, such as amplitude and / or velocity and / or travel frequency, may be varied, thereby changing the speed at which clusters are propelled through the de-clustering device.

[0087] As an alternative to, or otherwise to, a voltage source 13 can be used to generate an electrostatic field that applies a potential difference along the axial length of the de-clustering device to push the clusters through the device. The magnitude of the electric field can vary over time, thereby altering the speed at which the clusters are pushed through the de-clustering device.

[0088] Alternatively, or as an alternative to other techniques described herein for altering the amount of declustering, an airflow can be used to propel clusters through the declustering device, and the airflow velocity can vary over time, thereby altering the speed at which clusters are propelled through the declustering device.

[0089] Alternatively, or as an alternative to other techniques described herein for altering the amount of declustering, it is conceivable that, for example, by applying voltage to upstream and downstream portions of the declustering device, clusters can be axially captured within the declustering device, and the amount of declustering can be altered by changing the time it takes for clusters to be captured before being released from the declustering device.

[0090] Alternatively, or as an alternative to other techniques described herein for changing the amount of declusification, the composition or pressure of the background gas within the declusification apparatus can be altered to change the amount of declusification.

[0091] The declustering device can operate in a second, different mode in which no declustering AC voltage is applied, preventing clusters from being declustered. For example, in this mode, only a DC voltage (and no AC / RF voltage) can be applied to the upper and lower electrodes 1 to confine ions in the y-axis. The DC voltage (i.e., potential) applied to the upper and lower electrodes 1 can be higher than the DC bias voltage of the side electrode 3 (applying an RF voltage to confine ions in the x-direction). However, in a less preferred embodiment, it is contemplated that, in addition to or instead of the DC voltage, an RF voltage can be applied to the upper and lower electrodes 1 to confine ions in the y-axis (without causing declustering). Clusters can be radially confined by electrodes 1 and 3 and guided through the conduit. In the second mode, ions 5 enter the declustering device in the z-direction.

[0092] As the clusters flow through the conduit, the de-clustering device can switch between two modes by turning the de-clustering AC voltage on or off.

[0093] Figure 1B The diagram shows the differences from the above description except for the arrangement of side electrode 3. Figure 1A Another embodiment of the same declustering device as described. Figure 1B In the depicted embodiment, each sidewall of the declustering device includes a plurality of electrodes 3, each extending in the y-axis, i.e., in the direction between the upper electrode and the lower electrode 1. These electrodes 3 may be planar and may have principal surfaces substantially orthogonal to the principal surfaces of the upper and lower electrodes 1. The electrodes 3 in each sidewall may be spaced apart from each other in the z-axis direction. The electrodes 3 in each sidewall may be arranged parallel to each other. In use, an RF voltage is applied to these side electrodes 3 to generate an AC electric field and a pseudo-barrier that radially confines ions between the sidewalls (in the x-axis). For example, opposite phases of the RF voltage may be applied to electrodes adjacent to each other within each sidewall. Side electrodes 3 adjacent and opposite to each other in the x-axis may remain in the same RF phase. Less preferably, instead of applying an RF voltage to the electrodes in each sidewall, it is envisioned that a DC voltage be applied to the electrodes 3 in each sidewall to confine ions in the x-axis.

[0094] As mentioned above, various techniques can be used to modify the amount of declustering, such as altering the characteristics of the transient DC voltage that repeatedly travels along the declustering device, or changing the magnitude of the electrostatic field across the axial length of the declustering device. It should be understood that by applying the current at different times... Figure 1B A transient DC voltage is applied to different electrodes 3 in the process, and the transient DC voltage can travel along the de-clustering device. Similarly, an electrostatic field can be arranged by simultaneously applying different DC voltages to different electrodes 3.

[0095] Although the upper and lower electrodes 1 are described as parallel to each other in the above embodiments, it is contemplated that they may be angled relative to each other, for example, such that when a DC and / or AC voltage is applied to these electrodes, ions are driven through the means between the planar electrodes. Alternatively or additionally, although each of the upper and lower electrodes 1 is described as a single electrode, it is contemplated that one or each of these electrodes may be replaced by an array of multiple RF electrodes (or DC electrodes), for example, so that the upper and / or lower walls are formed by multiple electrodes in a manner corresponding to the formation of one of the sidewalls.

[0096] To avoid any doubt, although electrode 1 has been described as an upper electrode and a lower electrode, the y-axis can be arranged in any orientation and is not necessarily vertical.

[0097] In mass spectrometry, analyte ions are typically generated by a relatively high-pressure ion source, such as an atmospheric pressure ion source. These ions are then necessary to be transferred to the vacuum region of the mass spectrometer, as ion processing or analysis requires relatively low vacuum pressure.

[0098] Figure 2 A known arrangement is schematically illustrated, comprising an electrospray ionization (ESI) probe 2 disposed in an atmospheric pressure region 4, a low-pressure vacuum chamber 6 of a mass spectrometer, and a medium-pressure chamber 8 disposed between the atmospheric pressure region 4 and the vacuum chamber 6 of the mass spectrometer. A cone 10 is disposed between the atmospheric pressure region 4 and the medium-pressure chamber 8 such that the medium-pressure chamber 8 can be maintained at a pressure lower than that of the atmospheric pressure region 4, and a differential pumping orifice 12 is disposed between the vacuum chamber 6 and the medium-pressure chamber 8 such that the vacuum chamber can be maintained at a pressure lower than that of the medium-pressure chamber. An ion guide 14, such as an ion tunnel or a multipolar ion guide, is disposed in the medium-pressure chamber 8 to guide ions received by the cone 10 toward and through the differential pumping orifice 12.

[0099] In operation, the intermediate-pressure chamber 8 is pumped to a pressure lower than that of atmospheric pressure region 4, and the vacuum chamber 6 is pumped to a pressure lower than that of intermediate-pressure chamber 8. The analyte solution is then delivered to the capillary 16 of the ESI probe 2 and ejected from its tip, thereby generating analyte ions 18 in atmospheric pressure region 4. The analyte ions 18 then pass through cone 10 and enter ion guide 14 in intermediate-pressure chamber 8. Ion guide 14 guides the ions through the intermediate-pressure chamber and through differential pumping orifice 12 into vacuum chamber 6. The ions can then split in vacuum chamber 6, or further downstream of the spectrometer, in a vacuum chamber that can be pumped to even lower pressures. In conventional declustering techniques, a voltage can be selectively applied to cone 10 to accelerate the clusters through the background gas in atmospheric pressure region 4 at a certain velocity, thus declustering the clusters.

[0100] According to an embodiment of the invention, the declustering device can be arranged downstream of the ESI probe 2 to receive clusters. For example, the declustering device can replace the ion guide 14. Alternatively, the declustering device can be arranged inside, upstream or downstream of the ion guide 14 to decluster the analyte ions before they enter the vacuum chamber 6 through the differential pumping port 12.

[0101] Figure 3A A portion of another embodiment of the invention is shown, which is similar to Figure 2 The illustrated embodiment differs in that ion director 14 has been replaced by another type of ion director that guides ions along a first axial path and then onto a second axial path displaced from the first axial path, guiding ions along the second axial path. A voltage can be applied to the ion director to push ions downstream along the ion director.

[0102] exist Figure 3A In this embodiment, cone 20 separates a relatively high-pressure region 22 (such as an atmospheric pressure region) from the first vacuum chamber 24. An electrospray ionization (ESI) probe or other ion source may be arranged in the high-pressure region 22. A differential pumping orifice 26 is arranged between the first vacuum chamber 24 and the second vacuum chamber 28, allowing the second vacuum chamber 28 to be maintained at a lower pressure than the first vacuum chamber 24. An ion guide is arranged in the first vacuum chamber 24 to guide ions received by cone 20 toward and through the differential pumping orifice 26, as will be described in more detail below. A mass analyzer 29, such as an orthogonal accelerated time-of-flight mass analyzer, may be arranged in the second vacuum chamber 28 to analyze ions transported through the differential pumping orifice 26.

[0103] The ion guide includes a first portion 30 for guiding ions along a first axial path, a second portion 32 for guiding ions along a second axial path (which may be parallel to and displaced from the first axial path), and a transition portion 33 for transferring ions from the first axial path to the second axial path. In the depicted embodiment, each of the first ion guiding portion 30 and the second ion guiding portion 32 includes a plurality of axially separated porous electrodes (e.g., annular electrodes) for radially confining ions along their respective axial paths. An RF voltage is applied to these electrodes to radially confine the ions. For example, different (e.g., opposite) phases of the RF voltage source can be applied to adjacent porous electrodes in a known manner to radially confine the ions.

[0104] Figure 3BThree cross-sectional views of the electrode arrangement in the ion guide at different axial points along the ion guide are shown. View 30 shows the electrode arrangement near the cone 20, where ions are confined to a first axial path by perforated electrodes 34 in the first portion 30 of the ion guide. View 32 shows the electrode arrangement near the differential pumping orifice 26, where ions are confined to a second axial path by perforated electrodes 35 in the second portion 32 of the ion guide. View 33 shows the electrode arrangement in the transition region 33 of the ion guide, where ions are transferred from the first axial path of the first ion guide portion 30 to the second axial path of the second ion guide portion 32. This transfer can be achieved by providing one or more electrodes 36 in the transition region, each electrode partially surrounding the first axial path and having a radial opening (e.g., an arcuate electrode) on its side facing the second axial path; providing one or more electrodes 37 in the transition region, each electrode partially surrounding the second axial path and having a radial opening (e.g., an arcuate electrode) on its side facing the first axial path; and pushing ions from the first axial path through the radial openings in the electrodes to the second axial path. This ion drive can be performed by providing a potential difference, for example by providing a potential difference in the radial direction by applying a voltage to the electrodes in the transition region.

[0105] Figure 3C A perspective view of the electrode arrangement in the transition region is shown.

[0106] Return to reference Figure 3A The first ion guiding portion 30 may be arranged in the first vacuum chamber 24 such that the hole of the cone 20 is aligned (e.g., coaxial) with the first axial path defined by the first ion guiding portion 30. The second ion guiding portion 32 may be arranged in the first vacuum chamber 24 such that the hole in the differential pumping hole 26 is aligned (e.g., coaxial) with the second axial path defined by the second ion guiding portion 32.

[0107] A vacuum pump is provided for evacuating the first vacuum chamber 24 through the gas pumping port 38. The opening of the gas pumping port 38 can be aligned (e.g., coaxial) with the first axial path of the first ion guiding portion 30. The end of the ion guide formed by the second portion 32 can be physically isolated from the gas pumping port 38 by a barrier 40.

[0108] In operation, ions are generated in high-pressure region 22. The pressure difference between high-pressure region 22 and first vacuum chamber 24 causes gas and ions to pass through cone 20 and enter first vacuum chamber 24, whereby gas and ions tend to expand into low-pressure region. Ions enter first portion 30 of ion guide and are thus radially confined, but can diffuse relatively, as shown in ion cloud 42. Ions are driven at least partially axially along first portion 32 of ion guide by gas flow toward gas pump port 38. When ions reach transition portion 33 of ion guide, they are propelled radially onto a second axial path defined by second portion 32 of ion guide, as shown in ion trajectory 43. As described above, this is likely due to the application of a potential difference in the radial direction. As a result, ions migrate from first ion guide portion 30 to second ion guide portion 32. Conversely, as shown by arrow 44, most of the gas flow continues substantially along the axis defined by first ion guide portion 30 toward and through gas pump port 38. Therefore, ions are radially confined in the second ion guiding portion 32 and travel along the second axial path toward the differential pumping port 26, while most of the gas is guided in a different direction toward the gas pumping port 38. At least a portion of the second portion 32 of the ion guide can be isolated from the pumping port by the barrier 40, such that the gas flow toward the pumping port 38 is guided away from the second axial path of the second ion guide portion 32.

[0109] The second ion guiding section 32 may have a smaller radial cross-section than the first section 30, such that the ions are radially compressed in the second section compared to the first section, as shown by the ion beam 46. The ions are then guided by the second ion guiding section 32 through the differential pumping port 26 and into the second vacuum chamber 28.

[0110] Clusters and other ions can be propelled along the ion guide by a static DC electric field. For example, a DC voltage gradient can be arranged in the first vacuum chamber 24 between a point toward the cone 20 and a point toward the differential pumping orifice 26, for example, by applying different DC voltages to the electrodes of the ion guide. The DC voltage gradient can be arranged along the first and / or second axis of the ion guide (and / or transition region 33), for example, by applying different voltages to the electrodes of the ion guide at different axial positions. Alternatively or additionally, clusters and other ions can be propelled along the ion guide by repeatedly traveling one or more DC barriers along the first guide portion 30 and / or the second ion guide portion 32. This can be performed by continuously applying one or more transient DC voltages to the electrodes along the ion guide. One or more DC barriers can repeatedly travel along the ion guide.

[0111] As described below, the declustering device can be located upstream, downstream, or inside the ion director. It has been recognized that the ion director in the above arrangement is capable of handling relatively high gas loads (e.g., since the ion director initially directs ions along with the gas flow toward the pump port and then removes the ions from the gas flow), and the ion director thus allows the first vacuum chamber 24 to operate at relatively high pressures. This is advantageous because the relatively high pressure provides a suitable background gas in which the declustering device can cause the clusters to oscillate, thereby declustering them.

[0112] As mentioned above, the declustering device can be located upstream, downstream, or inside the ion guide. Figure 4A and Figure 4B The location of the declustering device within the ion guide is schematically shown in one embodiment.

[0113] Figure 4A The above shows about Figures 3A to 3C A schematic side view of the ion director shown and described. The declustering device may be located within or downstream of the ion director. For example, the declustering device may be located downstream of the transition region 33 of the ion director, within portion 32 of the ion director (e.g., Figure 3A (As shown). This location is... Figure 4A The circled portion is shown in the image.

[0114] Figure 4B It shows Figure 4A A schematic perspective view of the circular portion. This shows a declustering device arranged within portion 32 of the ion guide. The declustering device shown is related to... Figure 1B The declustering device in the embodiment is the same, but other embodiments of the declustering device can be used. The printed circuit board 35 can be arranged on opposite sides of the ion guiding and declustering device and connected to the electrodes 1, 3, 34-37 of these components for applying various voltages to the electrodes.

[0115] Figure 5 A to Figure 5 C shows the mass spectrometry data used to analyze the alcohol dehydrogenase protein complex, which was dissolved in 200 mM ammonium acetate to 10 μM and infused at 5 μL / min via a standard flow electrospray ionization source on a Q-ToF mass spectrometer. No desalting procedure was used prior to infusion.

[0116] Figure 5 Figure A shows mass spectrometry data obtained using a Synapt G2-Si mass spectrometer, where a relatively low voltage of 50 V was applied to the sampling cone. This causes ions to be accelerated through the device at a relatively low rate, resulting in relatively low-energy collisions between the clusters and the background gas. Figure 5As can be seen from A, the observed multi-charge peaks are relatively broad due to the ion clusters. Figure 5 B shows mass spectrometry data obtained using a Synapt G2-Si mass spectrometer, where a relatively high voltage of 200 V was applied to the sampling cone. This causes ions to pass through the device at a relatively high rate, resulting in relatively high-energy collisions between clusters and the background gas, causing declustering of analyte ions and adducts. Figure 5 B shows that, with Figure 5 Compared to A, the observed mass spectrum peaks are relatively narrower, and the signal-to-noise ratio is improved.

[0117] Figure 5 C illustrates mass spectrometry data obtained according to an embodiment of the present invention, wherein mass spectrometry data is used. Figure 5 A has a relatively low cone voltage, but provides a declustering device as described herein. The declustering AC voltage applied to the device has a square wave waveform, a frequency of 45 kHz, and a peak-to-peak voltage of 280 V. From Figure 5 As can be seen from C, even with a relatively low cone voltage, the observed mass spectrum peaks exhibit relatively high clarity and signal-to-noise ratio when a declustering AC voltage is applied, for example, compared to... Figure 5 Compared to B.

[0118] Further experimental data were obtained by mass analysis of the alcohol dehydrogenase protein complex dissolved in 200 mM ammonium acetate to 10 μM and infused at 5 μL / min via a standard flow electrospray ionization source. No desalting procedure was used prior to infusion.

[0119] Figures 6A to 6C The graphs show mass spectrometry data obtained using known cone voltage declustering techniques and those obtained using embodiments of the present invention. Figure 6A The image shows mass spectrometry data obtained when a relatively low voltage of 50 V was applied to the sampling cone. This causes the ions to be accelerated through the device at a relatively low rate, resulting in relatively low-energy collisions between the clusters and the background gas. Figure 6A It can be seen that the m / z peaks are relatively broad; for example, the FWHM of the peak closest to m / z = 5466 is 35 m / z. Figure 6B The image shows mass spectrometry data obtained when a relatively high voltage of 200 V was applied to the sampling cone. This causes ions to be accelerated through the device at a relatively high rate, resulting in relatively high-energy collisions between clusters and the background gas, causing declustering of analyte ions and adducts. Figure 6B It can be seen that the observed mass spectrum peaks are narrower (e.g., the FWHM of the peak closest to m / z = 5466 is 18 m / z), and the signal-to-noise ratio is improved. Figure 6CMass spectrometry data obtained according to an embodiment of the invention are shown, where a relatively low cone voltage of 50V is used, but a declustering apparatus as described herein is provided. The declustering AC voltage applied to the apparatus has a substantially sinusoidal waveform, a frequency of 60kHz, and a peak-to-peak voltage of 280V. From Figure 6C It can be seen that even with a relatively low cone voltage, the observed m / z peaks still exhibit high clarity (e.g., the FWHM of the peak closest to m / z = 5466 is 16 m / z) and signal-to-noise ratio when a declustering AC voltage is applied.

[0120] Figures 7A to 7D It shows the correspondence as about Figure 6C The difference lies in the curve of mass spectrometry data obtained as described in the implementation scheme. Figure 7A The spectrum data obtained using a declustered AC voltage at a frequency of 55 kHz is shown. Figure 7B The spectrum data obtained using a declustered AC voltage at a frequency of 60 kHz is shown. Figure 7C The spectral data obtained using a declustered AC voltage at a frequency of 65 kHz is shown, and Figure 7D The spectral data obtained using a declustered AC voltage at a frequency of 70 kHz is shown. Figures 7A to 7D It can be seen that the observed peak width decreases as the frequency of the declustering AC voltage decreases. For example, the FWHM peak closest to m / z = 5466 is... Figure 7A The value is 15 m / z. Figure 7B The value is 16 m / z. Figure 7C The value is 18 m / z and in Figure 7D The value is 23 m / z.

[0121] Figures 8A to 8E As shown in the example about Figure 6C The difference lies in the curve of mass spectrometry data obtained as described in the implementation scheme. Figures 8A to 8E The spectral data obtained using a declustering AC voltage at a frequency of 55 kHz are shown, and the peak-to-peak amplitude of the declustering AC voltage is shown for... Figures 8A to 8E Each curve in the different curve graphs is different. Figure 8A The graph shown is obtained when the de-clustering AC voltage has a peak-to-peak amplitude of 280V. Figure 8B The graph shown is obtained when the de-clustering AC voltage has a peak-to-peak amplitude of 260V. Figure 8C The graph shown is obtained when the de-cluster AC voltage has a peak-to-peak amplitude of 175V. Figure 8D The graph shown is obtained when the de-cluster AC voltage has a peak-to-peak amplitude of 90V. Figure 8E The graph shown is obtained when the de-cluster AC voltage has a peak-to-peak amplitude of 0V.

[0122] Figures 9A to 9E As shown in the example about Figure 6C The difference lies in the curve of mass spectrometry data obtained as described in the implementation scheme. Figures 9A to 9E The spectral data obtained using a declustering AC voltage at a frequency of 70 kHz are shown, and the peak-to-peak amplitudes of the declustering AC voltage are shown for... Figures 9A to 9E Each curve in the different curve graphs is different. Figure 9A The graph shown is obtained when the de-clustering AC voltage has a peak-to-peak amplitude of 280V. Figure 9B The graph shown is obtained when the de-clustering AC voltage has a peak-to-peak amplitude of 260V. Figure 9C The graph shown is obtained when the de-cluster AC voltage has a peak-to-peak amplitude of 175V. Figure 9D The graph shown is obtained when the de-cluster AC voltage has a peak-to-peak amplitude of 90V. Figure 9E The graph shown is obtained when the de-cluster AC voltage has a peak-to-peak amplitude of 0V.

[0123] from Figures 8A to 8E It can be seen that once the amplitude of the declustering AC voltage has increased to a certain level, the signal-to-noise ratio of the ions of interest (e.g., the peak closest to m / z = 5466) increases significantly, and the peak width decreases with further increases in amplitude. Figures 9A to 9E The same trend can be observed in [the context].

[0124] Figure 10 Four graphs are shown illustrating how the width of the peak closest to m / z = 5466 varies as a function of the amplitude of the declustered AC voltage for four declustered AC voltages at four different frequencies. Curve A is for a declustered AC voltage at 70 kHz, curve B is for a declustered AC voltage at 65 kHz, curve C is for a declustered AC voltage at 60 kHz, and curve D is for a declustered AC voltage at 55 kHz.

[0125] Figure 11 Four graphs are shown illustrating how the peak area between m / z = 5440 and 5540 varies as a function of the declustered AC voltage amplitude for four declustered AC voltages with four different frequencies. Curve A represents the declustered AC voltage at 70 kHz, curve B represents the declustered AC voltage at 65 kHz, curve C represents the declustered AC voltage at 60 kHz, and curve D represents the declustered AC voltage at 55 kHz.

[0126] The frequency and amplitude of the declustering AC voltage determine the location of the low m / z cutoff. To maximize declustering, the low m / z cutoff can be set just above the m / z of the ion of interest. However, this results in some ion loss. Transmission is not enhanced, but spectral quality is improved (lower FWHM).

[0127] As can be seen from the figures above, increasing the amplitude of the declustering AC voltage generally increases the signal-to-noise ratio and decreases the FWHM and peak area. Furthermore, as the frequency of the declustering AC voltage decreases, the observed peak width generally decreases. This is because the declustering AC voltage causes the device to function, to some extent, as a high-pass filter. Generally, ions with relatively low mass-to-charge ratios tend to have relatively high mobility, and therefore these ions tend to oscillate with a larger amplitude by the declustering AC voltage than ions with higher mass-to-charge ratios. Therefore, when the declustering AC voltage is set to the frequency and amplitude necessary for declustering ions with relatively high mass-to-charge ratios, this generally results in ions with lower mass-to-charge ratios oscillating with a relatively large amplitude and being lost to the electrodes of the declustering device (e.g., by impacting the electrodes to which the declustering AC voltage is applied). This effect can be seen in Figures 8 and 9. For example, ion swarms with relatively low mass-to-charge ratios near m / z = 1000 disappear as the amplitude of the declustering AC voltage increases. These ions impact the upper and lower electrodes 1 of the declustering device and do not propagate through the device. As the declustering AC voltage further increases (e.g., from...), Figures 8C to 8B The ion cluster with a mass-to-charge ratio of approximately m / z = 3500 disappears, leaving only the ions of interest with a mass-to-charge ratio of approximately m / z = 5500. However, if the amplitude of the declustering AC voltage is increased too much, some ions of interest may be lost, such as in... Figure 8A In this process, ions with a mass-to-charge ratio of approximately m / z = 5500 are lost. Similarly, changing the frequency of the de-clustering AC voltage alters the ions transported by the de-clustering device.

[0128] Therefore, it may be necessary to change the amplitude and / or frequency of the declustering AC voltage in order to optimize the declustering and / or transport of ions of interest.

[0129] For example, the amplitude and / or frequency of the declustering AC voltage can vary over time. In these embodiments, clusters can be separated by mass-to-charge ratio or ion mobility before being transferred to the declustering device, and the amplitude and / or frequency of the declustering AC voltage can vary over time based on the mass-to-charge ratio or ion mobility of the clusters transferred to the declustering device. For example, clusters can be separated by a scanning / stepping mass filter (e.g., a quadrupole mass filter), a mass-selective ion trap, or other separator device to transfer ions with different mass-to-charge ratios to the declustering device at different times. The separator can be scanned / stepped in this manner over a period of time, and the variation of the declustering AC voltage can be synchronized with this period of time, so that clusters with different mass-to-charge ratios experience different AC amplitudes and / or frequencies in the declustering device. Similarly, clusters can be separated by an ion mobility separator to transfer ions with different mobilities to the declustering device at different times. The separator can separate ions over a period of time, and the variation of the declustering AC voltage can be synchronized with this period of time, so that clusters with different mobilities experience different AC amplitudes and / or frequencies in the declustering device.

[0130] Alternatively or additionally, multiple different de-clustering AC voltages with different amplitudes and / or frequencies can be applied at different axial locations along the length of the de-clustering device. The de-clustering AC voltages applied at progressively downstream axial locations of the de-clustering device can have progressively lower amplitudes. This is advantageous because the mobility of clusters tends to increase as they move along the de-clustering device and cause adducted ions to detach; therefore, reducing the amplitude of the de-clustering AC voltages along the de-clustering device helps prevent these ions from oscillating with larger amplitudes, which would result in the loss of these ions to the electrodes of the de-clustering device. For the same purpose, the different de-clustering AC voltages can (alternatively or additionally) have different frequencies.

[0131] A mass filter, such as a desorption quadrupole mass filter, can be provided downstream of the declustering device. The mass filter has a mass-to-charge ratio transport window, and ions with mass-to-charge ratios within this window are transported, while ions with mass-to-charge ratios outside this window are not transported and are filtered out by the mass filter. This window can be scanned over time, allowing the mass filter to transport different mass-to-charge ratios (only) at different times. The amplitude and / or frequency of the declustering AC voltage applied to the declustering device can vary over time in conjunction with the mass-to-charge ratio transport window of the mass filter. For example, the amplitude and / or frequency of the declustering AC voltage can vary synchronously with the mass-to-charge ratio transport window of the mass filter, for example, so as to transport only ions that have already been declustered in a relatively large quantity. An example of this will be referred to... Figures 12A to 12D Describe it.

[0132] Figures 12A to 12C A schematic graph showing how the mass spectrum changes as the amplitude of the declustering AC voltage increases is shown. Figure 12AThe mass spectra obtained using a relatively low-amplitude declustering AC voltage on the declustering apparatus are shown. The ions represented by the peak with the lowest mass-to-charge ratio have been relatively well declustered, as indicated by the relatively narrow peaks. However, the two higher m / z peaks are relatively broad, which is due to the relatively poor declustering of these ions. Figure 12B The mass spectrum obtained using a higher amplitude declustering AC voltage on the declustering device is shown. Due to the aforementioned reasons (i.e., lower m / z ions oscillate to higher amplitudes and are thus lost to the electrode as the amplitude of the declustering AC voltage increases), the intensity of the lowest m / z peak has significantly decreased because the increased amplitude of the declustering AC voltage leads to an increase in the low-mass cutoff of the declustering device. The ions represented by the two higher m / z peaks have been declustered to a greater extent, which is why the intensity of these two peaks is higher than that of the lower m / z peaks. Figure 12A The reason for its narrower shape. It can be seen that... Figure 12B The middle m / z peaks are relatively well declustered and narrow, while the highest m / z peak remains relatively wide and is not well declustered. Figure 12C It shows the use of ratio Figure 12B Mass spectra obtained with higher amplitude declustering AC voltages. The lowest m / z peak has disappeared, and the intensity of the middle m / z peaks has decreased significantly. This is because the increase in the amplitude of the declustering AC voltage leads to an increase in the low-mass cutoff of the declustering device. The ions represented by the highest m / z peak have been declustered to a relatively high degree, which is the peak ratio. Figure 12A and Figure 12B The reason for the narrower width in the middle. Alternatively or additionally, the frequency of the de-cluster AC voltage can be varied over time to achieve this.

[0133] As described above, a mass filter can be provided downstream of the declustering device, the mass filter having a mass-to-charge ratio transport window that scans in sync with the amplitude (and / or frequency) of the declustering AC voltage applied to the declustering device. The mass filter can scan in sync with the declustering AC voltage such that essentially only declustered ions are transported downstream through the mass filter. The upper and lower limits of the mass-to-charge ratio transport window are... Figures 12A to 12C The line shown is a vertical dashed line. From Figure 12A It can be seen that at the first moment when the de-cluster AC voltage has a first (low) amplitude, the mass-to-charge ratio transfer window of the mass filter is set to transfer ions with a first (low) mass-to-charge ratio range. Thus, only ions from the first (low) mass-to-charge ratio range can be transferred. Figure 12A The lowest m / z peak indicates that highly declustered ions are transported. From Figure 12B As can be seen, at the second time when the de-cluster AC voltage has a relatively high (medium) amplitude, the mass-to-charge ratio transport window of the mass filter is set to transport ions with a relatively high (medium) mass-to-charge ratio range. Thus, only ions from the high (medium) mass-to-charge ratio range can be transported. Figure 12B The middle m / z peak in the image indicates that highly declustered ions are being transported. From Figure 12CIt can be seen that at the third time when the de-cluster AC voltage has a high (maximum) amplitude, the mass-to-charge ratio transport window of the mass filter is set to transport ions with a high (maximum) mass-to-charge ratio range. Thus, only ions from the high mass-to-charge ratio range can be transported. Figure 12C The highest m / z peak in the spectrum indicates that highly declustered ions are transported.

[0134] Figure 12D The combined mass spectra obtained by scanning the mass filter in this manner are shown. By comparison... Figure 12D and Figure 12A As can be seen, this significantly reduces the width of the m / z peak and provides higher resolution mass spectrometry data.

[0135] Removing adducted ions removes low m / z ions, thereby reducing the amount of charge entering the mass analyzer and thus reducing the potential harmful space charge effect in the instrument.

[0136] Although the invention has been described with reference to preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the invention as set forth in the appended claims.

[0137] For example, although the de-clustered AC voltage has been described above as having a basically square or sinusoidal waveform, it is conceivable that it can have any other waveform shape, such as a triangle. Ideally, the peak positive amplitude and peak negative amplitude of the waveform are the same, that is, the waveform is symmetrical, but it is conceivable that these peak amplitudes can be different and the waveform can be asymmetrical.

[0138] The de-clustering apparatus described herein can be located in an atmospheric pressure region or a vacuum chamber, utilizing the background gas therein to provide collisions during de-clustering mode. Alternatively, the de-clustering apparatus can be provided in a gas chamber (e.g., its own dedicated gas chamber), and the gas pressure and / or gas composition in said gas chamber can be controlled, for example, to optimize for de-clustering and / or non-de-clustering modes. The gas chamber can maintain different gas pressures and / or gas compositions in the two different modes.

[0139] Although the declustering device has been described as part of or immediately downstream of the ion source, it is envisioned that it could be placed at other downstream locations within the spectrometer.

Claims

1. A method of mass spectrometry and / or ion mobility spectrometry, comprising: providing an ion guide comprising a plurality of electrodes and having a background gas in the ion guide; applying an RF voltage to the electrodes of the ion guide to radially confine ions in the ion guide; transmitting clusters of analyte ions and adduct species into the ion guide; in a first mode, applying one or more AC voltages to the ion guide so as to oscillate the clusters, such that the clusters collide with molecules of the background gas, and such that adduct species in the clusters separate from the analyte ions, wherein the one or more AC voltages have an amplitude and / or a frequency that is different from an amplitude and / or a frequency of the RF voltage; and (i) varying a speed at which the clusters are pushed along the ion guide during the first mode; and / or (ii) varying the amplitude and / or the frequency of the one or more AC voltages as the clusters travel along the ion guide.

2. The method of claim 1, wherein the amplitude and / or the frequency of the one or more AC voltages varies over time.

3. The method of claim 2, comprising transmitting ions from the ion guide into a mass filter and mass filtering the ions in the mass filter; wherein a mass-to-charge ratio or range of mass-to-charge ratios selectively transmitted by the mass filter varies over time in synchrony with the variation of the amplitude and / or the frequency of the one or more AC voltages over time; or transmitting ions from the ion guide into a mobility filter and mobility filtering the ions in the mobility filter; wherein a mobility or range of mobilities selectively transmitted by the mobility filter varies over time in synchrony with the variation of the amplitude and / or the frequency of the one or more AC voltages over time.

4. The method of claim 2 or 3, comprising mass analyzing ions from the ion guide in a mass analyzer; wherein operation of the mass analyzer varies over time so as to vary a mass-to-charge ratio or range of mass-to-charge ratios that the mass analyzer can analyze or is optimized to analyze; and wherein this mass-to-charge ratio or range of mass-to-charge ratios varies over time in synchrony with the variation of the amplitude and / or the frequency of the one or more AC voltages.

5. The method of claim 2 or 3, comprising separating the clusters by mass-to-charge ratio or ion mobility prior to transmitting the clusters into the ion guide, and varying the amplitude and / or the frequency of the one or more AC voltages over time based on the mass-to-charge ratio or ion mobility of the clusters transmitted into the ion guide.

6. The method of claim 1 or 2, wherein the one or more AC voltages are a plurality of different AC voltages having different amplitudes and / or frequencies, and wherein the different AC voltages are applied at different axial locations along a length of the ion guide.

7. The method of claim 6, wherein the AC voltages applied at progressively more downstream axial positions of the ion guide have progressively lower amplitudes.

8. The method of claim 1 or 2, wherein the electrodes of the ion guide define a conduit through which the cluster is guided, wherein in the first mode, the RF voltages applied to the electrodes radially confine the ions and push the ions towards a central axis through the conduit, and wherein the AC voltages cause the cluster to oscillate around the central axis.

9. The method of claim 8, wherein the AC voltages cause the ions to oscillate around the central axis such that the cluster has the same average oscillation amplitude on either side of the axis.

10. The method of claim 1 or 2, wherein the step of varying the speed of pushing the cluster along the ion guide comprises repeatedly travelling a transient DC voltage along the ion guide in order to push the cluster along the ion guide; and wherein the amplitude of the transient DC voltage and / or the speed and / or frequency at which the transient DC voltage moves along the ion guide varies over time, thereby varying the speed of pushing the cluster along the ion guide in the first mode.

11. The method of claim 1 or 2, wherein the step of varying the speed of pushing the cluster along the ion guide comprises generating an axial electric field along the ion guide by simultaneously applying different DC voltages to different electrodes of the ion guide, and varying the different DC voltages in order to vary the magnitude of the electric field, and thereby vary the speed of pushing the cluster along the ion guide in the first mode.

12. The method of claim 1 or 2, wherein during the first mode, the background gas is maintained at a pressure of between 0.01 mbar and 10 mbar.

13. The method of claim 1 or 2, comprising operating the ion guide in a second mode in which the one or more AC voltages are not applied to the ion guide.

14. The method of claim 13, comprising switching between the first mode and the second mode when the cluster is passing through the ion guide.

15. The method of claim 1 or 2, comprising ionizing an analyte solution in order to generate the cluster, wherein the analyte solution comprises a membrane protein dissolved in a solvent using a detergent, and wherein the analyte ions in the cluster are membrane protein ions and the adduct species in the cluster are detergent molecules.

16. The method of claim 1 or 2, comprising performing mass analysis and / or ion mobility analysis on the analyte ions and any remaining cluster downstream of the ion guide in order to obtain mass peaks and / or mobility peaks of the analyte ions and the remaining cluster, respectively.

17. The method of claim 16, comprising determining a width and / or signal-to-noise ratio of one or more of the peaks, and varying the frequency and / or amplitude of the one or more AC voltages during the first mode so as to vary the width and / or signal-to-noise ratio of peaks of analyte ions and clusters subsequently analysed.

18. The method of claim 1 or 2, wherein the method is performed on a mass spectrometer or a mobility spectrometer, the mass spectrometer or mobility spectrometer comprising: a first vacuum chamber having an inlet aperture; a second vacuum chamber adjacent the first vacuum chamber; and a differential pumping aperture separating the first and second vacuum chambers; wherein the ion guide is arranged in the first vacuum chamber.

19. The method of claim 18, wherein the first vacuum chamber comprises an ion guide device having a first portion that guides ions along a first axial path, a second portion that guides ions along a second axial path, and a transition portion that pushes ions from the first axial path onto the second axial path, wherein the second axial path is different from the first axial path.

20. The method of claim 19, wherein the ion guide is part of, or downstream of, the ion guide device.

21. A mass or mobility spectrometer comprising: an ion guide comprising a plurality of electrodes and a background gas in the ion guide; an RF voltage source for applying an RF voltage to the electrodes of the ion guide to radially confine ions in the ion guide; one or more AC voltage sources for applying one or more AC voltages to the ion guide in a first mode so as to oscillate clusters of analyte ions and adduct species, so that the clusters collide with molecules of the background gas, and so that adduct species in the clusters separate from the analyte ions, wherein the AC voltages have an amplitude and / or frequency that is different from that of the RF voltage; and control circuitry configured to control the spectrometer so as to: (i) vary a speed at which the clusters are pushed along the ion guide during the first mode; and / or (ii) vary the amplitude and / or frequency of the one or more AC voltages as the clusters travel along the ion guide.

22. The spectrometer of claim 21, comprising a DC voltage source connected to the electrodes of the ion guide, wherein the control circuitry controls the DC voltage source to apply a DC voltage successively to different ones of the electrodes, thereby repeatedly travelling a transient DC voltage along the ion guide; wherein an amplitude of the transient DC voltage and / or a speed and / or frequency at which the transient DC voltage moves along the ion guide varies with time, to vary a speed at which the clusters are pushed along the ion guide.

23. The spectrometer of claim 21 or 22, comprising a DC voltage source connected to electrodes of the ion guide, wherein the control circuitry controls the DC voltage source to simultaneously apply different DC voltages to different ones of the electrodes to produce an axial electric field along the ion guide, and to vary the different DC voltages over time so as to vary a magnitude of the electric field to vary a speed of pushing the cluster along the ion guide.

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