Radio frequency free electrostatic magnetostatic cell for improving electron based fragmentation of biomolecules

Abstract

In some examples, an electronic-based ExD cell can include a container for enclosing a filament cartridge and a first inlet configured to deliver a gas into the container. The ExD cell can further include a second inlet configured to deliver ions to the filament cartridge, and an ion outlet opening.

Description

Cross-reference to related applications

[0001] This application claims priority to co-pending U.S. Provisional Patent Application Serial No. 63 / 568,295, filed March 21, 2024, entitled “RADIOFREQUENCY-FREE ELECTROMAGNETOSTATIC CELL TO IMPROVEELECTRON-BASED FRAGMENTATION OF BIOLOGICAL MOLECULES”, the disclosure of which is incorporated herein by reference in its entirety. Background Technology

[0002] Electron-based fragmentation (ExD) can be used for comprehensive biomolecular characterization, such as in biopharmaceuticals. ExD can refer to electron-based fragmentation techniques, such as electron capture dissociation (ECD), electron-induced dissociation (EID), electron transfer dissociation (ETD), and electron de-dissociation (EDD). Attached Figure Description

[0003] The features of this disclosure are illustrated by way of example, not limitation, in the following figures, in which the same numbers denote the same elements, wherein:

[0004] Figure 1 A cross-sectional view is shown of a first embodiment of an electron-based fragmentation (ExD) cell placed after a collision cell (e.g., a collision hexapole) and before an ion beam compressor (IBC), according to an example of this disclosure.

[0005] Figure 2 Examples of this disclosure are shown. Figure 1 The operational principles associated with the ExD pool;

[0006] Figure 3 Examples of demonstrations based on this disclosure text are shown. Figure 1 A schematic diagram of the operation of the ExD pool;

[0007] Figure 4 Examples of demonstrations based on this disclosure text are shown. Figure 1 Another schematic diagram illustrating the operation of the ExD pool;

[0008] Figure 5 Examples of adding gas to a filament cassette, as shown in this disclosure, are illustrated to illustrate this. Figure 1 A schematic diagram of the operation of the ExD pool;

[0009] Figure 6The electron capture dissociation (ECD) efficiency of a double-charged peptide, as illustrated in the examples of this disclosure, increases with increasing gas pressure.

[0010] Figure 7 A cross-sectional view showing a second implementation of an ExD pool with a roughly centered configuration, as illustrated in the examples in this disclosure;

[0011] Figure 8 The results are shown based on the addition of CID energy to the protein analyte before it enters the ExD pool, according to an example of this disclosure.

[0012] Figure 9 A side view, an isometric front view, and a rear view of a filament box according to an example of this disclosure are shown;

[0013] Figure 10 This illustrates, in the example text of this disclosure, the gas emitted before and after leaving the filament box and into the ExD cell to illustrate... Figure 1 A schematic diagram of the operation of the ExD pool;

[0014] Figure 11 This demonstrates, according to examples in this disclosure, how gas from the filament box is directed to the collision pool and IBC to illustrate... Figure 1 Operations on the ExD pool;

[0015] Figure 12 Examples of collision pools, ExD pools, and IBC shielding structures based on this disclosure are shown to illustrate... Figure 1 Operations on the ExD pool;

[0016] Figure 13 The example collision cell according to this disclosure and the ExD cell with an electrically separated RF shield added to the exit lens are shown to illustrate... Figure 1 Operations on the ExD pool; and

[0017] Figure 14 The fragment strength is shown in the example according to this disclosure text to illustrate... Figure 1 Operations on the ExD pool. Detailed Implementation

[0018] For simplicity and illustrative purposes, this disclosure is described primarily through examples. Numerous specific details are set forth in the following description to provide a thorough understanding of this disclosure. However, it will be readily apparent that the practice of this disclosure may not be limited to these specific details. In other instances, some methods and structures have not been described in detail to avoid unnecessarily obscuring this disclosure.

[0019] Throughout this disclosure, the terms “a” and “an” are intended to mean at least one of a particular element. As used herein, the term “comprising” means including but not limited to, and the term “including” means including but not limited to. The term “based on” means at least partially based on.

[0020] As disclosed herein, electron-based fragmentation (ExD) can be used for comprehensive biomolecular characterization, such as in biopharmaceuticals. In some cases, utilizing and tuning ExD cells can be technically challenging. In this regard, this paper discloses a radio-frequency (RF)-free electrostatic magnetostatic cell (e.g., an ExD cell) that improves electron-based fragmentation of biomolecules. With the ExD cell disclosed herein, the electron cloud within the ExD cell can be maintained in an RF-free electromagnetic field.

[0021] Typically, an ExD cell may include one or more magnets, a filament, and several electrostatic lenses around the ion flight path along a central axis. In one example, the ExD cell may be positioned after a quadrupole and directly mounted to a collision cell. This type of ExD cell can produce efficient fragmentation of peptides and proteins, for example, in the range of 200 Daltons to 160 kilodaltons. However, the operation of this type of ExD cell can depend on the characteristics of the ion optics upstream of the ExD cell (e.g., RF lenses and quadrupoles). The dynamic nature of these upstream elements may require the user to have a high degree of understanding of the tuning and operation of both the ExD cell and the associated instrument. Furthermore, analyte ions entering the ExD cell from the quadrupole and the associated Brubaker post-filter can have a relatively broad energy distribution and can be divergent. Moreover, in an ExD cell, fragmentation efficiency may be relatively low with respect to small peptides (e.g., those carrying two positive charges).

[0022] To at least address the aforementioned technical challenges, the ExD cell disclosed herein can be positioned between the collision cell (CC) and the ion beam compressor (IBC), and can be referred to as CC-ExD-IBC, in which the collision gas is directly fed into the filament box. The ExD cell disclosed herein can utilize the gas to cool electrons and allow for more complete electron distribution within the ExD cell.

[0023] Based on the examples disclosed herein, such as the ExD pool disclosed herein, peptide fragmentation efficiency is improved to the same level as CID, and further produces cleaner spectra while revealing improved analytical information that may not be available through CID fragmentation.

[0024] Based on the examples disclosed herein, such as the ExD cell disclosed herein, electron capture dissociation (ECD) efficiency can be improved while simplifying operation by maintaining ion transport and resolution independently of the MS1 / MS2 (e.g., tandem mass spectrometry) transition and upstream optics setup.

[0025] Based on the examples disclosed herein, such as the ExD cell, improved ECD fragmentation efficiency is provided because operating the filament with a collision gas facilitates thermalization and electron redistribution. In one example, the improved ECD efficiency is approximately 2–10 times.

[0026] Based on the examples disclosed herein, such as the ExD cell disclosed herein, the original CID fragmentation capability and standard mass spectrometer functionality are retained.

[0027] Based on the example ExD cell disclosed herein, a gas can be added to an RF-free ExD cell. In some cases, adding a gas may result in ion scattering through the ExD cell.

[0028] Based on the example of the ExD cell disclosed herein, regarding the gas addition leading to improved ECD efficiency, enhanced ECD can be induced by electron thermal cooling to very low energies, where electrons are effectively trapped to initiate ECD. Furthermore, since the transient electron / gas molecule complex has a larger mass than electrons, this complex can facilitate the wider movement of electrons in the magnetic field, allowing for more efficient filling of the ExD cell with low-energy electrons. This increases the probability of electrons interacting with analyte ions to enhance ECD. These two distinct mechanisms can work synergistically to improve ECD, for example, by 2–10 times.

[0029] Based on the example of the ExD cell disclosed herein, analyte ions are not trapped and pass through the ExD cell within microseconds without cooling. Regarding electron transfer dissociation (ETD) or electron activated dissociation (EAD) techniques, analyte ion trapping occurs on a millisecond timescale within an RF-associated ion trap using a cooling gas. With this extended trapping time, large analyte ions (e.g., peptides and proteins) may collapse into folded structures held together by non-covalent interactions. Even when these collapsed folded structures are fragmented by ETD or EAD, the fragments do not separate. This results in the ET-no-D (no dissociation) phenomenon.

[0030] Based on the example of the ExD cell disclosed herein, in one example, RF confinement devices can be added before and after the ECD cell. In this regard, the RF ion guide that delivers ions to the ExD cell can shape and cool the ion beam to focus it on the axis, and the ions have similar energies and trajectories as they pass through the ExD cell. Therefore, with respect to the ExD cell disclosed herein, a collision cell (e.g., a gas-filled RF ion guide) can be used to add energy to protein analytes to unfold the proteins and thus prevent ET-no-D.

[0031] Based on the example of the ExD cell disclosed herein, in one instance, for a lens as disclosed herein, an off-axis aperture can be cut into the ExD cell to direct the gas exiting the filament box away from the central axis of the ExD cell and out of the ion path. Properly positioned vents can allow the gas to undergo electron cooling and redistribution while minimizing ion scattering or stagnation. In this example, the off-axis aperture also allows the gas to be supplied to the collision cell more uniformly.

[0032] Based on the example of the ExD cell disclosed herein, electronic control of the lens element can achieve ion transport without an ECD. Furthermore, the ExD cell disclosed herein includes a combination of ECD and CID, as well as higher-energy electron fragmentation.

[0033] Based on the example ExD cell disclosed herein, the RF shielding at the cell's inlet or outlet may include a thin copper sheet connected to ground to maintain an RF-free region inside the ExD cell. An electrostatic lens may be fitted within a hole in the RF shielding to prevent the RF shielding from interfering with ion transport through the ExD cell.

[0034] Based on the examples disclosed herein, an ExD cell may include a filament box and a gas container for sealing the filament box. A gas inlet may be configured to deliver gas into the gas container. The gas container may include an ion inlet opening and an ion outlet opening. The gas may be a pure gas or a mixture of, for example, nitrogen, argon, helium, or xenon. Other gases may also be used.

[0035] Based on the examples disclosed herein, for the aforementioned ExD pool, the gas inlet can be configured to deliver gas along the central axis adjacent to the ExD pool.

[0036] Based on the examples disclosed herein, for the aforementioned ExD cell, the gas inlet can be configured to deliver gas in a region where electrons are constrained by a static (RF-free) electromagnetic field.

[0037] Based on the examples disclosed herein, for the aforementioned ExD cell, the gas inlet can be configured to deliver gas to the filament box. For example, the gas inlet can be configured to deliver gas to the RF-free area within the ExD cell.

[0038] Based on the examples disclosed herein, for the aforementioned ExD cell, the filament box may include an inlet for the filament and a gas inlet (e.g., a filament inlet as disclosed herein). The filament inlet may be configured to deliver gas directly to an area adjacent to the filament.

[0039] Based on the examples disclosed herein, for the aforementioned ExD cell, the filament assembly may include a column for providing electrical contact to the filament. The ExD cell may further include a filament inlet for gas, wherein the filament inlet is configured to direct the gas toward the column to uniformly distribute the gas. In this respect, the inlet gas may be directed toward the filament column to disperse gas molecules within the cell. Alternatively, the gas may flow in a different manner at low pressures within the mass spectrometer and may form a molecular jet.

[0040] According to the examples disclosed herein, for the aforementioned ExD cell, the ExD cell may further include at least two electrostatic lenses disposed on opposite sides of the filament box. In this regard, the at least two electrostatic lenses may include vent holes to allow gas to exit the ExD cell.

[0041] According to the examples disclosed herein, for the aforementioned ExD cell, the vents can be symmetrically arranged in the at least two lenses. In other examples, the vents can be symmetrically arranged in one of the at least two lenses.

[0042] According to the examples disclosed herein, for the aforementioned ExD cell, the vent holes can be asymmetrically arranged in the at least two lenses.

[0043] Based on the examples disclosed in this article, for the above-mentioned ExD pool, a radio frequency (RF) shielding structure can be added to maintain an RF-free environment within the ExD pool.

[0044] Based on the examples disclosed herein, the ExD cell can also be positioned between other types of ion optics. In other examples, the ExD cell can be positioned before the isolated quadrupole in a QqQ, Q-ToF, Q-orbitrap, or Q-FTICR mass spectrometer.

[0045] Based on the examples disclosed herein, for the above-described components, the ExD pool may include a gas inlet configured to deliver gas adjacent to the central axis of the ExD pool.

[0046] Based on the examples disclosed herein, for the aforementioned components, the ExD cell may include a gas inlet configured to deliver gas in a region where electrons are confined by an electromagnetic field.

[0047] Based on the examples disclosed herein, for the above-described components, the ExD cell may include a filament box and a gas inlet configured to deliver gas into the filament box.

[0048] Based on the examples disclosed herein, for the aforementioned components, the filament box may include a filament and a filament inlet for gas. The filament inlet may be configured to deliver gas directly to an area adjacent to the filament.

[0049] Based on the examples disclosed herein, for the above-described components, the filament box may include a filament and a post for providing electrical contact to the filament. The ExD cell may further include a filament inlet for gas, wherein the filament inlet is configured to direct gas toward the post for uniform gas distribution.

[0050] Based on the examples disclosed herein, for the aforementioned components, the ExD cell may include a gas container for sealing the filament box. The gas container may include an ion inlet opening and an ion outlet opening.

[0051] According to the examples disclosed herein, for the above-described components, the ExD cell may include at least two electrostatic lenses disposed on opposite sides of the filament box. The at least two electrostatic lenses may include vent holes to allow gas to exit the ExD cell.

[0052] Based on the examples disclosed herein, an ExD cell may include a filament box and a sealed filament box, and a gas container for supplying gas to the ExD cell. The gas container can function as a box holder to directly supply gas to the filament box. The gas container can provide removal of the filament box to replace a burnt-out filament without interrupting the gas supply.

[0053] Based on the example of the ExD pool disclosed in this article, the gas can be retained in a gas container.

[0054] Based on the example of the ExD cell disclosed in this article, the gas delivered to the ExD cell can then be delivered to the collision cell or the IBC.

[0055] Based on the example of the ExD cell disclosed herein, additional orifices or paths may be provided for off-axis delivery of gas to the collision cell or IBC (e.g., by diverting the gas away from the ion path).

[0056] Based on the example of the ExD cell disclosed in this paper, an oxygen scrubber can be placed in the gas path. In this respect, making the gas as oxygen-free as possible improves the filament's working life.

[0057] Based on the examples disclosed herein, an ExD cell may include a container for sealing the filament box, and a first inlet configured to deliver gas into the container. The ExD cell may further include a second inlet configured to deliver ions into the filament box, and an ion outlet opening.

[0058] Based on the example of the ExD pool disclosed herein, the first inlet can be configured to deliver gas adjacent to the central axis of the ExD pool.

[0059] Based on the example of the ExD cell disclosed herein, the first inlet can be configured to deliver gas in a region where electrons are confined by an electromagnetic field.

[0060] Based on the example of the ExD cell disclosed in this article, the first inlet can be configured to deliver gas into the filament box.

[0061] Based on the example of the ExD cell disclosed herein, the filament box may include a filament and a filament inlet for gas. The filament inlet may be configured to deliver gas directly to an area adjacent to the filament.

[0062] Based on the example of the ExD cell disclosed herein, the filament box may include a filament, a post for providing electrical contact to the filament, and a filament inlet for gas. The filament inlet may be configured to direct gas toward the post for uniform gas distribution.

[0063] According to the example of the ExD cell disclosed herein, the ExD cell may further include at least two electrostatic lenses disposed on opposite sides of the filament box. The at least two electrostatic lenses may include vent holes to allow gas to exit the ExD cell.

[0064] Based on the example of the ExD cell disclosed herein, vents can be symmetrically arranged in the at least two electrostatic lenses.

[0065] Based on the example of the ExD pool disclosed in this article, the ExD pool can further include a radio frequency (RF) shielding structure to maintain an RF-free environment.

[0066] Based on the example of the ExD pool disclosed in this article, the pressure in the container does not decrease substantially. In this respect, the pressure in the container can increase or remain substantially constant.

[0067] According to the examples disclosed herein, the components may include a collision cell, a downstream ion optics device, and an ExD cell disposed between the collision cell and the downstream ion optics device.

[0068] Based on the examples of components disclosed herein, the downstream ion optics device is an ion beam compressor.

[0069] Based on the examples of components disclosed herein, an ExD cell may include a gas inlet configured to deliver gas adjacent to the central axis of the ExD cell, a gas inlet configured to deliver gas in a region where electrons are confined by an electromagnetic field, or a gas inlet configured to deliver gas into a filament box.

[0070] Based on the examples of components disclosed herein, a filament box may include a filament and a filament inlet for gas. The filament inlet may be configured to deliver gas directly to an area adjacent to the filament.

[0071] Based on the examples of components disclosed herein, a filament box may include a filament, a post for providing electrical contact to the filament, and a filament inlet for gas. The filament inlet may be configured to direct gas toward the post for uniform gas distribution.

[0072] Based on the examples of components disclosed herein, the ExD cell may include a gas container for sealing the filament box, and the gas container may include an ion inlet opening and an ion outlet opening.

[0073] Based on the examples of components disclosed herein, the ExD cell can be positioned between the collision cell and the downstream ion optics by being arranged at approximately equal distances.

[0074] Based on the examples of components disclosed herein, the ExD cell may include at least two electrostatic lenses disposed on opposite sides of the filament box. The at least two electrostatic lenses may include vent holes to allow gas to exit the ExD cell.

[0075] Figure 1 A cross-sectional view is shown of a first embodiment of an electron-based fragmentation (ExD) cell (hereinafter “ExD cell 100”) placed after a collision cell (CC) 102 (e.g., a collision hexapole) and before an ion beam compressor (IBC) 104, according to an example of this disclosure. The combination of ExD cell 100, CC 102, and IBC 104 may also be referred to herein as a component. ExD cell 100 may include an inlet conduit 106 for a gas (such as an inert gas).

[0076] refer to Figure 1 The ExD cell 100 can be positioned after CC 102 and before IBC 104. The gas present in CC 102 enhances ECD efficiency. In this regard, up to 20 mTorr of nitrogen, argon, or another inert gas can be supplied through gas inlet 106 to the central axis of the ExD cell 100, where electrons are generated by a filament as disclosed herein and radially confined by a magnetic field generated by two permanent magnets. The gas can be applied anywhere within the non-RF region of the ExD cell 100. Thus, supplying gas in this electron-containing non-RF region improves ECD.

[0077] The applied gas can be applied at any pressure up to a certain limit (e.g., up to 100 mTorr). Exceeding this limit may cause ion scattering and reduce ion transport. As disclosed herein, RF shielding can be used to prevent the RF field from the IBC from penetrating into the ExD cell.

[0078] Figure 2 The operating principle associated with ExD pool 100 is demonstrated in the example of this disclosure.

[0079] refer to Figure 2Setup 200 may include an annular filament 202. Ions can pass through the center of the electrically heated annular filament and the center of the permanent annular magnet 206. Setup 200 may further include an aperture 208 within the magnet 206, wherein the diameter of aperture 208 may be, for example, 3 mm. An adjustable DC potential can be applied when the annular filament 202 is negative relative to the permanent annular magnet 206 to draw electrons away from the filament and into the region of aperture 208. For setup 200, V is a low voltage, and the high-current filament power supply may be electrically floating. To achieve high ECD efficiency, the gas helps cool the electron cloud and distribute it more widely. This can be accomplished by increasing the gas pressure in the ExD cell.

[0080] Figure 3 A schematic diagram illustrating the operation of ExD pool 100 is shown, based on an example of this disclosure text.

[0081] refer to Figure 3 The diagram shows an ExD cell 300 without a supplied gas. The ExD cell 300 may include a metal box 302 surrounding a filament 304, wherein the metal box 302 is positively charged relative to the filament to help draw electrons away from the filament and into the central axis 306 of the magnet. The region 308, defined by the arrow shown, indicates the RF-free region of the ExD cell. In this respect, electrons can be trapped throughout this region. The electrostatic lens 310 and the magnet 312 may have a DC potential applied thereto.

[0082] Figure 4 Another schematic diagram illustrating the operation of ExD pool 100 is shown, based on an example from this disclosure.

[0083] refer to Figure 4 , Figure 4 An ExD cell 100 is shown, comprising a cylinder 400 (e.g., a container or gas container as disclosed herein). Gas can be supplied by a source (not shown) at 402 (e.g., also referred to herein as a first inlet). Orifices 404 and 406 (e.g., about 3 mm in diameter; also referred herein as a second inlet and ion outlet opening) can be provided to allow ion entry and debris exit, respectively. Gas escaping from the orifices may reduce the vacuum in the mass spectrometer and impose an additional load on the turbopump. In this regard, the cylinder 400 can provide a shell to increase the gas pressure within the ExD cell 100. Orifices 404 and 406 can be formed narrow enough to restrict gas exit and wide enough to allow analyte ions to pass through. The gas can thus be delivered in an adjacent region near the filament 408 to maximize electron cooling and redistribution.

[0084] Figure 5A schematic diagram is shown to illustrate the operation of the ExD cell 100 by adding gas to the filament box, according to an example of this disclosure.

[0085] refer to Figure 5 , Figure 5 An ExD cell 100 including a filament box 500 is shown. In this respect, a gas can be added to the filament box 500 to maximize the amount of gas near the filament, thereby producing an effective ECD. The gas can escape through central ion orifices 502 and 504 at each end. In this respect, the ExD cell may include a container for sealing the filament box 500 (e.g., an airtight container as disclosed herein), and a first inlet 106 configured to deliver gas into said container. The ExD cell may further include a second inlet (e.g., a through-hole 502) and an ion outlet opening (e.g., a through-hole 504) configured to deliver ions into the filament box.

[0086] Figure 6 The electron capture dissociation (ECD) efficiency of a double-charged peptide, as illustrated in the examples of this disclosure, increases with increasing gas pressure.

[0087] refer to Figure 6 The dependence of ECD efficiency on gas pressure is shown at 600 (measured as the abundance of ECD fragments at m / z 624 relative to the 2+ precursor at m / z 674). In this regard, the improvement in ECD efficiency for the 11-amino acid peptide P(2+) is shown. The x-axis refers to the pressure applied to the gas inlet pipe (e.g., at...). Figure 4 The sum of gas pressures (in PSI) at point 402. The dashed line at point 602 shows the increase in ECD efficiency as the gas pressure applied to the gas inlet of the filament box increases.

[0088] Although ECD efficiency increases proportionally with gas pressure, higher gas pressures may begin to cause analyte ion scattering as the gas exits through the central orifice. This may limit the practical improvement in ECD efficiency, but can be overcome by considering how the gas is directed out of ExD cell 100.

[0089] To address analyte ion scattering, additional venting can be provided before and after the filament box via magnets and surrounding lenses. This is because the increased fragmentation efficiency due to the gas largely occurs in the immediate vicinity of the filament and adjacent magnets.

[0090] Furthermore, the aperture size of various lens elements can be adjusted to help guide gas flow. For example, assuming a 3 mm diameter aperture is typically used, the aperture size on the filament box can be reduced to 2 mm to retain the gas within the filament box. Other apertures can be increased to 4 mm to facilitate gas emission away from the ion path. In one example, the aperture size can be set to approximately 3 mm. By changing the DC voltage applied to the lens, variations in electrostatic properties can be compensated for for different aperture diameters.

[0091] The operation of ExD cell 100 benefits from the cooling and condensation of analyte ions within the collision cell before they enter ExD cell 100. The DC voltage required to operate ExD cell 100 can be independent of the operation of the quadrupole on other ion optics elements in Q-ToF, and thus facilitates the tuning and operation of ExD cell.

[0092] Adding extra gas to the filament box at high flow rates can increase the gas pressure within the mass spectrometer, leading to reduced quadrupole isolation efficiency, decreased Time-of-Flight (ToF) resolution, and other adverse effects. In quadrupole time-of-flight (Q-ToF) instruments, a gas (e.g., nitrogen) can be supplied to the collision cell to achieve CID fragmentation (collision-induced dissociation). To minimize the addition of extra gas to the mass spectrometer, the gas inlet can be moved from the collision cell to deliver all collision gas directly to the filament box. Escape gas from the orifice in the filament box can be directed to fill the collision cell to maintain its normal pressure. Enhanced electron fragmentation can be achieved without adding an extra gas load to the mass spectrometer by appropriately selecting the lens orifice in the electrostatic cell and appropriately venting gas to the collision cell and IBC. Therefore, collision-induced dissociation (CID) and ion-cooled time-of-flight (ToF) resolution in the IBC can be maintained without imposing an additional gas load on the Q-ToF.

[0093] For ExD cell configurations operating next to the IBC, RF filtering or shielding structures can be used to minimize interference from the IBC hexapole. RF filtering or shielding structures can also protect the electron cloud inside the electrostatic cell by keeping it RF-free.

[0094] When CID energy is applied, analyte ions can be accelerated by an increased DC voltage applied to the ion optics elements in front of the collision cell. The analyte ions can be accelerated as they enter the collision cell, where they are initially heated through internal collisions with the collision gas. For larger proteins, heating can aid in protein unfolding, promoting improved dissociation of fragments generated in the ExD cell. The application of CID energy increases the internal energy of large analytes such as proteins.

[0095] Regarding the operation of the ExD cell, when ions first enter the collision cell and travel several millimeters, the ions can be heated. Further within the collision cell, the kinetic energy of the ions is transferred to gas molecules, which cool the ion beam and focus it into the ExD cell. The internal energy of the protein, enhanced by the added collision energy, remains sufficiently high for efficient fragmentation.

[0096] Figure 7 A cross-sectional view of a second embodiment of an ExD pool 700, centered (e.g., with approximately equal distances between CC and IBC), is shown, according to an example in this disclosure.

[0097] refer to Figure 7 A second shortened collision pool can be added, followed by an ExD pool of 700. Figure 7 The configuration allows for the application of additional energy to enhance dissociation or alternative fragmentation. In this respect, Figure 7 The configuration can be represented as an intermediate pool. The gas supplied to the ExD pool can be used to fill two collision pools with collision gas.

[0098] Figure 8 The results 800 are shown as an example based on adding CID energy before the protein analyte enters the ExD pool, according to an example of this disclosure.

[0099] refer to Figure 8 , Figure 8 Results 800 show the values ​​of CID energy added before the protein analyte enters the ExD pool 100. In this respect, results 800 show that, with the increase of CID activation, ECD fragmentation increases significantly on the left side (e.g., at 802 and 804).

[0100] Figure 9 Side view, isometric front view and rear view of a filament box (e.g., filament box 500) according to an example of this disclosure are shown.

[0101] refer to Figure 9 The images show different views of a filament box 500 with an opening or inlet to directly supply gas into a chamber surrounding the filament. For example, the views show a side view of the filament box at 902, isometric front views at 904 and 906, and a rear view at 908. The isometric front view at 906 includes a removed cover compared to the isometric front view at 904. The filament box 500 may include a gas filament inlet 910. The filament inlet 910 may directly supply gas to an electron-generating filament 914.

[0102] The filament box 500 may include an aperture 912, which includes a filament 914. Resilient fingers 916 and 918 may be insertable or otherwise slide into corresponding channels (not shown) in the ExD pool 100 to provide engagement of the filament box 500 in the ExD pool 100 when the enlarged protrusion 920 engages with a corresponding enlarged recess (not shown) in the ExD pool 100. In this way, the filament box 500 can be releasably positioned and / or locked into the ExD pool 100.

[0103] As shown at 922, columns 924 and 926 can retain filament 914 therebetween.

[0104] A corresponding aperture 928 can be positioned opposite aperture 912. Apertures 912 and 928 can be arranged along a central axis to allow analyte ions to pass through the filament box 500 and enter the interior of the hot filament ring of the filament 914. Apertures 912 and 928 also allow gas to fill the ExD cell 100.

[0105] According to the examples disclosed herein, the gas can be directly supplied to the filament box 500. To improve ECD efficiency, the gas pressure can be in the range of 1 to 200 millitors, and in one example, between 20 and 100 millitors.

[0106] Based on the examples disclosed herein, the gas lines typically supplied to the collision cell can be used to feed the filament box 500. Therefore, all the gas required for collision cell operation can flow out from the small central orifice in the filament box 500.

[0107] Figure 10 A schematic diagram illustrating the operation of ExD cell 100 is shown, illustrating the gas emitted before and after leaving filament box 500 in an example according to this disclosure.

[0108] refer to Figure 10 Before and after the filament box 500, additional venting can be provided through magnetic lenses 1000 and 1002 and surrounding lenses 1004, 1006, 1008, and 1010. In this regard, upper and lower vents, positioned off-axis (e.g., at a specified distance from the central axis 1016 of the ExD cell 100) as shown in 1012 and 1014, allow gas to exit the ion path from the ExD cell 100. Lenses 1004, 1006, 1008, and 1010 may include vents at 1012 and 1014 with various geometries. For example, as shown... Figure 10 As shown, the vent can include four holes for each lens, wherein in Figure 10In terms of orientation, two holes are located in the upper region of the lens and two holes are located in the lower region of the lens. Vent holes 1012 and 1014 may have the same dimensions as shown, or different diameters, to adjust the amount of gas flowing out to the pre-collision cell relative to the IBC. Lenses 1004, 1006, 1008, and 1010 may also include a central aperture for ion entry and exit, such as... Figure 10 As shown.

[0109] After exiting the filament box 500 and flowing into the ExD cell 100, the gas can be discharged as indicated by the flow path arrows (e.g., at 1018, 1020, etc.). Vents at 1012 and 1014 allow gas to escape between the elements. This reduces the gas flow present in the ion path through the ExD cell 100 (e.g., along the central axis 1016), as shown by the alternative paths for gas movement indicated by the various flow path arrows (e.g., at 1018, 1020, etc.).

[0110] Figure 11 The example shown in this disclosure illustrates how gas from filament box 500 is directed to collision pool 102 and IBC 104 to illustrate the operation of ExD pool 100.

[0111] refer to Figure 11 By appropriately selecting the lens aperture in the electrostatic magnetostatic cell and appropriately venting the gas into CC102 and IBC104, the benefits of enhanced electron fragmentation are possible without adding an additional gas load to the mass spectrometer. In this respect, to... Figure 1 In a similar way, Figure 11 This is shown where CC 102 is on the left and IBC 104 is on the right relative to ExD pool 100 (in Figure 11 The arrangement (in the orientation) is as follows. CC 102 and IBC 104 can utilize a hexapole with RF to confine ions and utilize gas to cool the ions. The conduction of gas moving forward or backward can be modified by small changes in the orifice diameter. For example, changing the diameter from 3 mm to 2 mm will limit the gas flow rate by more than half. These diameter changes can be used to balance the gas flow to CC 102 and IBC 104.

[0112] With regard to this article Figure 1 and Figure 10 In a similar manner as disclosed, gas from the filament box 500 can be directed to CC 102 and IBC 104, which can utilize a pressure of 10-20 millitor. Based on this configuration, relatively high gas pressure can be achieved near the filament to maximize ECD, while using the escaped gas to supply CC 102 and IBC 104.

[0113] Figure 12 The CC 102, ExD pool 100, and IBC shielding structure, as shown in the examples of this disclosure, are illustrated to illustrate the operation of ExD pool 100.

[0114] refer to Figure 12 Next to the IBC, CC 102, ExD cell 100, and exit lens are shown as RF shielding. The capacitor allows RF current induced on the external electrostatic lens to be transferred to instrument ground.

[0115] IBC 104 is shown as two distinct RF-dependent hexapoles. The electrical mounting bracket adjacent to ExD cell 100 induces an asymmetric RF field on the last lens within ExD cell 100. In this respect, capacitors can transfer the induced RF current on the exit lens to ground to maintain an RF-free region within ExD cell 100 for confining electrons.

[0116] Figure 13 An example collision pool according to this disclosure and an ExD pool with an electrically separated RF shield added to the exit lens are shown to illustrate the operation of ExD pool 100.

[0117] refer to Figure 13 The diagram illustrates CC 102, ExD cell 100 with RF shielding, and IBC shielding. Regarding the IBC, the RF shielding structure, as shown at 1300, reduces the induced voltage in the ExD cell to maintain an RF-free field for confining electrons.

[0118] Figure 14 The fragment intensity is shown in the example according to this disclosure text to illustrate the operation of ExD pool 100.

[0119] refer to Figure 14 The top subplot 1400 shows an ECD cell operated with gas and observes the fragmentation of 2% of substance P (e.g., a peptide of 11 amino acids). Desired ECD fragments are shown at 1402, and unwanted, unassigned fragments are shown at 1404. Without the addition of gas, the ECD cell will only produce 2-5% ECD.

[0120] The subplots at 1406 and 1408 show CID fragments at impact energies of 25 V and 30 V, respectively. Typical CID fragments are shown at 1410 and 1412, although many other fragments are generated. At 1402, the intensity of CID fragments is lower than that of ECD fragments. The entire spectrum at 1406 and 1408 is filled with numerous fragments that may be difficult to assign or misclassified when observing unknown samples.

[0121] The examples described and illustrated herein are examples, along with some variations thereof. The terminology, descriptions, and figures used herein are illustrative only and are not intended to be limiting. Many variations are possible within the spirit and scope of the subject matter, which is intended to be defined by the following claims and their equivalents, wherein, unless otherwise stated, all terms are to be used in their broadest reasonable sense.

Claims

1. An electron-based fragmentation (ExD) cell, comprising: Containers used to seal filament boxes; A first inlet, configured to deliver gas into the container; A second inlet, configured to deliver ions to the filament box; as well as Ion outlet opening.

2. The ExD pool according to claim 1, wherein, The first inlet is configured to deliver the gas adjacent to the central axis of the ExD pool.

3. The ExD pool according to claim 1, wherein, The first inlet is configured to deliver the gas in a region where electrons are confined by an electromagnetic field.

4. The ExD pool according to claim 1, wherein, The first inlet is configured to deliver the gas into the filament box.

5. The ExD pool according to claim 4, wherein, The filament box includes: filament; and A filament inlet for the gas, wherein the filament inlet is configured to deliver the gas directly to an area adjacent to the filament.

6. The ExD pool according to claim 4, wherein, The filament box includes: filament; A post for providing electrical contact to the filament; and A filament inlet for the gas, wherein the filament inlet is configured to direct the gas toward the column to distribute the gas evenly.

7. The ExD pool according to claim 1, further comprising: At least two electrostatic lenses are disposed on opposite sides of the filament box. The at least two electrostatic lenses include vents to allow the gas to exit the ExD cell.

8. The ExD pool according to claim 7, wherein, The vents are symmetrically arranged in the at least two electrostatic lenses.

9. The ExD pool according to claim 1, further comprising: Radio frequency (RF) shielding structure, used to maintain an RF-free environment.

10. The ExD pool according to claim 1, wherein, The pressure in the container does not decrease substantially.

11. A component comprising: Collision pool; Downstream ion optics; as well as An electron-based fragmentation (ExD) cell is disposed between the collision cell and the downstream ion optics.

12. The component of claim 11, wherein, The downstream ion optical device is an ion beam compressor.

13. The component of claim 11, wherein, The ExD pool includes: A gas inlet configured to deliver gas adjacent to the central axis of the ExD pool; A gas inlet configured to deliver the gas in a region where electrons are confined by an electromagnetic field; or It is configured to deliver the gas to the gas inlet in the filament box.

14. The component of claim 13, wherein, The filament box includes: filament; and A filament inlet for the gas, wherein the filament inlet is configured to deliver the gas directly to an area adjacent to the filament.

15. The component of claim 13, wherein, The filament box includes: filament; A post for providing electrical contact to the filament; and A filament inlet for the gas, wherein the filament inlet is configured to direct the gas toward the column to distribute the gas evenly.

16. The component of claim 11, wherein, The ExD cell includes a gas container for sealing the filament box, wherein the gas container includes an ion inlet opening and an ion outlet opening.

17. The component of claim 11, wherein, The ExD cell is positioned between the collision cell and the downstream ion optics by being arranged at approximately equal distances.

18. The component of claim 11, wherein, The ExD pool includes: At least two electrostatic lenses are positioned on opposite sides of the filament box. The at least two electrostatic lenses include vents to allow gas to exit the ExD cell.

19. An electron-based fragmentation (ExD) cell, comprising: A gas container that encloses the gas being delivered to the ExD pool.

20. A component comprising the ExD pool of claim 19, wherein, The ExD cell is positioned before the isolation quadrupole.

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