Ion guide, method of manipulating ions using ion guide, mass spectrometry method, mass spectrometer, and computer software

Abstract

An ion guide is provided, the ion guide comprising: an inlet region for receiving ion packets; a first exit region for ejecting ions; a second exit region for ejecting ions; and a beam separation electrode arranged to direct a first portion of the ion packet toward the first exit region and a second portion of the ion packet toward the second exit region.

Description

Field of the Invention

[0001] The field of the present invention is liquid chromatography mass spectrometry (LC-MS). Specifically, the present invention relates to tandem mass spectrometry in which precursor ions and fragment ions are analyzed. More specifically, the present invention relates to improving the dynamic range of MS1 scans in tandem mass spectrometry. The present invention relates specifically but not exclusively to advanced hybrid mass spectrometers having multiple analyzers. Background Art

[0002] Standard tandem liquid chromatography mass spectrometry (LC-MS) methods include performing an "MS1" scan, in which ions having a wide m / z range are analyzed by a mass analyzer to produce an MS1 spectrum (containing information about precursor ions). Methods of operating an LC-MS also involve isolating and fragmenting ions from eluted analyte species to perform an "MS2" scan, in which ions within a narrow m / z range are isolated (e.g., using a quadrupole mass filter), those ions are fragmented, and the fragment ions are mass analyzed to produce an MS2 spectrum, which contains structural and quantitative information about the fragment ions.

[0003] In an LC-MS method, multiple MS2 scans are typically performed during chromatographic separation, where the m / z isolation range is different for each MS2 scan. The MS2 (or "MS / MS") spectrum is supported by an MS1 (or "MS" or "full MS") survey scan, which provides high-quality peak information such as accurate mass data and precursor intensities of a wide range of unfragmented precursor ions.

[0004] For data-independent acquisition (DIA) methods, the MS1 scan is optional and can be skipped to allow time to generate additional MS2 spectra. The list of m / z targets for each of the multiple MS2 scans can be a stepwise increasing / decreasing m / z list across the m / z range of interest. One such example is described in EP 3,410,463, which is incorporated herein by reference. The resulting precursor information can be used for quantification, while the fragment information can be used for identification.

[0005] In data-dependent acquisition (DDA), an MS1 step is required to generate a list of precursor targets for MS2 analysis. The list of m / z targets for each of the multiple MS2 scans corresponds to the list of precursor ions identified in the MS1 scan.

[0006] To perform mass analysis of ions, the ions (fragment ions or precursor ions) are typically first accumulated in an ion trap and then the accumulated ions are ejected as an ion packet into a mass analyzer for mass analysis. Accumulation improves the sensitivity of the instrument, but care is required during the fill time of the ion trap for the ions (so-called "automatic gain control" AGC) to avoid harmful space charge effects.

[0007] One problem with existing methods is that the dynamic range of the MS1 spectrum is limited. Depending on the sample, the concentration of digested peptides varies by more than 10 orders of magnitude, but the dynamic range of a single-shot spectrum in a Fourier transform mass analyzer such as an electrostatic Orbitrap mass analyzer (e.g., Orbitrap TM manufactured by Thermo Fisher Scientific TM FT mass analyzer) may be limited to about 4 orders of magnitude. Additionally, the number of ions that can be injected into the Orbitrap mass analyzer is limited to about 10 5 by the capacity of the accumulation C-trap. Thus, low-intensity peptide precursor signals may be suppressed. For DDA experiments, these problems are significant and may lead to precursor targets being missed. For DIA experiments, the lack of good precursor data hinders identification and quantification.

[0008] One way to improve the dynamic range of the MS1 spectrum is the "Boxcar" method (as described in Meier et al., Nature Methods, 2018, Vol. 15, pp. 440 - 448) and the high dynamic range (HDR) method described in UK Patent Application No. 2211790.7, which is incorporated herein by reference. In these methods, the wide mass range being analyzed is subdivided into multiple narrower isolation windows. For each isolation window, a separate injection is made into the C-trap, with different fill times depending on the ion current. By this method, densely populated m / z regions are attenuated while sparsely populated m / z regions are amplified. Thus, the detection sensitivity of relatively weak peaks is improved, and the effective dynamic range of the scan is increased. However, the use of these methods significantly increases the time required to accumulate ions, especially when a large number of isolation windows are needed. Due to the long fill times for low-level windows and the time required to switch the quadrupole and ion source voltages, HDR scans may require a large amount of additional time and affect the rate at which MS2 scans can be performed. Thus, these methods can reduce the time available for MS2 scans in fast LC-MS methods, which is limited by the time it takes for the sample to elute from the column.

[0009] The multi-window HDR method also has the drawback of discarding a large number of ions due to quadrupole isolation. The loss of ions can be addressed via a pre-accumulation process. For example, a trapped ion mobility device can be used to pre-accumulate ions before the quadrupole and release the ions in a mass / mobility-correlated manner synchronized with the mass filter, thus greatly reducing ion loss (as described in Meier et al., Molecular Cellular Proteomics, 2018, Vol. 17, pp. 2524 - 2545).

[0010] Differential mobility filtering can be used to improve proteomics performance by removing analytically less useful singly charged ions from the aggregated population (as described in Hebert et al., Anal. Chem., 2018, Vol. 90, pp. 9529-9537). This can be advantageous in low sample or single cell experiments where the analyte signal is small compared to the singly charged solvent background signal.

[0011] In DIA experiments, the MS2 spectra can retain a proportion of unfragmented precursor ions (in an amount not sufficient to be analytically useful). Some instruments offer an optional feature called "stepped collision energy" described in US 9,536,717. This feature provides multiple separate injections into the collision cell (also known as the "fragmentation chamber") at a range of collision energies, and then the summed ion population is transferred to a C-trap / Fourier transform mass analyzer and analyzed together. This variation in energy increases the probability that one of the energies used is optimal for fragmenting the precursor ions. However, this method also uses many collision energies that are not optimal for fragmenting the precursor ions, along with the associated cost of ion beam time. The actual act of changing the collision energy and making a second or third injection in the collision cell may not be overly time-intensive, and in some cases, there may be an additional 1-3 ms overhead on a scan cycle of >40 ms.

[0012] US 8,686,350 describes a method where different types of ions can be accumulated in an ion trap before being ejected into a mass analyzer. In one example, a combination of two types of ions with the same narrow mass range is injected into the ion trap, one ion being fragmented and one ion remaining as an intact precursor. This allows for greater confidence that the precursor ions can be detected and mass measured accurately. However, quantification may be affected by the proportion of unfragmented precursors left by the MS2 injection. SUMMARY OF THE INVENTION

[0013] There is provided an ion guide comprising:

[0014] an inlet region for receiving a packet of ions;

[0015] a first outlet region for ejecting ions;

[0016] a second outlet region for ejecting ions; and

[0017] a beam separation electrode arranged to direct a first portion of the packet of ions towards the first outlet region and a second portion of the packet of ions towards the second outlet region.

[0018] There is provided an ion deflector that separates a beam according to beam width. This provides a branched beam path where ions can be guided along two paths simultaneously (without forcing a choice between them). The ion deflector can be used to facilitate simultaneous filling of SIM injection for HDR MS1 scans in conjunction with a series of MS2 scans.

[0019] Many other uses of separating ion beams are also possible. For example, separating ions for any other purpose, such as alternative fragmentation.

[0020] A second part of the ion grouping can be the remainder of the ion grouping after a first part of the ion grouping has been separated. In other words, the ion grouping can consist of a first part and a second part.

[0021] The ion deflector can be an ion beam splitter. In other words, the ion deflector can be used to separate a continuous ion beam into two separate continuous ion beams. In cases where ions “groups” are mentioned herein, this will illustrate the operation of the ion deflector, and the groups can be considered part of a continuous beam. The present invention is not limited to processing discrete ion groupings (although discrete ion groupings can be separated in some examples).

[0022] The ion deflector can be an ion deflector for a mass spectrometer.

[0023] The ion deflector can be configured to receive an ion grouping, direct a first part of the received ions to a first ion storage device and direct a second part of the ion grouping to a second ion storage device or a mass analyzer.

[0024] The inlet region can include an inlet hole.

[0025] The first outlet region can include a first outlet hole.

[0026] The second outlet region can include a second outlet hole.

[0027] The ion deflector can further include one or more electrodes (preferably multiple electrodes) for applying a DC gradient (and the ion deflector can be configured to apply a DC gradient). The DC gradient can urge ions away from the inlet region and towards the first outlet region and / or the second outlet region.

[0028] One or more electrodes can be RF electrodes arranged to generate a pseudopotential surface (e.g., a so-called “blanket electrode”). In other words, the RF electrodes can provide a DC gradient and a pseudopotential surface, and can be arranged on a first surface of the ion beam splitter.

[0029] One or more electrodes can be PCB printed electrodes.

[0030] One or more electrodes for applying a DC gradient may be provided on a first surface of the ion beam splitter.

[0031] The first surface may extend between an entrance region and a first and a second exit region.

[0032] The ion guide may further include a DC repeller provided on a second surface of the ion beam splitter that is opposite (and facing) the first surface. The entrance region, the first and second exit regions (and all corresponding apertures) may be between the first and second surfaces.

[0033] Alternatively, the ion guide may further include one or more electrodes provided on a second surface of the ion beam splitter that is opposite (and facing) the first surface. The entrance region, the first and second exit regions (and all corresponding apertures) may be between the first and second surfaces.

[0034] The beam separation electrode may be substantially wedge-shaped or pointed.

[0035] The beam separation electrode may include a vertex between the first and second exit regions.

[0036] The vertex may be offset from the central longitudinal axis of the ion guide. In other words, the vertex may be closer to one of the first and second output regions than to the other of the first and second output regions. The relative proportion of a first portion of the ions guided to the first exit region and a second portion of the ions guided to the second exit region may depend at least in part on the position of the vertex relative to the central longitudinal axis of the ion guide (i.e., the perpendicular distance from the central longitudinal axis to the vertex). If the vertex is positioned towards one exit region, the relative proportion of the ions guided to the other exit region may be greater.

[0037] The beam separation electrode may further include a base. The base of the beam separation electrode may be provided between the first and second exit regions.

[0038] The beam separation electrode may be aligned with the central longitudinal axis of the ion guide (even if the vertex is offset). In other words, the base of the beam separation electrode may be aligned with the central longitudinal axis of the ion guide.

[0039] The ion guide may further include one or more guard electrodes (e.g., DC electrodes) arranged to prevent ions from entering or leaving the ion beam splitter, except via the entrance and exit regions.

[0040] The ion entrance region (and in some examples, the ion entrance aperture of the ion entrance region) may be configured to receive an ion beam.

[0041] Multiple radio frequency electrodes may be parallel to each other.

[0042] The ion guide may further include a radio frequency voltage source configured to apply an alternating radio frequency phase to each of the multiple radio frequency electrodes.

[0043] The ion guide may further include a DC potential source configured to apply a DC gradient across the radio frequency surface. The DC gradient may be configured to direct an ion beam from an inlet region to a first outlet region and a second outlet region. In this way, ions may be trapped within a large volume on the radio frequency surface. The ions may be gently directed to the first outlet region and the second outlet region by the DC gradient.

[0044] Due to the low mean free path of ions, a DC gradient is particularly desirable in systems operating at higher pressures (or lower vacuum). Ions may stop flying due to excessive collisions with the background gas. Then, these ions may not reach the analyzer in a timely manner, resulting in losses or causing the ions to reach the analyzer for incorrect measurements. Ions remaining in the ion guide may form an unwanted space charge effect on other ions in flight. The DC gradient may help ensure that ions are removed from the ion guide and reach the analyzer, thereby reducing transmission losses and transit time losses.

[0045] The DC gradient may include a transverse component and a longitudinal component. In this way, the DC gradient may use the transverse component to adjust the proportion of the ion beam traveling to the first outlet region and the second outlet region, and may use the longitudinal component to direct the ion beam from the inlet region at one end of the ion guide to the outlet region at the other end of the ion guide. The transverse component may be orthogonal to the longitudinal component (and is sometimes referred to as the orthogonal component). The longitudinal component may be in the direction of the longitudinal axis of the ion guide (and is sometimes referred to as the axial component).

[0046] The radio frequency electrodes may include elongated electrode plates arranged such that the plane of each plate is parallel to the plane of an adjacent plate. Advantageously, the electrode plates may prevent ions from approaching the first surface.

[0047] The radio frequency electrodes may be arranged in a grid. In this way, the radio frequency electrodes may be used to apply a DC gradient or a traveling wave in the axial direction and the orthogonal direction.

[0048] The ion guide may include a top plate on a second surface opposite the first surface, the top plate being configured to apply a repulsive voltage that repels the ion beam towards the first surface. In this way, these ions may be compressed close to the first surface.

[0049] The top plate may include a DC potential source, where the DC potential source may be configured to apply a DC gradient to the top plate. In this way, the top plate may be configured to apply a DC gradient.

[0050] The top plate may include a PCB and a plurality of DC electrodes printed on the PCB. Advantageously, the DC electrodes may be printed in a shape that permits the application of a DC gradient. If the top plate is configured to apply a repulsive voltage and the top plate includes DC electrodes printed on the PCB, the repulsive voltage prevents ions from approaching the PCB.

[0051] The plurality of DC electrodes may be arranged in a grid. In this way, a two-dimensional DC gradient can be applied.

[0052] The plurality of DC electrodes may be arranged in a horseshoe configuration, where the prongs of the horseshoe are adjacent to the first and second exit regions.

[0053] The plurality of DC electrodes may be connected by resistors. In this way, a DC gradient can be applied.

[0054] The DC potential source may include a plurality of auxiliary DC electrodes, where each auxiliary DC electrode is positioned between the RF electrodes. In this way, both the RF electrodes and the DC potential source can be arranged on or adjacent to the first surface.

[0055] The plurality of auxiliary DC electrodes may include elongated electrode plates, and the RF electrodes may include elongated electrode plates that are arranged such that the plane of each plate is parallel to the plane of an adjacent plate, where the plane of the plates of the DC electrodes is parallel to the plane of the plates of the adjacent RF electrodes. In this way, the DC electrodes can be mounted between the RF electrodes, and a DC gradient can be applied that is strong enough to reach the center of the ion guide.

[0056] The auxiliary DC electrodes may include elongated electrode plates that are wedge-shaped in the plane of the plate.

[0057] The RF electrodes may include: elongated electrode plates that are arranged such that the plane of each plate is parallel to the plane of an adjacent plate, and the first surface may include a PCB, where the auxiliary DC electrodes include printed electrodes located between the RF electrodes.

[0058] The ion guide may include a second surface facing the first surface, the second surface including a plurality of RF electrodes and a plurality of auxiliary DC electrodes arranged on the surface, each of the plurality of auxiliary DC electrodes being mounted between the RF electrodes. Both the first surface and the second surface may include electrodes that provide a pseudopotential surface and apply a DC gradient.

[0059] The first surface may be inclined relative to the second surface such that the distance between the first surface and the second surface decreases closer to the exit region. In this way, as the ions travel from the entrance region towards the exit region, the ion beam can spread spatially.

[0060] The ion guide may further include: a first side guard plate positioned on a first side of the RF surface; and a second side guard plate positioned on a second side of the RF surface, the second side being opposite to the first side. In this way, leakage of ions from the sides of the ion guide can be prevented. The first side guard plate and the second side guard plate may be configured to prevent ions from leaving the ion guide via the first side or the second side, and / or to shape the ion cloud.

[0061] The first side guard plate and the second side guard plate may be parallel to each other.

[0062] The first side guard plate and the second side guard plate may include a first wall and a second wall. In this way, leakage of ions and buffer gas from the sides of the ion guide can be physically prevented.

[0063] The first side guard plate and the second side guard plate may include a first protection electrode and a second protection electrode.

[0064] The first protection electrode and the second protection electrode may be configured to receive a repulsive DC voltage or an attractive DC voltage. If a repulsive DC voltage is applied, then ions can be repelled from the sides of the ion guide to keep the ions within the main volume of the ion guide as the ions travel between the inlet region and the outlet region. If an attractive DC voltage is applied, the ion cloud can be pulled towards the edges of the ion guide, thereby helping to spread the ion beam. In this example, an RF potential may be applied to inhibit the escape of ions from the device.

[0065] The width of the ion guide may be greater than the width of the inlet region (and / or the inlet aperture). The width of the ion guide may be defined as the distance between the first side guard plate and the second side guard plate. In some examples, the width of the ion guide may be at least twice or three times the width of the inlet region.

[0066] The first outlet region and the second outlet region may each be located between a respective one of the first side guard plate and the second side guard plate and the beam separation electrode. The width of each of the first outlet region and the second outlet region (and / or the first outlet aperture and the second outlet aperture) may be defined as the distance (measured perpendicular to the longitudinal axis) between the corresponding side guard plate and the base of the beam separation electrode.

[0067] The width of the base of the beam separation electrode may be sufficient such that the width of each of the outlet regions is narrow relative to the width of the ion guide. In some examples, the width of the ion guide may be at least twice or three times the width of each of the outlet regions.

[0068] The width of the base of the beam separation electrode may be less than the width of the ion guide.

[0069] An ion beam can enter via an entrance region as a focused beam (having a relatively narrow width) and can spread across the width of the ion guide as it travels through the ion guide, thereby widening the beam before beam separation by a beam separation electrode. Widening the beam can improve the precision of separating the beam into two parts in a desired ratio.

[0070] Diffusion of the ions can occur in an intermediate region of the ion guide between the entrance region and the exit region. The width of the intermediate region can be synonymous with the width of the ion guide (i.e., defined by the distance between the side guards). The length of the intermediate region can be defined by the distance between the entrance region and the apex of the beam separation electrode (or the projection of the apex onto the central longitudinal axis, where the apex is off-center).

[0071] The length of the intermediate region can be sufficient to allow the ion beam to spatially spread before reaching the beam separation electrode.

[0072] The ion guide can be configured to allow the ions to spatially spread in a direction orthogonal to the central longitudinal axis of the ion guide as the ions travel from the entrance region towards the exit region. The central longitudinal axis can be aligned with the main direction of ion travel. The exit region can be symmetrically disposed with respect to the central longitudinal axis of the ion guide. The entrance region can be located on the central longitudinal axis of the ion guide.

[0073] The beam separation electrode can first be used to divide the ion beam into two parts (e.g., across the apex of the beam separation electrode). Then, the beam separation electrode can converge each part to a corresponding exit region, narrowing the beam of each part (e.g., using the angled sides of a wedge-shaped beam separation electrode).

[0074] A method of manipulating ions using an ion guide is also provided. The ion guide includes: an entrance region; a first exit region; a second exit region; and a beam separation electrode. The method includes:

[0075] injecting a packet of ions via the entrance region; and

[0076] using the beam separation electrode to direct a first part of the packet of ions towards the first exit region and a second (remaining) part of the packet of ions towards the second exit region.

[0077] In some examples, the first part and the second part are non-zero such that at least some ions travel via the first exit region and at least some ions travel via the second exit region.

[0078] The first exit region can include a first exit hole. The method can also include ejecting the first part of the ions via the first exit hole.

[0079] The second exit region can include a second exit hole. The method can also include ejecting the second part of the ions via the second exit hole.

[0080] The inlet region may include an inlet orifice. Injecting an ion packet via the inlet region may include injecting the ion packet via the inlet orifice.

[0081] The ion deflector may further include one or more electrodes (preferably a plurality of electrodes). The one or more electrodes may be RF blanket electrodes and / or auxiliary DC electrodes. The one or more electrodes may be disposed on a first surface of the ion beam splitter.

[0082] The method may further include applying a radio frequency signal to the one or more electrodes to generate a pseudopotential surface.

[0083] The method may further include applying a longitudinal DC gradient (or traveling wave) to the one or more electrodes to urge the ions away from the inlet region and towards the first outlet region and / or the second outlet region.

[0084] The method may further include applying a transverse DC gradient (or traveling wave) to the one or more electrodes to urge the ions away from the first outlet region and towards the second outlet region (or vice versa) to adjust the relative proportion of the ion packets that make up the first portion and the second portion.

[0085] The transverse DC gradient may be orthogonal to the longitudinal DC gradient.

[0086] The method may further include applying a transverse DC gradient (or traveling wave) to the one or more electrodes to urge the ions away from the first outlet region and towards the second outlet region (or vice versa) such that substantially all of the ions in the ion packet are directed via the second outlet region. In other words, a negligible amount of ions may make up the first portion.

[0087] Also provided is a mass spectrometry method. The method includes the following steps for each of a plurality of sub-ranges selected from a total m / z range:

[0088] Configuring an ion beam splitter (e.g., an ion beam splitter according to the above examples) to direct ions towards a first ion destination and a second ion destination, wherein the first ion destination is

[0089] a first ion storage device;

[0090] Accumulating a sample of precursor ions to be analyzed in the first ion storage device, the precursor ions having an m / z value within the sub-range.

[0091] In option a), the second ion destination is a first mass analyzer, and the method further includes (for each of the plurality of sub-ranges) injecting a sample of fragmented precursor ions into the first mass analyzer, wherein the sample of fragmented precursor ions is formed by fragmentation of precursor ions having an m / z value within the sub-range.

[0092] In option b), the second ion destination is a second ion storage device, and the method further includes (for each of a plurality of sub-ranges) accumulating in the second ion storage device a sample of fragmented precursor ions for analysis in a first mass analyzer, wherein the sample of fragmented precursor ions is formed by fragmentation of precursor ions having m / z values within the sub-range.

[0093] The proposed method facilitates separating an ion beam proportionally based on the spatial distribution of the ion beam. Separating the ion beam can be used to create parallel accumulation regions for simultaneously accumulating precursor ions for HDR MS1 scans (SIM injection) alongside a series of MS2 scans.

[0094] The methods described herein can be used to establish high-quality HDR scans (or equivalent precursor data from multiple MS1 scans, where each scan spans multiple sub-ranges) using a Fourier transform mass analyzer.

[0095] The proposed method is suitable for quantification over a wide dynamic range of analyte ions. Normally performing separate MS1 and MS2 scans would involve extended delays to switch the ion source and quadrupole to scan through the mass range independently of the DIA cycle scans used to obtain fragment data. The number of ions to be processed for HDR scans is high and thus always consumes a significant proportion of the ion beam time.

[0096] The fragmented precursor ions in the sample of fragmented precursor ions can be formed by fragmentation of precursor ions having m / z values within the sub-range. More specifically, the sample of fragmented precursor ions can consist of fragmented precursor ions formed by fragmentation of precursor ions having m / z values within the sub-range.

[0097] The sample of precursor ions can consist of precursor ions having m / z values within the sub-range.

[0098] Samples of precursor ions for each of the plurality of sub-ranges can be combined together in a first ion storage device such that the first ion storage device contains precursor ions having m / z values from the total m / z range. In other words, ions from each sub-range can be present in the first ion storage device.

[0099] The method can further include analyzing the combined sample of precursor ions having m / z values from the total m / z range in a first mass analyzer or a second mass analyzer. In other words, multiple SIM injections can be combined together in the first ion storage device and analyzed together in the mass analyzer. While the precursor accumulation step is performed for each sub-range, the step of analyzing the combined sample of precursor ions can be performed once for the total m / z range.

[0100] In a first example, the method further includes ejecting ions from a first ion storage device into a second mass analyzer. In other words, there can be two mass analyzers in series, both downstream of the ion beam splitter.

[0101] In a second example, the first ion storage device is an intermediate ion storage device, where the method further includes configuring the ion beam splitter to transfer precursor ions accumulated in the first ion storage device to a third ion storage device for analysis in the second mass analyzer.

[0102] The third ion storage device can be between the ion filter and the ion beam splitter (specifically, the input region of the ion beam splitter). Ions can be transferred from the ion filter to the ion beam splitter via the third ion storage device.

[0103] In this case, ions can be transferred from a first “exit” region to an “entrance” region. Thus, in the step of transferring precursor ions accumulated in the first ion storage device to the third ion storage device, the first exit region can be used as an entrance, and the first entrance region can be used as an exit. In other words, the ions travel in a direction opposite to the normal.

[0104] The method can further include ejecting ions from the third ion storage device into the second mass analyzer.

[0105] The precursor ions transferred from the first ion storage device to the third ion storage device can include samples of precursor ions for each of a plurality of sub-ranges.

[0106] In other words, in the second example, there can be two mass analyzers in series, the first downstream of the ion beam splitter (for analyzing fragment ions, such as a time-of-flight mass analyzer) and the second upstream of the ion beam splitter (for analyzing precursor ions, such as a Fourier transform mass analyzer).

[0107] After samples of fragmented precursor ions for each of a plurality of sub-ranges have been accumulated in the second ion storage device, the precursor ions can be transferred from the first ion storage device to the third ion storage device. In other words, after all fragment ions have been accumulated or analyzed, the precursor ions can be transferred out of the intermediate ion storage device. This prevents the precursor ions accumulated in the intermediate ion storage device from mixing with other ions in the ion beam splitter during the transfer.

[0108] The third ion storage device can be a curved linear ion trap (also known as a “c-trap”).

[0109] The ion beam splitter can provide sufficient ion transmission to eliminate ion mixing between sub-ranges.

[0110] In some examples, the third ion storage device is disposed upstream of the ion beam splitter. In this case, ions can first accumulate in the third ion storage device (such as in a C-trap) before the ion beam splitter. This configuration can allow for the parallelization of the accumulation (in the C-trap) and the ion transport via the immediately preceding sub-range ion beam splitter. In other words, after all the precursor ions from the first sub-range have been ejected from the third ion storage device, the accumulation of precursor ions from the next sub-range in the third ion storage device can begin while the precursor ions from the first sub-range are being transported via the ion beam splitter. This improves the overall performance even if the time taken to clear the ions from the ion beam splitter is relatively slow.

[0111] The second mass analyzer can be a Fourier transform mass analyzer. This applies to the first and second examples above.

[0112] In a third example, the first ion storage device can be an intermediate ion storage device. The method may further include configuring the ion beam splitter to transfer the precursor ions accumulated in the first ion storage device to a second ion destination (e.g., the first mass analyzer in option a or the second ion storage device in option b).

[0113] In this case, ions can be transferred from the first “exit” region to the second exit region. Thus, in the step of transferring the precursor ions accumulated in the first ion storage device to the second ion destination, the first exit region can be used as an entrance.

[0114] In the case where the second ion destination is the second ion storage device (option b), the method may further include ejecting the precursor ions from the second ion storage device into the first mass analyzer.

[0115] The precursor ions transferred (transferred to the first mass analyzer or the second ion storage device) may include a sample of precursor ions for each of the plurality of sub-ranges.

[0116] The transferred precursor ions may include precursor ions having m / z values from the total m / z range. In other words, the transferred precursor ions can include a combined sample from each sub-range.

[0117] In the case where the second ion destination is the second ion storage device (option b), the method may further include ejecting a sample of fragmented precursor ions from the second ion storage device into the first mass analyzer.

[0118] After each accumulation (before accumulating the fragment ions formed by the fragmentation of the precursor ions from the next sub-range), a sample of the fragmented precursor ions can be ejected from the second ion storage device.

[0119] A sample of fragmented precursor ions can be ejected from the second ion storage device before the precursor ions are transferred to the second ion storage device.

[0120] After a sample of fragmented precursor ions in each of a plurality of sub-ranges has been ejected from the second ion storage device into the first mass analyzer, the precursor ions can be transferred from the first ion storage device to the second ion storage device.

[0121] The first ion storage device can be provided by a DC barrier adjacent to the first output region. In other words, the first ion storage device (intermediate ion storage device) can be integrated into the ion beam splitter.

[0122] In some examples, the ion beam splitter is between the ion filter and the first ion destination and between the ion filter and the second ion destination, and is configured to direct ions (simultaneously) from the ion filter to the first ion destination and the second ion destination.

[0123] For each sub-range, the step of accumulating a sample of precursor ions can be performed before the step of accumulating a sample of fragmented precursor ions.

[0124] Alternatively, for each sub-range, the step of accumulating a sample of fragmented precursor ions can be performed before the step of accumulating a sample of precursor ions.

[0125] The method can also include, for each of a plurality of sub-ranges, analyzing the precursor ion sample in the first mass analyzer or the second mass analyzer. In this alternative, the precursor ions of each sub-range are analyzed separately in a SIM scan, rather than injecting all SIM accumulations together in one scan and analyzing the precursor ions from the total m / z range. The SIM scan can be interleaved with the fragmentation scan, which can advantageously mean that no reconfiguration of the ion filter is required between the SIM scan and the fragmentation scan for each sub-range. The method can also include obtaining scan data related to the precursor ions of each sub-range. The method can also include combining the scan data related to the precursor ions for each sub-range to form a high-definition scan for the total m / z range. In other words, the SIM scan data is stitched together to provide a high-resolution MS scan. In this case, a pre-scan may not be required (since the sub-ranges are contiguous and cover the total m / z range).

[0126] In another example, an HDR MS scan can be separated into multiple scans, each of the multiple scans being associated with precursor ions from multiple consecutive sub-ranges. In this alternative, each scan includes the simultaneous analysis of multiple SIM injections. The method can also include combining the scan data from each of the multiple scans to form a high-definition scan of the total m / z range. A pre-scan may not be required in this case because a) the sub-ranges are consecutive and cover the total m / z range, and b) each sub-range within the sub-ranges is analyzed in a corresponding one of the multiple scans.

[0127] The ion beam splitter can be configured to operate under pure molecular flow conditions. Pure molecular flow conditions (also known as free molecular flow or Knudsen diffusion) are observed when the Knudsen number Kn is greater than 20 or more preferably Kn > 10.

[0128] The method can also include fragmenting the precursor ions to produce a sample of fragmented precursor ions.

[0129] In the case where the second ion destination is a second ion storage device (option b), the ions can be fragmented in the second ion storage device.

[0130] In other words, the precursor ions can be directed by the ion beam splitter to the second ion storage device, and then once the ions have been accumulated in the second ion storage device, they can be fragmented.

[0131] Alternatively, a multipole collision cell (e.g., an IRM collision cell) can be used to fragment the ions.

[0132] The multipole collision cell can be downstream of the ion beam splitter such that the ion beam splitter directs the precursor ions to the multipole collision cell, which then transfers the fragmented ions to the second ion destination (the first mass analyzer in option a, or the second ion storage device in option b). In other words, the multipole collision cell can be between the ion beam switch and the second ion destination.

[0133] In option b), the fragmented ions can be accumulated in the second ion storage device and then ejected from the second ion storage device into the first mass analyzer.

[0134] The method can also include configuring an ion filter for each of the multiple sub-ranges to transmit precursor ions having m / z values within that sub-range. A sample of precursor ions can be received from the configured ion filter. The sample of fragmented precursor ions can be formed by fragmenting the precursor ions received from the configured ion filter.

[0135] In other words, the ion filter can be configured once for each sub-range, and this configuration is adapted to fill the first ion storage device with precursor ions and send the fragmented precursor ions to a second ion destination (while using an ion beam splitter).

[0136] A sample of the fragmented precursor ions can be formed by fragmentation of precursor ions (a second part of the precursor ion sample) received from the configured ion filter via the ion beam splitter.

[0137] Configuring the ion filter can include setting the transmission window of the ion filter. The transmission window can be adjusted between each of the plurality of sub-ranges. For each sub-range, the transmission window for the step of accumulating a sample of precursor ions can be the same as the transmission window for the step of injecting a sample of the fragmented precursor ions into the first mass analyzer (in option a) or accumulating a sample of the fragmented precursor ions in the second ion storage device (in option b).

[0138] A front-end accumulation device can be used, such as an ion mobility separator, such as a trapped ion mobility separator (as incorporated in the Bruker TIMS-ToF series of mass spectrometers) (and would be advantageous for the methods described herein). Such a device can release ions in an m / z range synchronized with the quadrupole isolation window. Thus, ion transport is greatly facilitated. Also, as a result, the required injection time can be reduced (due to a brighter isolated ion beam). This reduction in injection time can be used to offset the additional time overhead of SIM injection.

[0139] The method can further include configuring the ion mobility separator to transfer precursor ions having m / z values within the sub-range to the ion filter.

[0140] The method can further include, for each of the plurality of sub-ranges in the total m / z range, controlling the ion mobility separator such that the precursor ions transferred to the ion filter correspond to the transmission window of the ion filter.

[0141] Accumulating a sample of precursor ions can include controlling the fill time of the precursor ions based on the relative abundances of the precursor ion species in the corresponding sub-range.

[0142] In the case where the second ion destination is the second ion storage device and a sample of the fragmented precursor ions is accumulated in the second ion storage device (option b), the method can further include, for each of the plurality of sub-ranges, ejecting the sample of the fragmented precursor ions from the second ion storage device into the first mass analyzer and analyzing the sample of the fragmented precursor ions in the first mass analyzer.

[0143] The plurality of sub-ranges can include a first sub-range and a second sub-range.

[0144] The step of analyzing a sample of fragmented precursor ions from the first sub-range can at least partially overlap (in time) with the step of accumulating a sample of fragmented precursor ions formed by fragmentation of precursor ions having m / z values within the second sub-range in a second ion storage device.

[0145] In the case where the second ion destination is a first mass analyzer and a sample of fragmented precursor ions is injected into the first mass analyzer (option a), the method may further include analyzing the sample of fragmented precursor ions in the first mass analyzer.

[0146] The step of analyzing a sample of fragmented precursor ions from the first sub-range can at least partially overlap (in time) with the step of accumulating a sample of precursor ions having m / z values within the second sub-range in a first ion storage device.

[0147] The first mass analyzer can be a time-of-flight ToF analyzer.

[0148] The first mass analyzer can be a multi-reflection time-of-flight MR-ToF analyzer.

[0149] The first ion storage device can be a curved linear trap (e.g., c-trap).

[0150] In the case where the second ion destination is a second ion storage device and a sample of fragmented precursor ions is accumulated in the second ion storage device (option b), the second ion storage device can be a linear trap (e.g., DP-R trap).

[0151] The method may further include ionizing the sample to generate precursor ions.

[0152] A sample of fragmented precursor ions can be formed by fragmentation of precursor ions having m / z values within the sub-range at multiple different collision energies.

[0153] The method may further include:

[0154] Configuring an ion filter to transmit precursor ions having m / z values from the total m / z range;

[0155] Transferring an initial sample of precursor ions having m / z values from the total m / z range to the first mass analyzer or the second mass analyzer;

[0156] Analyzing the initial sample of the precursor ions; and

[0157] Obtaining scan data of the total m / z range from the analysis of the initial sample of the precursor ions.

[0158] In other words, the method can include a pre-scan of the total m / z range (e.g., AGC pre-scan) before analyzing ions from multiple sub-ranges. Optionally, the method can include accumulating (in a single fill and in the first or second ion storage device) an initial sample of precursor ions having m / z values from the total m / z range. Alternatively, the initial sample can be directed to the first mass analyzer or the second mass analyzer. To deliver the initial sample of ions, the ion beam splitter can be configured such that substantially all of the ions are directed towards one exit region (and negligible ions towards the other exit region). Analyzing the initial sample of precursor ions having m / z values from the total m / z range can include obtaining scan data for the total m / z range (which can be low-resolution, full MS scan data rather than SIM scan data).

[0159] The method can further include determining the plurality of sub-ranges from the total m / z range based on scan data obtained from the analysis of the initial sample of precursor ions (having m / z values from the total m / z range). This can be referred to as a data-dependent acquisition (DDA) method.

[0160] Each of the plurality of sub-ranges can have the same width.

[0161] The plurality of sub-ranges can be contiguous.

[0162] The method can be performed over a period of time based on the chromatographic peak width when the sample elutes from the chromatographic system.

[0163] Also provided is a mass spectrometer configured to perform the above method.

[0164] Also provided is a computer software including instructions that, when executed by a processor of a computer, cause the computer to perform the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0165] The present invention can be implemented in various ways and specific embodiments will now be described by way of example only and with reference to the following drawings.

[0166] Figure 1 Schematic diagram showing a mass spectrometer adapted to perform the method according to an embodiment.

[0167] Figure 2 Showing a branched RF multipole that can be used as an ion beam switch.

[0168] Figure 3 Schematic diagram showing an ion guide including an RF surface according to an embodiment of the present disclosure, the RF surface including a plurality of RF electrodes including elongated electrode plates.

[0169] Figure 4Schematic diagram showing the RF electrode of an ion guide according to an embodiment of the present disclosure, wherein the RF electrode includes a notch forming a channel.

[0170] FIG. 5 shows a schematic cross-sectional view of an ion guide according to an embodiment of the present disclosure. Figure 5A The RF electrode, top plate, first side guard plate, and second side guard plate are shown. Figure 5B The RF electrode and top plate are shown, wherein the RF electrode includes a first side guard plate and a second side guard plate. Figure 5C The RF electrode and top plate are shown, wherein the RF electrode includes a first side guard plate, a second side guard plate, a first notch, and a second notch. Figure 5D The RF electrode, top plate, first DC electrode, and second DC electrode are shown, wherein the RF electrode includes a first side guard plate and a second side guard plate.

[0171] Figure 6 Schematic diagram showing an ion guide according to an embodiment of the present disclosure, wherein the ion guide includes a radio frequency (RF) surface and a top plate, the RF surface includes a plurality of RF electrodes, and the top plate includes a DC electrode structure.

[0172] Figure 7 Schematic diagram showing a DC electrode structure according to an embodiment of the present disclosure, wherein the DC electrode structure includes a grid of printed electrodes.

[0173] Figure 8 Schematic diagram showing a DC electrode structure according to an embodiment of the present disclosure, wherein the DC electrode structure includes a printed electrode in a horseshoe shape.

[0174] Figure 9 Schematic diagram showing an ion guide according to an embodiment of the present disclosure, wherein the ion guide includes a radio frequency (RF) surface, the RF surface includes a plurality of RF electrodes and a plurality of auxiliary DC electrodes mounted between the RF electrodes.

[0175] Figure 10 Schematic diagram showing an ion guide according to an embodiment of the present disclosure, wherein the ion guide includes a radio frequency (RF) surface, the RF surface includes a plurality of RF electrodes and a plurality of auxiliary DC electrodes mounted between the RF electrodes, and wherein the ion guide further includes a top surface, the top surface includes a plurality of RF electrodes and a plurality of auxiliary DC electrodes mounted between the RF electrodes.

[0176] Figure 11 Schematic cross-sectional view showing an auxiliary DC electrode according to an embodiment of the present disclosure.

[0177] Figure 12Schematic illustration of an ion guide according to an embodiment of the present disclosure, wherein the ion guide includes a radio frequency (RF) surface that includes a plurality of RF electrodes and a plurality of auxiliary DC electrodes printed between the RF electrodes.

[0178] Figure 13 Schematic illustration of a mass spectrometer incorporating an ion guide according to an embodiment of the present disclosure.

[0179] Figure 14 An ion beam separation device based on an RF blanket ion guide is shown.

[0180] FIG. 15 shows two alternative configurations of a hybrid mass spectrometer incorporating a beam separation device.

[0181] Figure 16 A MASIM 3D model of an RF surface with wedge-shaped beam splitter electrodes is shown.

[0182] Figure 17 A graph showing the simulated trajectories of the ions received across the wedge is shown.

[0183] Figure 18 A DIA method for parallel accumulation of SIM injections in a curved linear ion storage device is shown, constructed as a single HDR scan using a Fourier transform mass analyzer and interleaved between a series of MS2 scans in a time-of-flight mass analyzer. Detailed Description

[0184] Figure 1 A schematic arrangement of a mass spectrometer 1 suitable for performing the method according to an embodiment is shown. The mass spectrometer 1 can be a hybrid Fourier transform / multiple reflection time-of-flight mass spectrometer (MR-ToF) as described in US 10,699,888, which is incorporated by reference. Details of the mass analyzer are described in US 9,136,101, which is incorporated by reference herein.

[0185] In Figure 1 , a sample to be analyzed (e.g., from an autosampler) is supplied to a chromatographic device such as a liquid chromatography (LC) column ( Figure 1 not shown in TM ). One such example of an LC column is the ProSwift

[0186] Chromatograms can be generated by measuring the number of sample molecules eluted from an HPLC column over time using a detector, such as a mass spectrometer. The sample molecules eluted from the HPLC column will be detected as peaks above the baseline measurement on the chromatogram. When different sample molecules have different elution rates, multiple peaks on the chromatogram can be detected. Preferably, the individual sample peaks are separated in time from other peaks in the chromatogram such that different sample molecules do not interfere with each other.

[0187] On the chromatogram, the presence of a chromatographic peak corresponds to the period of time during which the sample molecules are present at the detector. Thus, the width of the chromatographic peak is equal to the period of time during which the sample molecules are present at the detector. Preferably, the chromatographic peak has a Gaussian shape distribution, or can be assumed to have a Gaussian shape distribution. Accordingly, the width of the chromatographic peak can be determined based on multiple standard deviations calculated from the peak. For example, the peak width can be calculated based on 4 standard deviations of the chromatographic peak. Alternatively, the peak width can be calculated based on the width at half the maximum height of the peak. Other methods known in the art for determining peak width may also be suitable.

[0188] Then, the sample molecules separated by liquid chromatography are ionized using an electrospray ionization source (ESI source) 2 at atmospheric pressure.

[0189] The sample ions then enter the vacuum chamber of the mass spectrometer 1 and are guided by a capillary 25 into a radio frequency (RF)-only S lens 3 (also known as an ion funnel). The ions are focused by the S lens 3 into an injection flat electrode 4 (also known as a quadrupole pre-filter) which injects the ions into a curved flat electrode 5 with an axial field. The curved flat electrode 5 guides (charged) ions along a curved path passing through it, while unwanted neutral molecules, such as entrained solvent molecules, are not guided along the curved path and are lost. For example, the curved path can be a 90-degree bend or an S-shaped wobble.

[0190] A TK lens 6 is located at the distal end of the curved flat electrode 5. The ions enter a downstream mass selector in the form of a quadrupole mass filter 7 from the curved flat electrode 5. The TK lens serves as an edge field corrector for the quadrupole mass filter 7. The quadrupole mass filter 7 is typically but not necessarily segmented and serves as a bandpass filter, allowing selected mass numbers or a limited mass range to pass while excluding ions of other mass-to-charge ratios (m / z). The filter can also be operated in an RF-only mode, in which the filter has no mass selectivity, i.e., it transmits substantially all m / z ions. For example, the quadrupole mass filter 7 can be controlled by a controller to select precursor ions within a certain range of mass-to-charge ratios to be allowed to pass through while filtering out (attenuating) other ions in the precursor ion stream. Alternatively, the S lens 3 can operate as an ion gate and the ion gate (TK lens) 6 can be an electrostatic lens.

[0191] Although Figure 1A quadrupole mass filter is shown, but those skilled in the art will understand that other types of mass selection devices may also be suitable for selecting precursor ions within the mass range of interest. For example, the ion separator depicted in US-A-2015287585, the ion trap depicted in WO-A-2013076307, the ion mobility separator described in US-A-2012256083, the ion gate mass selection device described in WO-A-2012175517, or the charged particle trap described in US799223, which is incorporated herein by reference. Those skilled in the art should understand that other methods of selecting precursor ions based on ion mobility, differential mobility, and / or transverse modulation are also suitable.

[0192] The isolation of multiple ions of different masses or mass ranges in an ion trap can also be performed using a method called synchronous precursor scan (SPS). Additionally, in some embodiments, more than one ion selection device or mass selection device may be provided. For example, an additional mass selection device may be provided downstream of the fragmentation chamber 12. In this way, MS 3 or MS n scans can be performed (usually using a ToF mass analyzer for mass analysis).

[0193] The ions then pass through a quadrupole exit lens / segmented lens arrangement 8, which acts as an ion gate to control the passage of ions into the first transfer multipole 9 optionally via a charge detector (not shown). The first transfer multipole 9 guides the mass-filtered ions from the quadrupole mass filter 7 into a curved linear ion trap (C-trap) 10. The C-trap (first ion storage device) 10 has a longitudinally extending curved electrode supplied with an RF voltage and end caps supplied with a DC voltage. The result is a potential well extending along the curved longitudinal axis of the C-trap 10. In a first operating mode, a DC end cap voltage is set for the C-trap such that the ions arriving from the first transfer multipole 9 are trapped in the potential well of the C-trap 10, where the ions are cooled. The injection time (IT) of the ions into the C-trap determines the number of ions (ion packet) subsequently ejected from the C-trap into the mass analyzer.

[0194] The cooled ions accumulate into a cloud towards the bottom of the potential well and are then orthogonally ejected from the C-trap towards the second mass analyzer 11. As Figure 1 shown, the second mass analyzer is a Fourier transform mass analyzer, such as an Orbitrap mass analyzer 11, for example, the Orbitrap sold by Thermo Fisher Scientific. TMMass analyzer. The Fourier transform mass analyzer 11 has an off-center injection hole, and ions are injected into the orbitrap mass analyzer 11 as coherent groups through the off-center injection hole. Then, the ions are trapped in the orbitrap mass analyzer by a super-logarithmic electric field, and the ions move back and forth in the longitudinal direction while orbiting around the inner electrode.

[0195] The axial (z) component of the movement of the ion packet in the orbitrap mass analyzer is (more or less) defined as simple harmonic motion, where the angular frequency in the z direction is related to the square root of the mass-to-charge ratio of a given ion species. Thus, over time, the ions are separated according to their mass-to-charge ratio.

[0196] Ions in the orbitrap mass analyzer are detected by using an image current detector (not shown), which generates a "transient" containing information about all ion species in the time domain when the ion species passes through the image current detector. Then, this transient is subjected to a fast Fourier transform (FFT), thereby generating a series of peaks in the frequency domain. Based on these peaks, a mass spectrum representing the abundance / ion intensity versus m / z can be generated.

[0197] In the above configuration, without fragmentation, sample ions (more specifically, a mass range segment of the sample ions within the mass range of interest selected by the quadrupole mass filter 7) are analyzed by the orbitrap mass analyzer 11. The resulting mass spectrum is represented as MS1.

[0198] Although Figure 1 the orbitrap mass analyzer 11 is shown, alternatively, other mass analyzers can be used, including other Fourier transform mass analyzers. For example, a Fourier transform ion cyclotron resonance (FTICR) mass analyzer can be used as the mass analyzer for MS1 scans. Mass analyzers (such as orbitrap mass analyzers and ion cyclotron resonance mass analyzers) can also be used in embodiments even when other types of signal processing different from Fourier transform are used to obtain mass spectrometry information from the transient signal (see, for example, WO 2013 / 171313, Thermo Fisher Scientific).

[0199] In the second operating mode of the C-trap 10, the ions that pass through the quadrupole exit lens / segmented lens arrangement 8 and the first transfer multipole 9 and enter the C-trap 10 can also continue their path through the C-trap and enter the fragmentation chamber 12, which can be an "ion routing multipole" (IRM) collision cell. Thus, the C-trap effectively acts as an ion guide in the second operating mode. Alternatively, the cooled ions in the C-trap 10 can be ejected axially from the C-trap into the fragmentation chamber 12. In Figure 1In the mass spectrometer 1, the fragmentation chamber 12 is a high-energy collision dissociation (HCD) device to which collision gas is supplied. The precursor ions arriving in the fragmentation chamber 12 collide with the collision gas molecules, thereby fragmenting the precursor ions into fragment ions.

[0200] Although the HCD fragmentation chamber 12 is shown in Figure 1 , other fragmentation devices employing methods such as collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), photodissociation, etc. may alternatively be used. Additionally, ion fragmentation may be performed in the high-voltage region of the extraction trap 14.

[0201] The fragmented ions are ejected from the fragmentation chamber 12 to the C-trap 10 at the relatively axial end. The ejected fragmented ions enter the second transfer multipole 13. The second transfer multipole 13 guides the fragmented ions from the fragmentation chamber 12 into the extraction trap (second ion trap) 14. The extraction trap 14 is a radio-frequency voltage-controlled trap containing buffer gas. For example, a suitable buffer gas is argon within a pressure range of 5×10 -4 mBar to 1×10 -2 mBar. The extraction trap is capable of quickly cutting off the applied RF voltage and applying a DC voltage to extract the trapped ions. A suitable flat extraction trap, also known as a rectangular ion trap, is additionally described in US 9,548,195, which is incorporated herein by reference. Alternatively, the C-trap is also suitable for use as the second ion trap.

[0202] The extraction trap 14 is provided to form ion packets of the fragmented ions before injection into the time-of-flight mass analyzer 15. The extraction trap 14 accumulates the fragmented ions before they are injected into the time-of-flight mass analyzer 15.

[0203] Although the extraction trap (ion trap) is shown in the Figure 1 embodiment, those skilled in the art will appreciate that other methods of forming ion packets of the fragmented ions will equally apply to the embodiment. For example, the aggregation of ions can be affected by the relatively slow transfer of ions through a multipole, and the ions can then be ejected as a single group into the ToF mass analyzer. Alternatively, the orthogonal displacement of ions can be utilized to form groups. Other details of these alternatives can be found in US2003 / 0001088, which describes the traveling-wave ion accumulation method and is incorporated herein by reference.

[0204] In Figure 1In this case, the time-of-flight mass analyzer 15 shown is a multi-reflection time-of-flight mass analyzer (MR-ToF) 15. The MR-ToF 15 is constructed around two opposing ion mirrors 16, 162 that are elongated in the drift direction. The mirrors face each other in a direction orthogonal to the drift direction. The extraction trap 14 injects ions into the first mirror 16, and then the ions oscillate between the two mirrors 16, 162. The ejection angles of the ions from the extraction trap 14 and additional deflectors 17, 172 allow control of the ion energy in the drift direction such that the ions are guided along the length of the mirrors 16, 162 during oscillation, thereby generating a Z-shaped trajectory. The mirrors 16, 162 are tilted relative to each other, thereby creating a potential gradient that delays the drift velocity of the ions and causes the ions to be reflected back and focused onto the detector 18 in the drift dimension. The tilting of the opposing mirrors generally has the negative side effect of changing the time period of ion oscillation as the ions travel along the drift dimension. This is corrected using a strip electrode 19 (as a compensation electrode) that changes the flight potential of the portion of the space between the mirrors that varies along the length of the opposing mirrors 16, 162. The combined variation in the width of the strip electrode 19 and the distance change between the mirrors 16, 162 allows ion reflection and spatial focusing onto the detector 18 as well as maintaining good time focusing. A suitable MR-ToF 15 for use in the embodiments is further described in US2015028197(A1), which is incorporated herein by reference.

[0205] In one example, an MS1 scan can be performed by the second mass analyzer (orbitrap mass analyzer 11). In a second example, precursor ions can be fragmented and an MS2 scan can be performed by the second mass analyzer (orbitrap mass analyzer 11) or the first mass analyzer (time-of-flight mass analyzer), depending on whether the fragmentation chamber is controlled to eject the ions back towards the C-trap 10 or forward into the second transfer multipole 13. In an additional operating mode, the second mass analyzer (time-of-flight mass analyzer 15) can perform an MS1 scan of the ions. In this operating mode, the ions are axially guided through the C-trap 10 to the fragmentation chamber, but do not have sufficient kinetic energy to cause fragmentation and the ions are guided to the second transfer multipole 13 without fragmentation. The ions can then be grouped in the extraction trap 14 as described above.

[0206] Once a predetermined number of ions have accumulated in the extraction trap, the ions accumulated in the extraction trap are injected into the MR-ToF analyzer 15 as ion packets. By ensuring that each packet of ions injected into the MR-ToF 15 has at least a predetermined (minimum) number of ions, the resulting ion packets reaching the detector will represent the entire mass range of interest of the MS1 or MS2 spectrum. A single group of precursor ions or fragmented ions is sufficient to obtain the MS1 or MS2 spectrum of the corresponding ions. For MS2, this represents an increased sensitivity compared to the conventional acquisition of the time-of-flight spectrum, in which multiple spectra are typically acquired and summed for each given mass range segment. Preferably, the minimum total ion current (TIC) in each mass window accumulates in the extraction trap before injection into the time-of-flight mass analyzer. In some examples, the time-of-flight mass analyzer acquires at least N spectra (scans) per second in the MS2 domain, where N = 50, or more preferably 100 or 200 or more.

[0207] Preferably, at least X% of the MS2 scans contain more than Y ion counts (where X = 30 or 50 or 70 or most preferably 90 or more, and Y = 200 or 500 or 1000 or 2000 or 3000 or 5000 or more). Most preferably, at least 90% of the MS2 scans contain more than 500 ion counts, or more preferably more than 1000 ion counts, and desirably more than 5000 ion counts. This provides an increased dynamic range of the MS2 spectrum. The desired ion count for each MS2 scan can be provided by adjusting the number of ions included in each group of fragmented ions. For example, in Figure 1 an embodiment, the accumulation time of the extraction trap can be adjusted to ensure that a sufficient number of ions have accumulated. Thus, the controller can be configured to determine that a suitable packet of fragmented ions has been formed when a predetermined number of ions are present in the extraction trap or when a predetermined time period has elapsed. The predetermined time period can be specified to ensure that the time-of-flight mass analyzer operates at the desired frequency when the ion current to the extraction trap is relatively low.

[0208] The mass spectrometer 1 is under the control of a controller that is configured, for example, to control the ejection timing of the trapping assembly; set appropriate potentials on the electrodes of the quadrupole, etc. to focus and filter ions; capture mass spectrometry data from the orbitrap device 11; obtain mass spectrometry data from the MR-ToF 15; control the sequence of MS1 and MS2 scans, etc. It should be understood that the controller can include a computer that can operate according to a computer program that contains instructions for causing the mass spectrometer to perform the method steps according to the embodiments.

[0209] It should be understood that Figure 1The specific arrangement of the components shown is not necessary for the methods described subsequently. In fact, other arrangements for performing the methods of the embodiments are also suitable. In some examples, all scans (MS1, MS2, and / or SIM) are performed by an MR-ToF analyzer, which is faster than an orbitrap analyzer.

[0210] A front-end accumulation device, such as an ion mobility separator (e.g., a trapped ion mobility separator TIMS), can be configured to release ions in the m / z range corresponding to the quadrupole isolation window. As a result, the ion transmission of the quadrupole filter is improved. Due to the ion mobility separator, the injection time required can also be reduced (since the isolated ion beam is brighter). This reduction in injection time can be used to at least partially offset the additional time overhead of SIM injection.

[0211] The ion mobility separator can include a stacked ring ion guide that applies a DC gradient in one direction to push ions and passes them in the opposite direction against a gas wind.

[0212] In one example of the ion mobility separator, an electric field barrier in the gas flow is used to block ions according to their ion mobility. As the ion mobility increases, the field barrier decreases, releasing the ions.

[0213] TIMS is described in detail in U.S. Patent 7,838,826, U.S. Patent 9,891,194, and Meier et al. (2018, Molecular Cellular Proteomics, Vol. 17, pp. 2534 - 2545), which are incorporated herein by reference.

[0214] The extended ion funnel consists of a plurality of segmented electrodes assembled around a common axis. The extended ion guide can be considered in three sections:

[0215] An entrance focusing section,

[0216] A mobility analysis section, and

[0217] An exit focusing section.

[0218] In the entrance focusing section, the distance between adjacent electrodes is approximately equal to the thickness of the electrodes. The diameter of the holes in the electrodes is a function of the position of the electrodes in the ion funnel assembly. For example, the segmented electrode with the largest holes is located at the entrance end of the ion funnel, and the segmented electrode with the smallest holes is located at the exit end of the ion funnel.

[0219] In some examples, the pore diameter can be a linear function of the position of the segmented electrodes. In other examples, the function can be non-linear. The angle formed between the common axis and the inner boundary of the ion funnel (i.e., formed by the inner edges of the segmented electrodes) can be about 19°. However, any angle between 0° and 90° can be used.

[0220] In the mobility analysis section of the ion funnel, the segmented electrodes can all have the same inner diameter. The space between adjacent electrodes can be filled with a dielectric or resistive pad. The thickness of the segmented electrodes should be less than their inner diameter, and the spacing between the electrodes should be less than the thickness of the segmented electrodes to maintain a uniform RF field, such that the axial DC field is uniform near the axis.

[0221] The pads or O-rings between the electrodes form a substantially airtight seal such that the pores in the electrodes form an airtight passage through which gas can flow. Gas enters the passage in the focusing section, forms a laminar flow that uniformly flows through the mobility analysis section, contracts by leaving the focusing section, and then exits through the pores in the final electrode. The pores are substantially cylindrically symmetric to maintain a cylindrically symmetric flow profile. In operation, the symmetric laminar flow of the gas means that all ions of a given type at a given position along the axis will experience a given force due to the gas flow, substantially independent of their lateral position relative to the axis.

[0222] The quadrupole ion filter includes four rods spaced equidistantly at a predetermined radius around a central axis. A radio frequency (RF) (e.g., 1 MHz sine wave) electric potential is applied between these rods. The electric potentials on adjacent rods are 180° out of phase. The rods on opposite sides of the axis of the quadrupole are electrically connected such that the quadrupole is formed as two pairs of rods. Ions travel along the axis of the quadrupole and exit the quadrupole through the pores. The RF electric potential applied between these rods tends to radially confine the ions. When only RF is applied between these rods, substantially all ions are transmitted through the quadrupole. Applying DC as well as RF electric potential between the rod pairs allows only ions within a limited mass range to be transmitted through the quadrupole. Ions outside this mass range are filtered out and do not reach the exit end.

[0223] The DC electric field strength varies as a function of the position along the axis. However, at some positions in the analysis section, the field strength reaches a maximum, thereby forming a potential barrier that ions must overcome in order to reach the exit end of the funnel. Near the position of this maximum field strength, the uniformity of the DC field is important because this is the point at which ions are selected based on their mobility. Therefore, the DC field should be cylindrically symmetric.

[0224] An example method of operation includes the following steps:

[0225] Form a DC potential barrier in the analysis section;

[0226] Apply an RF field to focus ions towards the axis;

[0227] Generate ions in an ion source;

[0228] Introduce ions in a carrier gas into an extended ion funnel;

[0229] Introduce ions into a focusing section by applying an electric potential to the electrodes of the focusing section and / or deflection electrodes;

[0230] Transfer ions into an analysis section by applying a DC potential to the electrodes of the focusing section;

[0231] Optionally, prevent additional ions from entering the analysis section by applying a DC potential to the deflection electrodes and / or the electrodes of the focusing section;

[0232] Use a pump located downstream of the exit end of the funnel to induce a carrier gas flow through the channel;

[0233] Gradually reduce the DC barrier in the analysis section to allow the carrier gas flow to push ions from the ion packet across the DC barrier in order of ion mobility; and

[0234] Focus ions through holes in the exit electrode.

[0235] In another example, a DC gradient is used to push ions out, against the gas wind. As the DC potential increases, ions are released in order of mobility.

[0236] In one exemplary method, a full mass scan is performed using a Fourier transform mass analyzer 11 with a long acquisition transient to generate a high-resolution MS1 spectrum. Meanwhile, the MR-ToF 15 analyzer performs a series of MS2 acquisitions at a very fast scan rate and high sensitivity.

[0237] Exemplary methods for combining ion injection and analysis are described in US 8,686,350, which is incorporated herein by reference.

[0238] The exemplary methods described herein utilize two injections to accumulate different types of ions: a first injection of fragmented ions and a second injection of intact precursor ions (the first and second ion injections can be performed in either order). The ions can be from the same ion source and have the same quadrupole isolation window but different fragmentation energies (the collision energy for precursor injection is zero). The fragment ions are analyzed in a mass analyzer to provide a fragment spectrum. The precursor ions are accumulated in an ion storage device and combined with precursor ions from other quadrupole isolation windows. The combined precursor ions are then analyzed in the mass analyzer to provide analytical scan data. Such scan data provides precursor information in addition to the fragment spectrum. Precursor ion accumulation can be performed quickly together with normal MS2 fragmentation analysis because there is no additional quadrupole switching time, and the additional injection time for the precursor ion (SIM) component should be lower than the time for fragmentation injection.

[0239] When using a hybrid instrument (such as Figure 1 the instrument shown), it is preferred to measure the SIM component (the unfragmented precursor ions for each sub-range) in a high-resolution MS1 scan. To do this, the unfragmented SIM component needs to be separated from the fragmented MS2 component and then collected for separate analysis. The path to the fragment analyzer (the first analyzer) must not be blocked by the accumulated SIM injection ions. One way to achieve this is to create a branched ion path such that the SIM ions can be separated and sent to one area while acquiring an MS2 scan in parallel.

[0240] In some examples, the ions can be separated between two separate ion destinations via the use of a beam switching device to create a branched ion path. A switchable path ion guide is described in UK Patent Application No. 2209555.8, which is incorporated herein by reference. Other suitable devices are described in Patent Publications US7829850B2, US20190103261A1, US8581181B2, and US9984861B2.

[0241] Ion beam switching devices exist in the prior art. Many of these involve switching in an RF gas-filled multipole, which is slow due to ion diffusion in the gas and cannot operate with sub-microsecond transition times. Such devices can be compatible with the proposed method. In this case, the ions are preferably decelerated before entering the guide filled with RF gas to reduce unwanted fragmentation in the guide. However, it is preferred to alternatively provide an ion beam switch that operates under pure molecular flow conditions (where Kn > 10 - 20).

[0242] In one example, after ion acceleration preferably in the range of 10 V - 50 V, a double plate gate can be used to switch to reduce ion loss. Preferably, such a gate is located in the pressure region of 10 -4 mbar to 10 -5 mbar and is spatially separated from the RF gas-filled guide. This reduces the probability of ion-molecule collisions and corresponding losses, while providing a reduced switching time important for strong ion beams from modern ion sources.

[0243] The branched RF multipole described in US7829850B2 may be suitable as an ion beam switch. Figure 2 The device shown is an RF quadrupole that can move ions down one channel or the other by switching the RF phase, amplitude, or by applying a DC gradient. The device is compatible with high vacuum, and thus ions can be transferred quickly without diffusion and thus have a minimum time loss due to path switching or ion migration.

[0244] Figure 2 A perspective view of the branched radio frequency multipole system described in US7829850B2 is shown. The branched radio frequency multipole system 50 includes branched electrodes 55A and 55B arranged parallel to each other. The branched radio frequency multipole system also includes orthogonal electrodes 60A, 60B, 60C, 60D, 60E, 60F, 65A and 65B. The orthogonal electrodes 60A - 60F, 65A and 65B are arranged orthogonally to the branched electrodes 55A and 55B such that the branched radio frequency multipole 50 includes a first ion channel between ports 70 and 75 of the branched radio frequency multipole 50 and a second ion channel between ports 70 and 80. Port 70 is an opening defined by the branched electrodes 55A and 55B and the orthogonal electrodes 60A and 60D. Port 75 is an opening defined by the branched electrodes 55A and 55B and the orthogonal electrodes 60C and 65A. Port 80 is an opening defined by the branched electrodes 55A and 55B and the orthogonal electrodes 60F and 65B. The first ion channel and the second ion channel overlap in a portion of the branched radio frequency multipole 50 adjacent to port 70 and fork at branch point 85 before continuing to port 75 and port 80 respectively.

[0245] The RF voltages applied to the orthogonal electrodes 60B, 60C and 65A can be controlled such that the first ion channel including the path between port 70 and port 75 is opened. Alternatively, the RF voltages applied to the orthogonal electrodes 60E, 60F and 65B can be controlled such that the second ion channel including the path between port 70 and port 80 is opened. Thus, the path of ions across the branched radio frequency multipole 50 can be controlled by selecting appropriate voltages.

[0246] In another example described in UK Patent Application No. 2209555.8, an ion guide having a switchable ion path includes a first ion transport aperture configured to receive an ion beam. The ion guide includes: an RF surface including a plurality of RF electrodes disposed on a first surface such that the plurality of RF electrodes are parallel to each other. The RF surface may also be referred to as an RF blanket. The ion guide further includes: an RF voltage source configured to apply an alternating RF phase to each of the plurality of RF electrodes. The ion guide further includes: a DC potential source configured to apply a DC gradient across the RF surface, wherein the DC gradient is configured to direct the ion beam via a first ion path or a second ion path. The ion guide further includes: a second ion transport aperture and a third ion transport aperture, wherein ions traveling in the first ion path are directed to the second ion transport aperture and ions traveling in the second ion path are directed to the third ion transport aperture.

[0247] Hereinafter, the term "DC potential source" refers to any DC potential source. A voltage can be applied to the DC potential source to generate a potential (or electric field). A DC voltage source can be used to apply the voltage. The DC potential source can include electrodes to which a voltage can be applied to generate a DC potential. The DC gradient can be applied using RF electrodes (such that the RF electrodes include the DC potential source) by applying a DC voltage gradient to the RF electrodes. Additionally, the DC gradient can be applied using auxiliary DC electrodes (where the auxiliary DC electrodes include the DC potential source).

[0248] In use, the ion guide can be configured to receive an ion beam via the first ion transport aperture. The DC gradient can be configured to direct the ion beam via a first ion path or a second ion path such that the ions of the ion beam exit the ion guide via the second ion transport aperture or the third ion transport aperture. The DC gradient can be configured to separate the ion beam into a first portion and a second portion and direct the first portion of the ion beam along the first ion path (such that the first portion exits the ion guide via the second ion transport aperture) and direct the second portion of the ion beam along the second ion path (such that the second portion exits the ion guide via the third ion transport aperture). Additionally, the ion guide can be configured to receive an ion beam via the second ion transport aperture and / or the third ion transport aperture. The DC gradient can be configured to direct ions entering the ion guide via the second ion transport aperture along the first ion path such that the ions are directed to the first ion transport aperture and exit the ion guide via the first ion transport aperture. The DC gradient can be configured to direct ions entering the ion guide via the third ion transport aperture along the second ion path such that the ions are directed to the first ion transport aperture and exit the ion guide via the first ion transport aperture.

[0249] For the sake of brevity, most of the following descriptions assume that the ion guide is configured to receive an ion beam via a first ion transport aperture and the ion beam exits the ion guide via a second ion transport aperture and / or a third ion transport aperture. The first ion transport aperture is referred to as the inlet, the second ion transport aperture is referred to as the first exit aperture, and the third ion transport aperture is referred to as the second exit aperture. However, any of the examples described below can be used in both directions (such that the ion beam travels from the first ion transport aperture to the second ion transport aperture and / or the third ion transport aperture, or in the opposite direction such that one or more ion beams travel from the second ion transport aperture and / or the third ion transport aperture to the first ion transport aperture). Additionally, a downstream (or upstream) stage may be adjacent to the ion guide such that the "ion transport apertures" of the ion guide would more accurately be considered "ion transport regions", such as an inlet region or an outlet region. For example, if a path leads to a trapping region built into the ion guide, a "hole" may not be provided. However, for simplicity, specific examples will be described below with reference to holes.

[0250] Reference Figure 3 , an ion guide 100 according to an embodiment of the present disclosure is shown. The ion guide 100 will be described with reference to Figure 3 the axis shown. The ion guide includes: a front end that includes a first ion transport aperture (inlet); and a rear wall 140 that includes a second ion transport aperture (first exit aperture) 120 and a third ion transport aperture (second exit aperture) 130. The front end may be open (such that the first ion transport aperture covers the entire front end) or may include a wall that includes the first ion transport aperture.

[0251] The RF surface 110 includes a plurality of RF electrodes arranged parallel to each other. In use, opposite radio frequency phases may be applied to alternating RF electrodes in series (such that each RF electrode has an RF phase opposite to that of its adjacent electrode), thereby forming a repulsive pseudopotential surface. In Figure 3 the embodiment shown, the RF electrodes include elongated electrode plates, wherein the plane of each of the plurality of plates is parallel to each other (and parallel to the z-x plane indicated by the Figure 3 axis in). The first three RF electrodes are labeled 111, 112, and 113 to illustrate the arrangement. The remaining RF electrodes are not labeled. There may be more or fewer RF electrodes than Figure 3 shown. In one embodiment, the RF surface 110 may include 50 RF electrodes that include elongated electrode plates. The first surface may be perpendicular to the plane of each of the plurality of plates (and thus parallel to the x-y plane) or may be at an angle A with the plane of each of the plurality of plates (such that the first surface is at an angle (90 - A)° with the x-y plane). The angle A may, for example, be between 45° and 90°, or may be any other angle.

[0252] In another embodiment, the RF surface may include a plurality of printed RF electrodes on a PCB. In another embodiment, the RF electrodes may include electrodes formed on a substrate, for example, by lithography.

[0253] In a specific example where the RF electrode includes an elongated plate, the RF electrode may include a thickness between 0.5 mm and 1.5 mm and a spacing between 0.5 mm and 1.5 mm. The RF electrode may include other thicknesses or spacings. The applied RF voltage may be between 20 V and 2000 V, where the frequency is between 1 MHz and 3 MHz. The applied RF voltage may have other magnitudes or frequencies. The internal volume of the ion guide may be approximately 100 cm 3 , where the dimensions are approximately 10 cm × 10 cm × 1 cm. However, this is a specific example and the ion guide may have any internal volume. In some embodiments where the RF electrode includes a PCB printed electrode (or an electrode formed on a substrate by means such as lithography), the electrodes may be smaller and closer spaced than the above-described electrodes. The thickness and spacing of the RF electrode may be about 10 μm, where the applied RF voltage may have a frequency of at least 10 MHz. The thickness and spacing of the RF electrode may be greater than 10 μm, for example, between 10 μm and 1 mm.

[0254] The ion guide 100 further includes a first exit hole 120 and a second exit hole 130. The ion guide may include a rear wall 140 that includes the first exit hole 120 and the second exit hole 130. In use, an ion beam may enter the ion guide 100 through an entrance at the front end of the ion guide 100 opposite the rear wall (the front end may be open or may include a hole through which the ion beam enters the ion guide 100). Ions may be directed to the first exit hole 120 or the second exit hole 130 by a DC gradient applied by a DC potential source. The RF surface 110 acts as an ion capture region while the DC gradient is superimposed on the RF field to direct the ions to the selected exit hole such that the ions are captured and directed within a large volume. The DC gradient may include a component (referred to as orthogonal DC) that directs the ion beam left or right (i.e., in either x direction) to follow a first ion path or a second ion path, but may also include a component (referred to as axial DC) that accelerates the ion beam from the front end of the ion guide towards the rear wall of the ion guide.

[0255] The first exit aperture 120 and the second exit aperture 130 may include physical apertures to define a maximum extent of an output channel of the ion beam. The first exit aperture 120 and the second exit aperture 130 may include physical apertures and may be further defined by an electric field such that the first exit aperture 120 and the second exit aperture 130 are defined by the physical apertures and the electric field. The first exit aperture 120 and the second exit aperture 130 may be defined by an electric field without physical apertures. In an embodiment where the first exit aperture 120 and the second exit aperture 130 are defined by an electric field without physical apertures, the rear wall may include an opening, where the opening may extend across all or part of the rear wall. The first exit aperture 120 and the second exit aperture 130 may also have a DC voltage applied to them. The DC voltage applied to the first exit aperture 120 and the second exit aperture 130 may be equal or different. The DC voltage may be configured to trap or admit ions, for example, according to the needs of downstream elements of the mass spectrometer. The DC voltage may be variable.

[0256] The ion guide 100 may further include a top plate 150 opposite the RF surface 110. The top plate 150 may be parallel to the RF surface 110 or at an angle to the RF surface 110. The top plate 150 may be parallel to the x-y plane or at an angle to the x-y plane. The top plate 150 may include a ground plate or a repelling plate. In the case where the top plate 150 includes a repelling plate, the repelling plate may be configured to confine the ion beam close to the RF surface. The repelling plate may include a repelling DC electrode (i.e., a DC electrode to which a DC voltage may be applied to repel the ion beam). The repelling plate may be configured to prevent the ion beam from approaching the repelling plate, thereby avoiding contamination and charging effects on the repelling plate. In one embodiment, the ion beam may be kept at least 5 mm away from the repelling plate.

[0257] The rear wall 140 may optionally further include a chamber 160. The chamber 160 may be positioned between the first exit aperture 120 and the second exit aperture 130. In the case where an ion beam is admitted into the ion guide 100 along with a flow of neutral particles and / or charged droplets or other unwanted materials, the chamber 160 may be configured to receive the flow of neutral particles and / or charged droplets or other unwanted materials. The chamber 160 may include a cylinder that is open at the ion guide end of the cylinder and closed at the opposite end of the cylinder such that the chamber 160 is configured to receive unwanted materials and retain the unwanted materials within the chamber 160. Additionally, the chamber 160 may include apertures or other exit components configured to receive unwanted materials and allow the unwanted materials to leave the ion guide 100. A pump may be used to assist in removing unwanted materials from the ion guide via the chamber 160.

[0258] In some embodiments, the ion guide 100 may include a first side guard and a second side guard. The first side guard and the second side guard may be configured to prevent ions from leaving the ion guide 100 via the first (left) side or the second (right) side. The first side and the second side each extend between a front end and a rear wall 140, and each of the first side and the second side may be open, closed, or partially open. The first side and the second side may be parallel to each other or at an angle to each other. The first side and the second side may be parallel to the z-axis. The first side guard and the second side guard may include a first protection electrode and a second protection electrode, respectively. The first protection electrode and the second protection electrode may be mounted at the first side and the second side of the ion guide 100. A small repulsive DC voltage may be applied to the first protection electrode and the second protection electrode to repel ions from the first side and the second side. The voltages applied to the first side guard and the second side guard may be used in combination with a DC gradient to define the maximum lateral displacement of the ion guide. The first side guard and the second side guard may include the first protection electrode and the second protection electrode, or may include a series of PCB printed electrodes separated by a resistor chain. The first side guard and the second side guard may be physically close to the first side and the second side to prevent gas from leaving the ion guide 100 via the first side and the second side. In other embodiments, the first side and the second side may be open, and the first side guard and the second side guard may use only electrodes to prevent ions from leaving. In some embodiments, the first side guard and the second side guard may be configured to prevent leakage using only physical closures, or only electrodes, or a combination of physical closures and electrodes. Figure 3 The illustrated embodiment shows the first side guard 170 and the second side guard 180 as physically enclosing the first side and the second side.

[0259] In some embodiments, the ion guide may be configured to increase the spatial focusing of the ion beam near the first exit hole and the second exit hole. For example, a downstream element of a mass spectrometer may have a narrow spatial acceptance, and thus it may be beneficial to focus the ion beam leaving the ion guide. The ion guide may be configured to gradually increase the spatial focusing of the ion beam as the ion beam approaches the first exit hole or the second exit hole.

[0260] In embodiments where the RF surface including the RF electrode includes an elongated electrode plate, the RF electrode may include channels that are configured to increase the spatial focusing (i.e., reduce the spatial spread) of the ion beam closer to the first exit hole and the second exit hole. Referring Figure 4 , the RF electrode may include notches that increase in depth closer to the rear wall of the ion guide. For simplicity, only a portion of the RF electrode is shown. In Figure 4Among the electrodes shown, the RF electrode 210 is closest to the front end of the ion guide, and the RF electrode 260 is closest to the rear wall of the ion guide. The front RF electrode 210 includes an elongated electrode plate without notches. The RF electrodes 220, 230, 240, 250, and 260 each include two notches (221, 222, 231, 232, 241, 242, 251, 252, 261, 262) in the top edge of the elongated electrode plate. The depth of the notches increases with the distance from the front end of the ion guide. The width of the notches may also increase with the distance from the front end of the ion guide. The notches follow a first ion path and a second path. The DC gradient is configured to direct the ion beam to follow the first ion path or the second path. In Figure 4 the example shown, the ion beam follows the left path. The direction of the orthogonal DC that directs the ion beam to the left path is indicated by arrow 270. The direction of the axial DC that accelerates the ion beam toward the rear wall is indicated by arrow 280. The ion beam shown by the dashed area passes through the notches of the RF electrodes and is compressed into the notches by a repulsive DC field that confines the ion beam close to the RF surface. As the ion beam passes over the larger notches, the focus of the ion beam thus narrows, and more of the ion beam is accommodated within the notches. The ion beam 213 passing over the RF electrode 210 is the widest and has a relatively flat cross-section. The ion beam 223 passing over the RF electrode 220 is slightly narrower. The ion beam 233 passing over the RF electrode 230 is narrower, and as the ion beam is compressed into the notch, the lower portion of the ion beam 233 has begun to assume the shape of the notch. The ion beam 233 still retains a wider and flatter portion above the RF electrode. The ion beams 243 and 253 passing over the RF electrodes 240 and 250 have a larger lower proportion located within the notch, and the size of the upper portion wider than the notch decreases. The ion beam 263 passing over the RF electrode 260 is the narrowest and does not have a portion wider than the notch. In Figure 4 between those electrodes shown, there may be more RF electrodes such that the size of the notches gradually increases. The notches are shown as circular arcs but may also be other shapes.

[0261] In some embodiments, in addition to or instead of being shaped to provide channels, the RF electrodes may be shaped to provide a first side guard and a second side guard. Figure 5A A cross-section of an ion guide is shown, which shows the RF electrode 311 and the DC repelling plate 312. The DC repelling plate 312 may be further configured to apply a DC gradient. The first side guard and the second side guard include a first DC side guard 313 and a second DC side guard 314. A repulsive DC voltage may be applied to the first DC side guard 313 and the second DC side guard 314. Figure 5BShows a cross-section of an ion guide, which shows the RF electrode 321 and the DC repelling plate 322. The RF electrode 321 bends upward at the ends to form a first side guard 323 and a second side guard 324. The first side guard 323 and the second side guard 324 can be perpendicular to the central portion of the RF electrode 321, angled with respect to the central portion of the RF electrode, or the RF electrode can bend at the ends to form the first side guard and the second side guard. Figure 5C Shows a cross-section of a configuration similar to Figure 5B where the ends of the RF electrode 331 form a first side guard 333 and a second side guard 334. As in Figure 5C the ion guide also includes a DC repelling plate 332. The RF electrode 331 also includes a first notch 335 and a second notch 336. The first notch 335 and the second notch 336 are configured to focus the ion beam, as described in reference to Figure 4 above. Figure 5D Shows a cross-section of an ion guide including a DC repelling plate 342 and an RF electrode 341 that bends at the ends to meet (or approach) the edges of the DC repelling plate 342. The ion guide also includes attracting DC electrodes 343 and 344. The attracting DC electrodes 343 and 344 can be configured to apply an attracting DC field that pulls the ion cloud towards the edges or corners of the RF electrode (advantageously, strongly pulling the ion cloud), which improves the focusing of the ion beam.

[0262] In some embodiments, the DC gradient can be applied by applying a DC voltage gradient to the RF electrode. In other embodiments, an auxiliary DC electrode can be used to apply the DC gradient. As will be described below, in some embodiments, the top plate can include an auxiliary DC electrode configured to apply a DC gradient. In other embodiments, the auxiliary DC electrode can be mounted between the RF electrodes. Both the axial and orthogonal components of the DC gradient can be applied using an auxiliary DC electrode, or both the axial and orthogonal components of the DC gradient can be applied using the RF electrode, or one component can be applied using an auxiliary DC electrode and the other component can be applied using the RF electrode.

[0263] In one embodiment, referring to Figure 6 , the top plate 150 includes a repelling plate. In the example shown in Figure 2 , the configuration of the RF surface is similar to that in Figure 3the same as that in, wherein the RF electrode includes an elongated electrode plate. As described above, the repelling plate may include a repelling DC electrode. The repelling plate may also be configured to apply one or both components of a DC gradient. The repelling plate may include a repelling PCB 410 having a series of printed electrodes configured to act as a repelling plate and apply a guiding DC gradient. The DC gradient may be superimposed on the repelling field such that ions are directed to the first exit hole 120 or the second exit hole 130. The DC gradient includes an orthogonal component and may also include an axial component. The orthogonal DC gradient may be configured to provide a guiding force in two orthogonal directions (left and right). Optionally, the DC gradient may be configured to provide a guiding force in only one orthogonal direction (e.g., push ions to the left or right), while the other direction may be provided by a DC series (by linking the RF electrode series with resistors and applying a DC voltage between the electrodes such that a series of DC steps between the RF electrodes forms a gradient), a traveling wave applied to the RF electrodes, or pulsed DC. The guiding force of the RF electrode may depend on the direction in which the RF electrode is mounted. The RF electrode may be mounted such that the plane of the elongated electrode plate is parallel to the z-x plane. Optionally, the RF electrode may alternatively be mounted such that the plane of the elongated electrode plate is parallel to the z-y plane. The RF electrode may be configured to provide the orthogonal component of the DC gradient, and the top plate may be configured to provide the axial component of the DC gradient. In another embodiment, the RF electrodes may be arranged in a grid including rows of electrodes parallel to the z-y plane and columns of electrodes parallel to the z-x plane such that a DC gradient or a traveling wave may be applied in the orthogonal and axial directions.

[0264] The repelling PCB configured to apply a DC gradient may include a series of printed electrodes separated by a resistor chain. Voltages may be applied at each end. A linear DC gradient may be generated by a linear one-dimensional series of electrodes. The ion guide may require DC gradients in two dimensions, providing an orthogonal DC gradient in one dimension to direct the ion beam to the first ion path or the second ion path, and an axial DC gradient in the second dimension to accelerate the ions from the front end of the ion guide to the back wall. Refer to Figure 7 , a grid of printed electrodes separated by resistors may be used to generate a diagonal DC gradient. The electrodes are shown as squares (e.g., 510). Each electrode in a row is separated by a resistor (e.g., 520), and the electrodes at the ends of each row are separated from the electrodes at the ends of the adjacent row by resistors (e.g., 530). A two-dimensional DC gradient requires four voltage inputs, one at each corner of the grid (V1, V2, V3, and V4).

[0265] In another embodiment, the top plate 150 may include a repelling plate 600 that includes DC electrodes arranged in a shape that defines a first ion path and a second ion path. Refer to Figure 8, the top plate 150 may include a horseshoe configuration of DC electrodes. The rear wall 140 is indicated to show the positions of the first exit hole 120 and the second exit hole 130. The bottom of the repelling plate 600 corresponds to the front end of the ion guide. In use, an ion beam enters the ion guide via the front end, and the polarity of the DC gradient determines which of the first exit hole 120 and the second exit hole 130 the ion beam is directed to. Printable DC electrodes. The DC electrodes are indicated by white rectangles and triangles (three of the DC electrodes 610, 620, and 630 are marked as examples in Figure 8 . The DC electrodes can be segmented differently to form a horseshoe shape. The remaining space around the horseshoe (indicated by the shading) is configured to repel ions. This can be achieved by using DC side guards and / or by other printed electrodes. The width of the channel narrows towards the first exit hole 120 and the second exit hole 130, thereby focusing the ion beam close to the exit holes and allowing a wide channel to approach the front end of the ion guide. The repelling plate 600 can be further configured to receive ions that are transferred back to the ion guide from a downstream element (such as ion optics) via one of the exit holes. The ions can be directed to another exit hole without changing the DC gradient while storing the ions within the ion guide.

[0266] As described above, the top plate 150 may include a repelling plate configured to apply a DC gradient in addition to applying a repelling field. Refer to Figure 9 , in one embodiment, the ion guide 700 may include a top plate 750 that includes a ground plate or a repelling plate but does not apply a DC gradient. The ion guide includes an RF surface 710 similar to Figure 3 , where the RF electrodes include elongated electrode plates (three example RF electrodes are marked as 711, 712, and 713). The ion guide 700 includes a rear wall 740 that includes a first exit hole 720 and a second exit hole 730. The ion guide also includes auxiliary DC electrodes (three example DC electrodes are marked as 761, 762, and 763) indicated by the shading and mounted between the RF electrodes. The static electric potential felt by the ion beam is the combination of the DC applied to the RF electrodes and the DC applied to the auxiliary DC electrodes. The axial DC gradient can be achieved by changing the height of consecutive DC electrodes or by connecting the auxiliary electrodes by a resistor chain. The orthogonal DC gradient can be achieved by having wedge-shaped auxiliary DC electrodes, as Figure 9 shown.

[0267] In one embodiment, Figure 9 the top plate 750 shown can be replaced by a second RF surface. Refer to Figure 10 , the ion guide 800 includes an RF surface similar to Figure 9The same first RF surface 810 as shown. The first RF surface includes RF electrodes including elongated electrode plates (three example RF electrodes are labeled 811, 812, and 813). The ion guide also includes auxiliary DC electrodes indicated by shading mounted between the RF electrodes of the first RF surface (three example DC electrodes are labeled 861, 862, and 863). The ion guide 800 includes a rear wall 840 that includes a first exit hole 820 and a second exit hole 830. The ion guide 800 includes a second RF surface 870 located at the top of the ion guide. The second RF surface includes RF electrodes including elongated electrode plates (three example RF electrodes are labeled 871, 872, and 873). The ion guide also includes auxiliary DC electrodes indicated by shading mounted between the RF electrodes of the second RF surface (three example auxiliary DC electrodes are labeled 881, 882, and 883). The static potential experienced by the ion beam is a combination of the DC applied to the RF electrodes of the first RF surface and the second RF surface and the DC applied to the auxiliary DC electrodes mounted between the RF electrodes of the first RF surface and the second RF surface. The axial DC gradient can be achieved by varying the height of successive DC electrodes or by connecting the auxiliary electrodes by a resistor chain. The orthogonal DC gradient can be achieved by having wedge-shaped auxiliary DC electrodes.

[0268] Figure 10 The illustrated embodiments do not include repelling plates, so ions are not compressed towards the top RF surface or the bottom RF surface. This arrangement increases the volume available for ions under space charge. However, as described above, it may be beneficial to focus the ion beam near the exit hole. In an embodiment, this can be achieved by tilting one or both of the RF surfaces such that the distance between the first RF surface 810 and the second RF surface 870 decreases closer to the rear wall 840. In other embodiments, the DC gradient can be configured to attract more strongly to the auxiliary DC electrodes in the first RF surface or the auxiliary DC electrodes in the second RF surface, thereby pulling the ions towards the surface with the more attractive DC gradient.

[0269] Figure 4 and Figure 5C RF electrodes are shown that include notches to form channels in the RF surface. In a similar manner, in embodiments where a DC gradient is applied by auxiliary DC electrodes mounted between the RF electrodes, the auxiliary DC electrodes can include peaks or valleys to define channels that are configured to improve the spatial focusing of the ion beam near the exit hole. This can be in addition to or in place of the notches in the RF electrodes. Refer to Figure 11, showing the auxiliary DC electrode 900, which includes a peak 910, a valley 920, and a ramp 930 between the peak and the valley to provide an orthogonal DC gradient. The peak 910 may correspond to the position of the first ion path and the valley 920 may correspond to the position of the second ion path. By selecting the polarity of the DC applied to the auxiliary DC electrode, ions are directed to the first ion path or the second ion path.

[0270] Note that any of the above features related to the spatial focusing of the ion beam can be used for the spatial focusing of the ion beam traveling from the first ion transport hole to the second ion transport hole or the third ion transport hole, or for the spatial focusing of the ion beam traveling from the second ion transport hole or the third ion transport hole to the first ion transport hole.

[0271] Reference Figure 9 and Figure 10 The described embodiments include auxiliary DC electrodes that include elongated electrode plates. The elongated electrode plates are mounted between the RF electrodes. In other embodiments, the ion guide may include auxiliary DC electrodes printed between the RF electrodes. Reference Figure 12 , the ion guide 1000 may include an RF surface 1010, a rear wall 1040 including a first exit hole 1020 and a second exit hole 1030, and a top plate 1050. The top plate 1050 may include a repelling plate or a ground plate. The ion guide 1000 may also include a plurality of auxiliary DC electrodes printed onto a PCB 1070. The RF surface 1010 may include RF electrodes mounted between the printed auxiliary DC electrodes. Three RF electrodes 1011, 1012, and 1013 are marked as examples, and three auxiliary DC electrodes 1061, 1062, and 1063 are marked as examples. The auxiliary DC electrodes may be separated by resistor chains, for example to form a two-dimensional grid similar to Figure 7 that shown. To reduce contamination, the RF electrodes may be suspended above the exposed portion of the PCB 1070 located between the RF electrodes and the auxiliary DC electrodes (where the exposed portion of the PCB 1070 may include a dielectric material).

[0272] For embodiments including auxiliary DC electrodes mounted between RF electrodes, where the auxiliary DC electrodes include elongated electrode plates, the height of the auxiliary DC electrodes relative to the RF electrodes can affect the performance of the ion guide. Preferably, the auxiliary DC electrodes may not protrude above the RF electrodes into the capture volume of the ion guide. In the case where the auxiliary DC electrodes are recessed below the RF electrodes, as the degree of recess of the DC electrodes relative to the RF electrodes increases, the proportion of the applied DC voltage reaching the center of the capture region decreases.

[0273] Reference Figure 13, the ion guide as described above can be used near the front of a complex hybrid mass spectrometer to separate the fast region from the slow or lossy regions. Figure 13 An example is shown in Figure 13 which shows a schematic of an instrument that combines fast MS2 operation through a fast path to a multi-reflection time-of-flight (MR-ToF) analyzer 1263 with a slow path to a Fourier transform mass analyzer 1274 for MS1, or with complex ion processing within an adjacent resolving ion trap (where MS1 can include analysis of unfragmented precursor ions and MS2 can include analysis of fragmented precursor ions). The instrument can include an electrospray ionization (ESI) source 1210, a lens 1220 (such as an S-lens including an ion funnel with increasing interpolation spacing between rings), an ion guide 1230, and a 90° ion guide 1240. The ion beam can then pass through a beam-switching ion guide 1250 according to an embodiment of the present disclosure, and the ion beam can be directed to the fast path or the slow path. The fast path can include a quadrupole mass filter 1261, a collision cell 1262, and an MR-ToF analyzer 1263. The slow path can include a C-trap 1271, a collision cell or resolving ion trap 1272, an ion guide 1273, and a Fourier transform analyzer 1274. The ion guides 1230, 1240, and 1273 are not beam-switching ion guides according to the present disclosure but direct the ion beam along a single path. Although the analyzers can be arranged in a single path, ion losses through the chain can reduce the sensitivity of the MR-ToF analyzer. The chain is blocked whenever the ion trap performs slower ion manipulations such as MS3 (involving fragmentation of fragment ions) or electron transfer dissociation (ETD). The MR-ToF analyzer also blocks the rear of the ion trap, preventing the possible installation of a laser for photodissociation fragmentation. At least for these reasons, the ability to switch between the fast path and the slow path is beneficial. Additionally, the fast path and the slow path can be arranged side by side to make the instrument more compact than a single long path.

[0274] Ion beam splitter

[0275] As described above, in the DIA method, for each injection performed with fragmentation, there is an additional injection with the same quadrupole isolation window and without fragmentation. As is normal for DIA, the target mass is scanned over a predetermined range and isolation step.

[0276] To further improve efficiency, it is preferred to eliminate the overhead due to the need to actively switch beam paths. One way to do this is to separate out a portion of the ion implantation. This can be achieved by differentiating the properties of the ions (such as position or energy). Then, a single (longer) implantation can be separated and used to supply two ion destinations. Even more preferably, these conditions are set such that a certain proportion of the ion beam is separated, either as a function of collision cooling or spatial distribution, and the separated ion beam is delivered to a separate ion destination. This then further saves the time overhead between implantations. Thus, a method of beam separation is provided by skimming off a portion of a broad ion packet. The ion beam is separated by passing the ions over a wedge electrode. The ions are separated based on the spatial distribution of the ion packet.

[0277] Figure 14 An ion beam splitter is shown. In this particular example, the ion beam splitter includes a pair of RF surfaces or a single RF blanket, where side guards and a wedge electrode (also referred to as a beam separation electrode or “beam limiter”) are positioned between an entrance hole and an exit hole. Ions are injected through the entrance hole and allowed to spread across the entire width of the deflector. The ions can be trapped during this period, for example, by guiding a DC gradient applied across the RF electrodes. After the ions have diffused across the width, the DC gradient or traveling wave pulls the ions towards the exit hole. The ions are repelled by the wedge electrode electric field but still attracted, causing the wedge to cut the ion distribution into two parts, and the resulting different ion populations are attracted to their nearest exit hole.

[0278] In the DIA method, when a series of MS2 scans are performed, most ions will be sent for analysis while a certain proportion will be separated and can be accumulated. The Fourier transform mass analyzer can be used to analyze this accumulation of ions in a manner similar to the “Boxcar” method described in the background art to provide an HDR scan.

[0279] The proposed ion separator is built on the principle of previously proposed beam switching devices (such as those described in UK Patent Application No. 2209555.8 or Patent Publications US7829850B2, US20190103261A1, US8581181B2, or US9984861B2) to create branched ion paths. Instead of switching the ion beam between destinations, the proposed beam splitter provides a proportional separation of the ion beam on the wedge electrode. Figure 14 The device of can be used to replace Figure 1 the ion routing multipole / fragmentation chamber 12 within a hybrid mass spectrometer of.

[0280] Once a portion of the precursor ions has been separated using the ion beam splitter, the separated ions must be stored somewhere. Therefore, there is a need for Figure 1The configuration of the instrument is further changed to incorporate an additional ion storage device.

[0281] Two possible configurations of an instrument incorporating a branched ion path (e.g., a hybrid Fourier transform / MR-ToF mass spectrometer) are shown in FIG. 15. These configurations show how a beam splitting device can be incorporated into Figure 1 the instrument. Both configurations provide the ability to perform parallel ion trapping at a first ion destination in a C-trap ( Figure 15A ) or an additional trapping region ( Figure 15B ) without blocking the ion beam path to a second ion destination (e.g., a linear trap or a time-of-flight mass analyzer).

[0282] In Figure 15A the first configuration shown, the ion beam splitter is located after the quadrupole mass filter and operates to direct MS2 injections via a first ion storage device (e.g., a linear ion storage device) to a first mass analyzer (e.g., a time-of-flight mass analyzer) and SIM injections via a second ion storage device (e.g., a curved linear ion storage device) to a second mass analyzer (e.g., a Fourier transform mass analyzer). While multiple MS2 injections and scans are being performed, the SIM injections are accumulated together in the first ion storage device. After a series of SIM injections have been accumulated in the first ion storage device, the ions can be ejected into the second mass analyzer (e.g., for long acquisition times). Advantageously, since the precursor ions from the SIM injections are accumulated in the curved linear ion storage device (e.g., a C-trap), they do not block the ion beam path to the linear ion storage device (e.g., a DP-R trap).

[0283] In the second configuration, the ion beam splitter device is located after the curved linear ion storage device (e.g., a C-trap) and thus alternatively separates ions between a second ion storage device (e.g., a linear trap) and an intermediate ion storage device (also referred to as the "parallel trapping region"). The intermediate ion storage device can be an ion trap or an IRM-type device. Alternatively, the intermediate ion storage device can be provided by a DC barrier, instead of the first exit aperture of the ion beam splitter, such that ions accumulate at a first exit region (e.g., at the end of the wedge of the ion beam splitter).

[0284] Once SIM ions from multiple sub-ranges have been accumulated in the parallel trapping region, the precursor ions can be returned via the ion beam splitter to a third ion storage device (e.g., a C-trap) and ejected from the third ion storage device into the second mass analyzer (e.g., a Fourier transform mass analyzer).

[0285] Alternatively, once SIM ions from multiple sub-ranges have accumulated in the parallel trapping region, the precursor ions can pass through the ion beam splitter to reach a second ion destination (e.g., a linear trap / MR-ToF).

[0286] To minimize overhead, it is important that the beam separation device is relatively fast, has voltage transitions and sufficient ion transport to eliminate mixing, preferably taking about 1 millisecond or less. If the process is relatively slow, ions can first accumulate before the switching region, such as in a C-trap, to allow parallelization of accumulation and subsequent ion transport.

[0287] In one example, the ion beam splitter creates a wide spatial distribution of ions in an ion guide. The distribution and the elongated ion cloud then pass through a sharp electric field that cuts through the distribution and divides it into two parts. The sharp electric field is preferably a sharp DC field. However, a sharp RF field can also be used in some examples.

[0288] Figure 14 One possible means for achieving this is shown. The ion guide consists of a wide RF tunnel or blanket, which is composed of a series of stacked RF electrodes to generate a pseudopotential surface. The opposing top surface can be a DC repeller or another matching RF surface.

[0289] Additional DC guard electrodes located around the perimeter can prevent ions from entering or leaving the system, except at the locations of the inlet and outlet regions (which can be holes).

[0290] A DC gradient can be applied across the RF electrode stack (which can be PCB printed). Alternatively or additionally, if the repelling plate is divided into a series of electrodes, a DC gradient can be applied along the repelling plate.

[0291] Figure 14 An elongated substrate electrode capable of pushing ions in only two directions (left or right) along one axis (the longitudinal axis) via a superimposed DC gradient is shown. However, in other examples, the RF electrodes can be further divided to form a grid such that orthogonal DC gradients can be applied to the electrodes in the longitudinal and transverse directions to push ions in any direction within the plane of the electrode surface / plate (the first surface of the ion beam splitter).

[0292] Alternatively, the top plate electrode can be set opposite and facing the substrate electrode. The top plate electrode (on the second surface of the ion beam splitter) can be divided along an axis orthogonal to the substrate electrode such that the substrate electrode can apply a DC gradient in the longitudinal direction and the top plate electrode can apply a DC gradient in the transverse direction (or vice versa, or in any pair of orthogonal directions). In this way, the two DC gradients can operate together to push ions in any direction within the plane of the ion beam splitter.

[0293] Further details regarding ways of generating RF blankets and DC forces in multiple directions are described with reference to the ion beam switch above. These methods are equally applicable to the ion beam splitters described herein.

[0294] The beam separation electrode is disposed at an end of the ion beam splitter opposite the inlet region, with exit regions on either side of the beam separation electrode. In some examples, the beam separation electrode is a wedge electrode. The beam separation electrode provides a small repulsive field to resist the approach of the ion packet being pushed towards it by the applied DC gradient. The beam separation electrode divides the ion cloud into two parts. In other words, the beam separation electrode acts as a knife to split the ion cloud while also guiding the two parts of the ion cloud to their respective exit regions. Preferably, the beam separation electrode is a DC electrode. Alternatively, the beam separation electrode can be an RF electrode.

[0295] In the case where the beam separation electrode is a wedge electrode, the apex (tip) of the wedge can be off - center. The relative position of the tip of the wedge electrode can define the proportion of ions being directed to each exit region (and thus the relative proportion of the ion packets being directed to the first ion destination and the second ion destination). The beam separation electrode can be depicted as a two - dimensional triangle (in a plan view), as Figure 14 shown. However, in some examples, the wedge beam separation electrode can be a three - dimensional triangular prism. Thus, the "tip" of the beam separation electrode can be shown as a point in two dimensions and can be a line (apex edge) of a three - dimensional triangular prism.

[0296] In some examples, a DC gradient is continuously applied in the longitudinal direction to push the ions towards the exit regions. In these examples, the length of the device should be sufficient for the ion packet to uniformly broaden in the transverse direction as it travels in the longitudinal direction, such that the ion cloud expands across the width of the channel before being separated (split into two parts by the beam separation electrode).

[0297] In other examples, a shorter device can be provided. After injecting the ion packet into the ion guide, the DC gradient in the longitudinal direction can initially be decreased, disabled, or reversed to allow the ions additional time to spread in the transverse direction. Alternatively, an elongated DC trapping well can be generated by applying a negative DC potential to one or more of the RF blanket electrodes. The operation of the device can be performed in two steps: an injection step, where the ion packet is injected into the ion guide and allowed to spread; and an extraction step, where the ion packet is pushed towards the beam separation electrode, split into two parts, and each part is transmitted via the respective exit region. Operating the ion beam splitter according to a two - step sequence may add some overhead time. However, the device can be more compact and can more precisely control the proportion of the ion packets being guided via the first exit region and the second exit region.

[0298] Many prior art ion beam switching devices involve switching in RF gas-filled multipoles, which is slow due to ion diffusion in the gas. In contrast, the proposed ion beam splitter can be configured to operate under pure molecular flow conditions (where Kn > 10-20).

[0299] A gas thickness of at least 0.2 mm*mbar in the ion guide is preferred.

[0300] The pressure in the ion guide should be relatively low (unless the device is additionally used as a collision cell). A pressure in the range of 2×10 -3 to 5×10 -3 mbar is preferred to facilitate faster ion separation.

[0301] The beam separation electrode can be a solid electrode or a PCB attached to the same surface as the RF electrode or repeller (if present).

[0302] The position offset along the width of the device or the apex position defines the proportion of ions in the two parts and can be offset away from the center in order to send the majority of ions in a group for MS2 analysis.

[0303] The DC potential on the adjustable guard electrode can be adjusted to affect the width or position of the ion beam, such that the relative proportion of ion packets that are tunably separated into a first part and a second part is adjusted (or the beam switching functionality is increased).

[0304] Additionally or alternatively, the segmentation of the RF electrode and / or the application of a transverse DC gradient can be applied to affect the width or position of the ion beam, such that the relative proportion of ion packets that are tunably separated into a first part and a second part is adjusted (or the beam switching functionality is increased).

[0305] Similarly, the beam separation electrode (or "beam limiter") can consist of more than one electrode, which can be activated via the application of a beam separation DC potential. The beam separation DC potential can be elevated relative to the general DC offset of the device. Selective application of DC potential to individual electrodes that make up the entire beam separation electrode can allow for an effective point offset of the wedge of the beam separation electrode. RF can also be applied to the beam separation electrode to correct for the mass dependence of the ratio of ions separated into two parts.

[0306] In other examples, the individual electrodes in the beam separation electrode can be activated via the application of a beam separation RF potential.

[0307] In some operating modes, a DC gradient in the transverse direction can be used to drive substantially all ions to one exit region or the other (such that the wedge no longer splits the ion packet but directs it to one exit or the other).

[0308] Construct a simulation model of a suitable beam splitter as described above within the MASIM3D software suite. The 3D model is shown in cross-section in Figure 16 and consists of two opposing stacks of 4 mm wide RF electrodes spaced 10 mm apart, where each electrode in series carries the opposite phase of the applied 2 MHz, 500 V peak-to-peak RF. Wedge electrodes are suspended near the distal end of the device and a small +2 V repulsive DC voltage is applied. A 10 V potential gradient is applied across the entire region and the pressure is set to 0.1 mbar N2.

[0309] Figure 17 A graph showing the ion trajectories across the beam splitter shown in Figure 14 and Figure 16 is shown. m / z 200 ions are generated in front of the device and allowed to drift under the DC gradient. In this example, the ions diffuse in the elongated potential well naturally created by the RF electrode stacks, obviating the need for an electrokinetic trapping stage. It can be seen that most of the ions are deflected by the wider side of the wedge electrode while only a few are skimmed to the other side. The passage of ions used for this simulation takes approximately 2 milliseconds. When the RF amplitude is reduced to 200 V, the passage of ions is reduced to approximately 1 millisecond.

[0310] Figure 18 A diagrammatic illustration of the DIA method that can be implemented on Figure 1 , Figure 13 and the mass spectrometer of Figure 15 is shown. In the DIA method according to some specific examples, for each injection performed with fragmentation, there is a simultaneous injection with the same quadrupole isolation window and reduced collision energy or no collision energy. As is normal for DIA, the target mass is scanned over a predefined range and isolation step. Figure 18 Such a scan sequence from m / z 300 Th - 900 Th is shown, with a 5 Th isolation window, as can be used for "bottom-up" measurements of protein samples for digestion. An optional AGC (automatic gain control) pre-scan (preferably in a time-of-flight mass analyzer) can be performed to help determine the appropriate ion injection time in subsequent scans. Alternatively, an optional MS1 full scan can be performed using a Fourier transform mass analyzer (e.g., an orbitrap mass analyzer). The MS2 injection is transferred to a linear trap and extracted to a time-of-flight (e.g., MR-ToF) mass analyzer for a series of scans (T1 to T61). SIM injections are accumulated together in an ion storage device, such as a curved linear ion storage device (C-trap) or other ion trapping device. An HDRMS1 scan can be generated by analyzing the combined precursor ions (e.g., in a Fourier transform mass analyzer).

[0311] The ion beam splitter allows for concurrent (simultaneous) execution of SIM and MS2 injections to minimize quadrupole and source switching time overhead, which could otherwise significantly reduce the time available for ion accumulation. A single "injection" of ions from the quadrupole mass filter into the ion beam splitter results in ion accumulation in both the first and second ion destinations. At the end of the cycle, the series of accumulated SIM injections are then extracted into a Fourier transform mass analyzer for long transient analysis, e.g., at 240K resolution.

[0312] Advantageously, AGC is used to control the number of ions in the curved linear ion storage device and the Fourier transform mass analyzer such that the total number of ions in each SIM injection does not add up to a level that overwhelms the ion storage device. For example, if there are 60 SIM injections per cycle, the number of ions in each SIM injection can be limited to approximately 1500 ions.

[0313] For longer cycles, additional scans using the Fourier transform mass analyzer can be performed in the middle of the cycle to increase the dynamic range of the precursor scan data at the cost of reducing the maximum transient time by half. In other words, the SIM injections from the first half of multiple sub-ranges can be analyzed together in the first scan, and the precursor ions of the SIM injections from the second half of multiple sub-ranges held in the first ion storage device can be analyzed together in the second scan.

[0314] More generally, rather than performing a single scan on precursor ions from the total m / z range, the method can include multiple scans of precursor ions, where each scan includes precursor ions from multiple sub-ranges.

[0315] Figure 18 The method can be performed on Figure 1 the combined Fourier transform mass analyzer and MR-ToF instrument shown, as modified according to Figure 14 and FIG. 15. Alternatively, the method can be performed on a single mass analyzer instrument, such as a ToF instrument only or a Fourier transform mass analyzer only (and potentially taking longer).

[0316] The timing of different operations can be configured such that certain operations are performed in parallel to reduce the total time taken. Figure 1 The Fourier transform / MR-ToF instrument shown in

[0317] is capable of running single injections at approximately 200 Hz with an injection time (also known as "fill time") of 3 ms and an overhead of 2 ms. Additional overhead will deplete the duty cycle and reduce the instrument sensitivity.

[0318] SIM / MS2 injections can be interleaved, depending on whether fragmentation occurs in the IRM 120 or in the high-pressure region of the extraction trap 140. The injection (also referred to as "loading") can be carried out via a branched ion path and directed to an ion storage device (also referred to as "extraction trap") or a mass analyzer, thereby minimizing additional overhead. Additional injections can involve precursor ions from an ion filter with the same m / z characteristics. During operation according to some example methods, the collision energy of the fragmentation chamber can be adjusted.

[0319] Ions from different injections should not be mixed before the fragmentation step. Since the ion beam splitter is delivering precursor ions to two ion destinations, fragmentation is performed downstream of the ion beam splitter (e.g., via the high-pressure region of the extraction trap). Before allowing ion packets from the next sub-range, the ion packets from one sub-range should be cleared from the ion beam splitter.

[0320] In Figure 18 In an additional variant of the DIA process shown, a stepped fragmentation energy can be applied to each MS2 scan. In other words, MS2 ions are added to the second ion storage device in multiple injections, each injection having a different fragmentation energy.

[0321] In some variants of the method, in the case of performing a pre-scan, it may be preferred not to perform SIM injections for the m / z sub-ranges of the total precursor mass range that have been densely loaded. This can be determined based on a full MS pre-scan, and the full MS scan should collect sufficient data for such regions. Omitting SIM injections for these sub-ranges can further reduce the time required for the overall method and improve the resolution of the scans for the combined SIM injections. The SIM injections can be omitted by operating the ion beam splitter in an operating mode such that substantially all ion packets are directed to the second exit region. The HDR MS1 scan can be obtained by combining the full MS pre-scan with the scans for the combined SIM injections.

[0322] Using a front-end accumulation device such as a trapped ion mobility separator (as incorporated in the Bruker TIMS-ToF series of mass spectrometers) or a structure for non-destructive ion manipulation (SLIM) can also be beneficial for the above examples. Such devices can release ions in an m / z range synchronized with the quadrupole isolation window. Thus, ion transmission can be improved. Also, as a result, the injection time required can be reduced (due to a brighter isolated ion beam). This reduction in injection time can be used to offset the additional time overhead of SIM injections.

[0323] To separate ion beams across the beam separation electrodes, the actual design of the beam separation electrodes should prevent any changes in contamination and m / z correlation.

[0324] In some examples, the wedge electrode may operate in a vacuum ion guide. In this case, the beam separation electrode may be a split gate with fast ions (>20V - 30V) and two symmetric output RF guides. The switching may be performed quickly using FET switches such that only the ions over the length of the gate (about 5mm - 6mm) are affected and potentially lost. The separation ratio may be controlled by PWM, as described in GB2585472, which is incorporated herein by reference. In a similar manner to WO2013092923, which is incorporated herein by reference, multiple outputs may be served by such a gate.

[0325] In other examples, the wedge electrode may operate in a gas-filled ion guide. In this case, the beam separation electrode relies on slow-moving ions with a strong correlation between z and m / z. Separation by gas flow is not preferred. Thus, a negligible gas velocity is assumed. Methods may be provided to overcome any surface and m / z-related effects. For example, the ion beam may be rasterized across the entrance to spread the ions more laterally. In another example, the mass range may be pre-shaped to avoid ejection in the DC / RF edge field.

[0326] In this document, the term mass may be used to refer to the mass-to-charge ratio m / z. Unless otherwise stated, the resolution of a mass analyzer is understood to refer to the mass analyzer resolution determined at a mass-to-charge ratio of 200.

[0327] In the description of the present invention herein, it should be understood that, unless otherwise implicitly or explicitly understood or stated, words in the singular form cover their plural counterparts, and words in the plural form cover their singular counterparts. Further, it should be understood that, unless otherwise implicitly or explicitly understood or stated, for any given component or embodiment described herein, any possible candidates or alternatives listed for that component may generally be used individually or in combination with each other. Additionally, it should be understood that, as shown in the figures herein, the figures are not necessarily drawn to scale, where only some elements may be drawn for the clarity of the present invention. Also, reference numerals may be repeated in the respective figures to show corresponding or similar elements. Additionally, it should be understood that, unless implicitly or explicitly otherwise understood or stated, any such list of candidates or alternatives is merely illustrative, and not restrictive.

[0328] Unless otherwise defined, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. It should be understood that an implied "about" precedes the quantitative terms recited in the description of the invention, such that minor and non-substantive deviations are within the scope of the invention. In this application, unless specifically stated otherwise, the use of the singular includes the plural. In addition, the use of "comprising", "including" and "containing" is not intended to be limiting and does not exclude the possibility of also including other elements. In the case of using the word "consisting", this is intended to mean the exclusion of other elements. As used herein, "a" may also refer to "at least one" or "one or more". In addition, the use of "or" is inclusive, such that the phrase "A or B" is true when A is true, B is true, or both A and B are true.

[0329] As used in this document, when used as a noun, the term "scan" means a mass spectrum, regardless of the type of mass analyzer used to generate and acquire the mass spectrum. When used as a verb herein, the term "scan" refers to generating and acquiring a mass spectrum by a mass analysis method, regardless of the type of mass analyzer or mass analysis used to generate and acquire the mass spectrum. As used herein, the term "full scan" refers to a mass spectrum that covers a range of mass-to-charge (m / z) values including multiple mass spectral peaks.

[0330] As used in this document, each of the terms "liquid chromatography" and "liquid chromatography method" (both abbreviated as "LC") and the term "liquid chromatography mass spectrometry" (abbreviated as "LC-MS") is intended to apply to any type of liquid separation system capable of separating a liquid sample carrying multiple analytes into various "fractions" or "isolates", where the chemical composition of each such "fraction" or "isolate" is different from the chemical composition of each other such fraction or isolate, where the term "chemical composition" refers to the quantity, concentration, and / or species of the various analytes in the fraction or isolate. Thus, the terms "liquid chromatography", "liquid chromatography method", "liquid chromatography mass spectrometry", "LC", and "LC-MS" are intended to include and refer to, but are not limited to, liquid chromatography, high performance liquid chromatography, ultra-high performance liquid chromatography, size exclusion chromatography, and capillary electrophoresis devices.

[0331] In addition to LC devices, any other separation device, including ion mobility devices, HPLC, GC, or ion chromatography, can be connected to a mass spectrometer. Any known fragmentation method (including collision-activated dissociation, photon-induced dissociation, electron capture, or electron transfer dissociation) also produces data suitable for the present invention.

[0332] Although embodiments in accordance with the present disclosure have been described with reference to a particular type of apparatus and application, specifically a mass spectrometer, and the embodiments have certain advantages in this context, as discussed herein, the methods in accordance with the present disclosure can be applied to other types of apparatus and / or applications. Specific manufacturing details and associated uses of ion guides are not only potentially advantageous (especially considering known manufacturing constraints and capabilities), but can also vary significantly to obtain devices with similar or identical operation. Unless otherwise stated, each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent, or similar purpose. Thus, unless otherwise stated, each feature disclosed is only one example of a series of equivalent or similar attribute features.

[0333] Unless otherwise stated, any and all uses of example or exemplary language (such as "for example," "such as," and similar language) herein are intended only to better illustrate the invention and do not limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to practicing the invention.

[0334] Unless otherwise specified or the context otherwise requires, any steps described in this specification may be performed in any order or simultaneously.

[0335] All aspects and / or features disclosed in this specification may be combined in any combination, except for combinations where at least some of such features and / or steps are mutually exclusive. As described herein, there may be specific combinations of aspects with additional benefits, such as aspects of ion guides for mass spectrometers and / or ion mobility spectrometers. Specifically, the preferred features of the invention apply to all aspects of the invention and may be used in any combination. Similarly, features described in non-essential combination form may be used alone (not in combination).

Claims

1. An ion deflector, comprising: An inlet region for receiving a packet of ions; A first outlet region for ejecting ions; A second outlet region for ejecting ions; A plurality of electrodes for applying a DC gradient, wherein the DC gradient urges the ions away from the inlet region and towards the first outlet region and / or the second outlet region, wherein the plurality of electrodes are disposed on a first surface of the ion deflector, and wherein the plurality of electrodes are radio frequency (RF) electrodes arranged to generate a pseudopotential surface; And A beam separation electrode arranged to direct a first portion of the packet of ions towards the first outlet region and a second portion of the packet of ions towards the second outlet region.

2. The ion deflector according to claim 1, wherein a DC repeller is disposed on a second surface of the ion deflector opposite the first surface, and wherein the inlet region, the first outlet region, and the second outlet region are between the first surface and the second surface.

3. The ion deflector according to claim 1, further comprising one or more electrodes disposed on a second surface of the ion deflector opposite the first surface, and wherein the inlet region, the first outlet region, and the second outlet region are between the first surface and the second surface.

4. The ion deflector according to any one of the preceding claims, wherein the beam separation electrode includes a vertex between the first outlet region and the second outlet region.

5. The ion deflector according to claim 4, wherein the vertex is off-center relative to a central longitudinal axis of the ion deflector.

6. A method of manipulating ions using an ion deflector, the ion deflector comprising: An inlet region; A first outlet region; A second outlet region; A plurality of electrodes disposed on a first surface of an ion deflector; And A beam separation electrode, The method comprising: Injecting a packet of ions via the inlet region; Applying a radio frequency signal to the plurality of electrodes to generate a pseudopotential surface; Applying a longitudinal DC gradient to the plurality of electrodes to urge the ions away from the inlet region and towards the first outlet region and / or the second outlet region; and Using the beam separation electrode to direct a first portion of the packet of ions towards the first outlet region and a second portion of the packet of ions towards the second outlet region.

7. The method according to claim 6, wherein the method further comprises: Applying a transverse DC gradient to the plurality of electrodes to urge the ions away from the first outlet region and towards the second outlet region to adjust a relative proportion of the packet of ions constituting the first portion and the second portion; And / or Applying a transverse DC gradient to the plurality of electrodes to urge the ions away from the first outlet region and towards the second outlet region such that substantially all of the ions in the packet of ions are directed via the second outlet region.

8. A mass spectrometry method, comprising the steps of: For each of a plurality of sub-ranges selected from the total m / z range: Configure the ion beam splitter according to claim 1 to direct ions towards a first ion destination and a second ion destination, wherein the first ion destination is a first ion storage device; Accumulate in the first ion storage device a sample of precursor ions to be analyzed, the precursor ions having an m / z value within the sub-range; Wherein Either of the following cases exists: a) The second ion destination is a first mass analyzer, and wherein the method further comprises injecting a sample of fragmented precursor ions into the first mass analyzer, or b) The second ion destination is a second ion storage device, and wherein the method further comprises accumulating in the second ion storage device a sample of fragmented precursor ions for analysis in a first mass analyzer, wherein the sample of fragmented precursor ions is formed by fragmentation of precursor ions having an m / z value within the sub-range.

9. The method according to claim 8, wherein the first ion storage device is an intermediate ion storage device, and wherein the method further comprises: i) Configure the ion beam splitter to transfer the precursor ions accumulated in the first ion storage device to a third ion storage device for analysis in a second mass analyzer; Or ii) Configure the ion beam splitter to transfer the precursor ions accumulated in the first ion storage device to the second ion destination.

10. The method according to claim 9, wherein the precursor ions transferred from the first ion storage device to i) the third ion storage device or ii) the second ion destination include a sample of the precursor ions for each of the plurality of sub-ranges.

11. The method according to claim 9 or claim 10, wherein the first ion storage device is provided by a DC barrier adjacent to a first output region.

12. The method according to any one of claims 8 to 11, wherein the ion beam splitter is configured to operate under pure molecular flow conditions.

13. The method according to any one of claims 8 to 12, wherein the method further comprises fragmenting the precursor ions to produce a sample of fragmented precursor ions, wherein the second ion destination is the second ion storage device, and wherein the ions are fragmented in the second ion storage device.

14. The method according to any one of claims 8 to 12, wherein the method further comprises fragmenting the precursor ions to produce a sample of fragmented precursor ions, wherein the ions are fragmented using a multipole collision cell.

15. The method according to any one of claims 8 to 14, further comprising for each of the plurality of sub-ranges: Configure an ion filter to transmit precursor ions having an m / z value within the sub-range; wherein the sample of precursor ions is received from the configured ion filter, wherein the sample of fragmented precursor ions is formed by fragmentation of the precursor ions received from the configured ion filter.

16. The method according to claim 15, wherein configuring the ion filter comprises setting a transmission window of the ion filter, wherein the transmission window is adjusted between each of the plurality of sub-ranges, and wherein for each sub-range, the transmission window for the step of accumulating the sample of the precursor ions is the same as the transmission window for the step of injecting the sample of the fragmented precursor ions into the first mass analyzer or accumulating the sample of the fragmented precursor ions.

17. The method according to claim 16 or claim 16, further comprising configuring an ion mobility separator to transfer precursor ions having m / z values within the sub-range to the ion filter.

18. The method according to claim 17, further comprising, for each of the plurality of sub-ranges within the total m / z range, controlling the ion mobility separator such that the precursor ions transferred to the ion filter correspond to the transmission window of the ion filter.

19. The method according to any one of claims 9 to 18, wherein accumulating the sample of the precursor ions comprises controlling the fill time of the precursor ions based on the relative abundances of the precursor ion species within the corresponding sub-range.

20. The method according to any one of claims 9 to 19, wherein any one of the following cases exists: The second ion destination is a second ion storage device, and the sample of the fragmented precursor ions is accumulated in the second ion storage device. The method further comprises, for each of the plurality of sub-ranges, ejecting the sample of the fragmented precursor ions from the second ion storage device into the first mass analyzer and analyzing the sample of the fragmented precursor ions in the first mass analyzer, wherein the plurality of sub-ranges includes a first sub-range and a second sub-range, and wherein the step of analyzing the sample of the fragmented precursor ions from the first sub-range at least partially overlaps with the step of accumulating the sample of the fragmented precursor ions formed by the fragmentation of precursor ions having m / z values within the second sub-range in the second ion storage device; or The second ion destination is the first mass analyzer, and the sample of the fragmented precursor ions is injected into the first mass analyzer. The method further comprises analyzing the sample of the fragmented precursor ions in the first mass analyzer, wherein the plurality of sub-ranges includes a first sub-range and a second sub-range, and wherein the step of analyzing the sample of the fragmented precursor ions from the first sub-range at least partially overlaps with the step of accumulating the sample of the precursor ions having m / z values within the second sub-range in the first ion storage device.

21. A mass spectrometer configured to perform the method according to any one of claims 8 to 20.

22. A computer software comprising instructions that, when executed by a processor of a computer, cause the computer to perform the method according to any one of claims 8 to 20.

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