WO2025199334A1 - Radiofrequency-free electromagnetostatic cell to improve electron-based fragmentation of biological molecules - Google Patents

Abstract

In some examples, an electron-based fragmentation (ExD) cell may include a container to enclose a filament cassette, and a first inlet that is configured to deliver gas into the container. The ExD cell may further include a second inlet that is configured to deliver ions to the filament cassette, and an ion exit opening.

Description

20230167-02 RADIOFREQUENCY-FREE ELECTROMAGNETOSTATIC CELL TO IMPROVE ELECTRON-BASED FRAGMENTATION OF BIOLOGICAL MOLECULES RELATED APPLICATIONS [0001] This application claims priority to co-pending U.S. Provisional Patent Application Serial No.63/568,295, filed March 21, 2024, titled “RADIOFREQUENCY-FREE ELECTROMAGNETOSTATIC CELL TO IMPROVE ELECTRON-BASED FRAGMENTATION OF BIOLOGICAL MOLECULES” the disclosure of which is hereby incorporated by reference in its entirety. BACKGROUND [0002] Electron-based fragmentation (ExD) may be utilized for comprehensive biomolecule characterization, for example, for biopharma. ExD may refer to electron-based fragmentation technologies such as electron capture dissociation (ECD), electron-induced dissociation (EID), electron transfer dissociation (ETD), electron detachment dissociation (EDD), etc.

20230167-02 BRIEF DESCRIPTION OF DRAWINGS [0003] Features of the present disclosure are illustrated by way of example and not limited in the following figure(s), in which like numerals indicate like elements, in which: [0004] Figure 1 illustrates a cross-sectional view of a first embodiment of an electron-based fragmentation (ExD) cell placed after a collision cell (e.g., collision hexapole) and before an ion beam compressor (IBC), in accordance with an example of the present disclosure; [0005] Figure 2 illustrates principles of operation associated with the ExD cell of Figure 1, in accordance with an example of the present disclosure; [0006] Figure 3 illustrates a diagrammatic view to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure; [0007] Figure 4 illustrates another diagrammatic view to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure; [0008] Figure 5 illustrates a diagrammatic view to illustrate adding of gas into a filament cassette to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure; [0009] Figure 6 illustrates electron capture dissociation (ECD) efficiency of a doubly charged peptide increases with greater gas pressure, in accordance with an example of the present disclosure; 20230167-02 [0010] Figure 7 illustrates a cross-sectional view of a second embodiment of a generally centrally disposed ExD cell, in accordance with an example of the present disclosure; [0011] Figure 8 illustrates results based on the addition of CID energy before protein analytes enter the ExD cell, in accordance with an example of the present disclosure; [0012] Figure 9 illustrates side, isometric front, and back views of a filament cassette, in accordance with an example of the present disclosure; [0013] Figure 10 illustrates a diagrammatic view of venting of gas after leaving the filament cassette to the front and back of an ExD cell to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure; [0014] Figure 11 illustrates how gas from the filament cassette is directed to the collision cell and the IBC to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure; [0015] Figure 12 illustrates a collision cell, an ExD cell, and IBC shielding to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure; [0016] Figure 13 illustrates a collision cell, and an ExD cell with an electrically separate RF shield added to the exit lens to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure; and 20230167-02 [0017] Figure 14 illustrates fragment intensity to illustrate operation of the ExD cell of Figure 1, in accordance with an example of the present disclosure.

20230167-02 DETAILED DESCRIPTION [0018] For simplicity and illustrative purposes, the present disclosure is described by referring mainly to examples. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. It will be readily apparent however, that the present disclosure may be practiced without limitation to these specific details. In other instances, some methods and structures have not been described in detail so as not to unnecessarily obscure the present disclosure. [0019] Throughout the present disclosure, the terms "a" and "an" are intended to denote at least one of a particular element. As used herein, the term "includes" means includes but not limited to, and the term "including" means including but not limited to. The term "based on" means based at least in part on. [0020] As disclosed herein, electron-based fragmentation (ExD) may be utilized for comprehensive biomolecule characterization, for example, for biopharma. In some cases, an ExD cell may be technically challenging to utilize and tune. In this regard, a radio frequency (RF)-free electromagnetostatic cell (e.g., ExD cell) is disclosed herein and improves electron-based fragmentation of biological molecules. For the ExD cell disclosed herein, an electron cloud within the ExD cell may be maintained in a RF-free electromagnetic field. [0021] Generally, an ExD cell may include one or more magnets, a filament and several electrostatic lenses surrounding an ion flight path on a central axis. In one example, an ExD cell may be positioned after a quadrupole and mounted directly to 20230167-02 a collision cell. This type of ExD cell may produce effective fragmentation of peptides and proteins, for example, in the 200 Dalton to 160 kiloDalton range. However, operation of this type of ExD cell may be dependent on the behavior of ion optic elements upstream of the ExD cell (e.g., RF lenses and quadrupole). The dynamic nature of these upstream elements may require a user to be highly knowledgeable with respect to tuning and operation of both the ExD cell and associated instrumentation. Yet further, analyte ions entering the ExD cell from a quadrupole and an associated Brubaker post filter may have a relatively wide energy distribution and may be divergent. Moreover, in ExD cells, with respect to the efficiency for small peptides (e.g., with two plus charges), fragmentation efficiency may be relatively low. [0022] In order to address at least the aforementioned technical challenges, the ExD cell as disclosed herein may be positioned between a collision cell (CC) and an ion beam compressor (IBC), and may be referred to as a CC-ExD-IBC, with collision gas delivered directly into a filament cassette. The ExD cell as disclosed herein may utilize gas to cool electrons and distribute the electrons more completely in the ExD cell. [0023] According to examples disclosed herein, the ExD cell as disclosed herein increases efficiency of peptide fragmentation to equivalent to CID, and further produces cleaner spectra while revealing improved analytical information that may not be accessible with CID fragmentation. 20230167-02 [0024] According to examples disclosed herein, the ExD cell as disclosed herein may increase electron capture dissociation (ECD) efficiency, while simplifying operation through maintaining ion transmission and resolution independently of MS1/MS2 (e.g., tandem mass spectrometry) transitions and upstream optics settings. [0025] According to examples disclosed herein, the ExD cell as disclosed herein provides increased ECD fragmentation efficiency because operating the filament with collision gas helps thermalize and redistribute electrons. In one example, the increased ECD efficiency is on the order of 2-10 fold. [0026] According to examples disclosed herein, the ExD cell as disclosed herein preserves original CID fragmentation capability and standard mass spectrometer functionality. [0027] According to examples of the ExD cell disclosed herein, gas may be added in an RF-free ExD cell. In some cases, the addition of gas may result in scattering of ions passing through the ExD cell. [0028] According to examples of the ExD cell disclosed herein, with respect to the addition of gas that results in increased ECD efficiency, the enhanced ECD may result from thermal cooling of the electrons to a very low energy where the electrons are efficiently captured to initiate ECD. Further, because the transient electron/gas molecule complex is more massive compared to an electron, the complex can help move electrons more widely across the magnetic field to more effectively fill the ExD cell with low energy electrons. This increases the probability 20230167-02 of electrons interacting with analyte ions to increase ECD. These two distinct mechanisms may operate in concert to increase ECD, for example, 2-10 fold. [0029] According to examples of the ExD cell disclosed herein, analyte ions are not trapped and pass through the ExD cell in microseconds without cooling. With respect to electron-transfer dissociation (ETD) or electron activated dissociation (EAD) technologies, trapping of analyte ions occurs on a timescale of milliseconds within RF-dependent ion traps using a cooling gas. With this prolonged trapping time, large analyte ions (e.g., peptides and proteins) may collapse into folded structures that are held together by non-covalent interactions. Even when these collapsed folded structures are fragmented with ETD or EAD, the fragments do not separate. This results in the phenomena of ET-no-D (no dissociation). [0030] According to examples of the ExD cell disclosed herein, in one example, RF confinement devices may be added before and after the ECD cell. In this regard, the RF ion guide delivering ions to the ExD cell may shape and cool the ion beam to be focused on an axis, and the ions have similar energies and trajectories when passing through the ExD cell. Thus, with respect to the ExD cell disclosed herein, the collision cell (e.g., a gas filled RF-ion guide) may be used to add energy to protein analytes to unfold proteins and thereby prevent ET-no-D. [0031] According to examples of the ExD cell disclosed herein, in one example, for a lens as disclosed herein, off axis holes may be cut in the ExD cell to let gas coming out of a filament cassette to be directed away from a central axis in the ExD cell and escape out of the path of ions. Appropriately placed vents may allow 20230167-02 the gas to perform electron cooling and redistribution while minimizing ion scattering or stalling. For this example, the off axis holes also allow for gas to be provided to the collision cell more uniformly. [0032] According to examples of the ExD cell disclosed herein, an electronic control of the lens elements may enable ion transmission without ECD. Further, the ExD cell as disclosed herein includes combined ECD and CID, as well as higher-energy electron fragmentation. [0033] According to examples of the ExD cell disclosed herein, an RF shield at entrance or exit of the cell may include a thin copper sheet connected to a ground to maintain an RF-free region inside the ExD cell. An electrostatic lens may be fitted inside a hole in the RF shield to prevent the RF shield from influencing ion transmission through the ExD cell. [0034] According to examples disclosed herein, an ExD cell may include a filament cassette, and a gas container to enclose the filament cassette. A gas inlet may be configured to deliver gas into the gas container. The gas container may include an ion entrance opening and an ion exit opening. The gas may be either pure or a mixture, for example, of nitrogen, argon, helium, or xenon. Other gases may also be utilized. [0035] According to examples disclosed herein, for the ExD cell described above, the gas inlet may be configured to deliver the gas adjacent to a central axis of the ExD cell. 20230167-02 [0036] According to examples disclosed herein, for the ExD cell described above, the gas inlet may be configured to deliver the gas in regions where electrons are restrained by a static (RF-free) electromagnetic field. [0037] According to examples disclosed herein, for the ExD cell described above, the gas inlet may be configured to deliver the gas into the filament cassette. For example, the gas inlet may be configured to deliver gas to an RF-free region in the ExD cell. [0038] According to examples disclosed herein, for the ExD cell described above, the filament cassette may include a filament, and an inlet (e.g., a filament inlet as disclosed herein) for the gas. The filament inlet may be configured to deliver the gas directly to a region next to the filament. [0039] According to examples disclosed herein, for the ExD cell described above, the filament assembly may include posts to provide electrical contacts to the filament. The ExD cell may further include a filament inlet for the gas, where the filament inlet is configured to direct the gas towards the posts to evenly distribute the gas. In this regard, the inlet gas may be directed towards the filament posts to scatter gas molecules within the cassette. Otherwise, gas may flow differently at the low pressures in the mass spectrometer and may form molecular jets. [0040] According to examples disclosed herein, for the ExD cell described above, the ExD cell may further include at least two electrostatic lenses disposed 20230167-02 on opposite sides of the filament cassette. In this regard, the at least two electrostatic lenses may include vent holes to allow gas to exit the ExD cell. [0041] According to examples disclosed herein, for the ExD cell described above, the vent holes may be symmetrically disposed in the at least two lenses. In other examples, the vent holes may be symmetrically disposed in one of the at least two lenses. [0042] According to examples disclosed herein, for the ExD cell described above, the vent holes may be asymmetrically disposed in the at least two lenses. [0043] According to examples disclosed herein, for the ExD cell described above, radio-frequency (RF) shielding may be added to maintain an RF-free environment within the ExD cell. [0044] According to examples disclosed herein, the ExD cell disclosed herein may also be disposed between other types of ion optical elements. In other examples, the ExD cell may be disposed prior to isolating quadrupoles in QqQ, Q- ToF, Q-orbitrap or Q-FTICR mass spectrometers. [0045] According to examples disclosed herein, for the assembly described above, the ExD cell may include a gas inlet that is configured to deliver gas adjacent to a central axis of the ExD cell. [0046] According to examples disclosed herein, for the assembly described above, the ExD cell may include a gas inlet that is configured to deliver gas in regions where electrons are restrained by an electromagnetic field. 20230167-02 [0047] According to examples disclosed herein, for the assembly described above, the ExD cell may include a filament cassette, and a gas inlet that is configured to deliver gas into the filament cassette. [0048] According to examples disclosed herein, for the assembly described above, the filament cassette may include a filament, and a filament inlet for the gas. The filament inlet may be configured to deliver the gas directly to a region next to the filament. [0049] According to examples disclosed herein, for the assembly described above, the filament cassette may include a filament, and posts to provide electrical contacts to the filament. The ExD cell may further include a filament inlet for the gas, where the filament inlet is configured to direct the gas towards the posts to evenly distribute the gas. [0050] According to examples disclosed herein, for the assembly described above, the ExD cell may include a gas container to enclose a filament cassette. The gas container may include an ion entrance opening and an ion exit opening. [0051] According to examples disclosed herein, for the assembly described above, the ExD cell may include at least two electrostatic lenses disposed on opposite sides of a filament cassette. The at least two electrostatic lenses may include vent holes to allow gas to exit the ExD cell. [0052] According to examples disclosed herein, an ExD cell may include a filament cassette and a gas container enclosing the filament cassette and gas 20230167-02 delivered into the ExD cell. The gas container may operate as a cassette holder to deliver gas directly to the filament cassette. The gas container may provide for removal of the filament cassette to replace burnt out filaments without disconnecting the gas. [0053] According to examples of the ExD cell disclosed herein, the gas may be retained in the gas container. [0054] According to examples of the ExD cell disclosed herein, gas delivered into the ExD cell may subsequently deliver gas into the collision cell or to the IBC. [0055] According to examples of the ExD cell disclosed herein, additional holes or paths may be provided for gas delivery to the collision cell or the IBC off axis (e.g., by diverting gas from the ion path). [0056] According to examples of the ExD cell disclosed herein, an oxygen scrubber may be provided in the path of the gas. In this regard, making the gas as free of oxygen as possible improves operational lifetime of the filament. [0057] According to examples disclosed herein, an ExD cell may include a container to enclose a filament cassette, and a first inlet that is configured to deliver gas into the container. The ExD cell may further include a second inlet that is configured to deliver ions to the filament cassette, and an ion exit opening. [0058] According to examples of the ExD cell disclosed herein, the first inlet may be configured to deliver the gas adjacent to a central axis of the ExD cell. 20230167-02 [0059] According to examples of the ExD cell disclosed herein, the first inlet may be configured to deliver the gas in regions where electrons are restrained by an electromagnetic field. [0060] According to examples of the ExD cell disclosed herein, the first inlet may be configured to deliver the gas into the filament cassette. [0061] According to examples of the ExD cell disclosed herein, the filament cassette may include a filament, and a filament inlet for the gas. The filament inlet may be configured to deliver the gas directly to a region next to the filament. [0062] According to examples of the ExD cell disclosed herein, the filament cassette may include a filament, posts to provide electrical contacts to the filament, and a filament inlet for the gas. The filament inlet may be configured to direct the gas towards the posts to evenly distribute the gas. [0063] According to examples of the ExD cell disclosed herein, the ExD cell may further include at least two electrostatic lenses disposed on opposite sides of the filament cassette. The at least two electrostatic lenses may include vent holes to allow the gas to exit the ExD cell. [0064] According to examples of the ExD cell disclosed herein, the vent holes may be symmetrically disposed in the at least two electrostatic lenses. [0065] According to examples of the ExD cell disclosed herein, the ExD cell may further include radio-frequency (RF) shielding to maintain an RF-free environment. 20230167-02 [0066] According to examples of the ExD cell disclosed herein, pressure in the container does not substantially drop. In this regard, the pressure in the container may increase or remain substantially constant. [0067] According to examples disclosed herein, an assembly may include a collision cell, downstream ion optics, and an ExD cell disposed between the collision cell and the downstream ion optics. [0068] According to examples of the assembly disclosed herein, the downstream ion optics is an ion beam compressor. [0069] According to examples of the assembly disclosed herein, the ExD cell may include a gas inlet that is configured to deliver gas adjacent to a central axis of the ExD cell, a gas inlet that is configured to deliver the gas in regions where electrons are restrained by an electromagnetic field, or a gas inlet that is configured to deliver the gas into a filament cassette. [0070] According to examples of the assembly disclosed herein, the filament cassette may include a filament, and a filament inlet for the gas. The filament inlet may be configured to deliver the gas directly to a region next to the filament. [0071] According to examples of the assembly disclosed herein, the filament cassette may include a filament, posts to provide electrical contacts to the filament, and a filament inlet for the gas. The filament inlet may be configured to direct the gas towards the posts to evenly distribute the gas. 20230167-02 [0072] According to examples of the assembly disclosed herein, the ExD cell may include a gas container to enclose a filament cassette, and the gas container may include an ion entrance opening and an ion exit opening. [0073] According to examples of the assembly disclosed herein, the ExD cell may be disposed between the collision cell and the downstream ion optics by being disposed generally equal distances between the collision cell and the downstream ion optics. [0074] According to examples of the assembly disclosed herein, the ExD cell may include at least two electrostatic lenses disposed on opposite sides of a filament cassette. The at least two electrostatic lenses may include vent holes to allow gas to exit the ExD cell. [0075] Figure 1 illustrates a cross-sectional view of a first embodiment of an electron-based fragmentation (ExD) cell (hereinafter “ExD cell 100”) placed after a collision cell (CC) 102 (e.g., collision hexapole) and before an ion beam compressor (IBC) 104, in accordance with an example of the present disclosure. The combination of the ExD cell 100, the CC 102, and the IBC 104 may also be referred to herein as an assembly. The ExD cell 100 may include an inlet tubing 106 for gas, such as inert gas. [0076] Referring to Figure 1, the ExD cell 100 may be placed after the CC 102 and before IBC 104. The gas present in the CC 102 enhances ECD efficiency. In this regard, nitrogen, argon or another inert gas up to 20 mTorr may be delivered through the gas inlet 106 into the central axis of the ExD cell 100 where electrons 20230167-02 are produced by a filament as disclosed herein, and constrained radially by a magnetic field produced by two permanent magnets. The gas may be applied anywhere within in a radio-frequency (RF)-free region of the ExD cell 100. Supplying gas in this RF-free region containing electrons thus increases ECD. [0077] The applied gas may be applied to any pressure up to some limit (e.g., up to 100 mTorr). Exceeding the limit may cause ion scattering and reduce ion transmission. As disclosed herein, an RF shield may be utilized to prevent RF fields from the IBC penetrating into the ExD cell. [0078] Figure 2 illustrates principles of operation associated with the ExD cell 100, in accordance with an example of the present disclosure. [0079] Referring to Figure 2, the setup 200 may include a loop filament 202. The ions may pass through the middle of an electrically heated loop filament and the center of a permanent ring magnet 206. The setup 200 may further include an aperture 208 within the magnet 206, where the aperture 208 may be, for example, 3 mm in diameter. An adjustable DC potential may be applied where loop filament 202 is made negative relative to permanent ring magnet 206 to pull electrons away from the filament and into the area of the aperture 208. For the setup 200, V is a low voltage, and a high current filament power supply may be electrically floated. In order to achieve high ECD efficiency, gas helps to cool the electron cloud and distribute the cloud more widely. This may be accomplished by increasing gas pressure in the ExD cell. 20230167-02 [0080] Figure 3 illustrates a diagrammatic view to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. [0081] Referring to Figure 3, an ExD cell 300 is shown without gas being provided. The ExD cell 300 may include a metal cassette 302 that surrounds a filament 304, with the metal cassette 302 being positively charged relative to the filament to help pull electrons off the filament and into central axis 306 of the magnets. Zone 308 defined by the arrows as shown indicates the RF-free region for the ExD cell. In this regard, electrons may be trapped throughout this region. Electronstatic lenses 310 and magnets 312 may have DC electrical potentials applied thereto. [0082] Figure 4 illustrates another diagrammatic view to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. [0083] Referring to Figure 4, Figure 4 shows the ExD cell 100 including a cylinder 400 (e.g., a container, or a gas container as disclosed herein). Gas may be provided by a source (not shown) at 402 (e.g., also referred to herein as first inlet). Apertures 404 and 406 (e.g., approximately 3 mm in diameter; also referred to herein as second inlet and ion exit opening) may be provided to allow ions to respectively enter and fragments to exit. Gas escaping from the apertures may reduce the vacuum in the mass spectrometer and put an added load on turbo pumps. In this regard, cylinder 400 may provide an enclosure to increase gas pressure within the ExD cell 100. The apertures 404 and 406 may be formed sufficiently narrow to restrict gas from exiting and wide enough to allow passage of 20230167-02 analytic ions. The gas may thus be delivered in the immediate regions near filament 408 to maximize the cooling and redistribution of electrons. [0084] Figure 5 illustrates a diagrammatic view to illustrate adding of gas into a filament cassette to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. [0085] Referring to Figure 5, Figure 5 shows the ExD cell 100 including a filament cassette 500. In this regard, gas may be added into the filament cassette 500 to maximize the amount of gas near a filament, resulting in efficient ECD. Gas may escape through central ion apertures 502 and 504 on each end. In this regard, the ExD cell may include a container (e.g., gas-tight container as disclosed herein) to enclose the filament cassette 500, and a first inlet 106 that is configured to deliver gas into the container. The ExD cell may further include a second inlet (e.g., through aperture 502) that is configured to deliver ions to the filament cassette, and an ion exit opening (e.g., through aperture 504). [0086] Figure 6 illustrates electron capture dissociation (ECD) efficiency of a doubly charged peptide increases with greater gas pressure, in accordance with an example of the present disclosure. [0087] Referring to Figure 6, ECD efficiency dependence (measured as abundance of m/z 624 ECD fragment relative to the m/z 6742+ precursor) on gas pressure is shown at 600. In this regard, the increase in ECD efficiency of the 11 amino acid peptide substance P (2+) is shown. The x-axis refers to the addition of gas pressure (in PSI) applied to the gas inlet tube (e.g., at 402 of Figure 4). The 20230167-02 dashed line at 602 shows the increase in efficiency of ECD with increasing gas pressure applied to the gas inlet to the filament cassette. [0088] Although ECD efficiency increases proportionally with gas pressure, higher gas pressures may begin to scatter analyte ions as the gas is flowing out through the central apertures. This may limit the practical increase in ECD efficiency, but can be overcome by consideration of how gas is directed out of the ExD cell 100. [0089] In order to address analyte ion scattering, additional venting may be provided through the magnets and surrounding lenses before and after the filament cassette. This is because most of the improvement of fragmentation efficiency with gas occurs in the immediate region of the filament and the adjacent magnets. [0090] Yet further, the aperture sizes of various lens elements may be adjusted to help direct gas flow. For example, assuming that 3 mm diameter apertures are generally utilized, the aperture sizes on the filament cassette may be reduced to 2 mm to retain gas inside the filament cassette. Other apertures may be increased to 4 mm to facilitate gas venting away from the ion paths. In one example, the aperture size may be set to approximately 3 mm. Changes in electrostatic behavior may be compensated for differing aperture diameter by altering the DC voltages applied to the lenses. [0091] Operation of the ExD cell 100 benefits from analyte ions being cooled and condensed within the collision cell before entering the ExD cell 100. The DC voltages needed to operate the ExD cell 100 may be independent of the operation 20230167-02 of the quadrupole on other ion optic elements in the Q-ToF, and thus facilitate tuning and operation of the ExD cell. [0092] Adding additional gas to the filament cassette at high flows may increase gas pressure within the mass spectrometer, thereby resulting in reduced quadrupole isolation efficiency, ToF resolution, and result in other adverse effects. In quadrupole time-of-flight (Q-ToF) instruments, gas (e.g., nitrogen) may be provided to the collision cell to enable CID fragmentation (collision-induced dissociation). In order to minimize the addition of extra gas to the mass spectrometer, the inlet for the gas may be moved from the collision cell to deliver all of the collision gas directly to the filament cassette. The escaping gas from the apertures in the filament cassette may be directed to fill the collision cell to maintain its normal pressure. With the proper choice of lens apertures in the electromagnetostatic cell and suitable venting of gas into the collision cell and to the IBC, enhanced electron fragmentation may be achieved without adding an additional gas load to the mass spectrometer. Hence, collision-induced dissociation (CID) and time-of-flight (ToF) resolution from ion cooling in the IBC may be maintained without an additional gas load being made to the Q-ToF. [0093] For an ExD cell configuration to operate next to the IBC, an RF filtering or shielding may be utilized to minimize interference from the IBC hexapole. The RF filtering or shielding may also protect the electron cloud inside the electromagnetostatic cell by keeping the cell RF-free. 20230167-02 [0094] When CID energy is applied, analyte ions may be accelerated by increased DC voltages applied to the ion optics elements in front of the collision cell. The analyte ions may be accelerated as they enter the collision cell, where the ions are thereby initially heated by internal collisions with the collision gas. For larger proteins, the heating can help unfold the protein to promote improved dissociation of fragments produced in the ExD cell. The application of CID energy raises the internal energy of large analytes (such as proteins). [0095] With respect to operation of the ExD cell, the ions may be heated when they first enter the collision cell for several millimeters. Further in the collision cell, the kinetic energy of the ions is transferred to gas molecules that are cooling the ion beam and making the beam focused to enter the ExD cell. The internal energy of proteins with added collision energy remains high enough for effective fragmentation. [0096] Figure 7 illustrates a cross-sectional view of a second embodiment of a centrally disposed (e.g., generally equal distances between the CC and IBC) ExD cell 700, in accordance with an example of the present disclosure. [0097] Referring to Figure 7, a second shortened collision cell may be added followed by the ExD cell 700. The configuration of Figure 7 allows for additional energy to be applied to increase dissociation or alternative fragmentation. In this regard, the configuration of Figure 7 may be denoted the middle cell. Gas provided to the ExD cell may be used to fill both collision cells with collision gas. 20230167-02 [0098] Figure 8 illustrates results 800 based on the addition of CID energy before protein analytes enter the ExD cell, in accordance with an example of the present disclosure. [0099] Referring to Figure 8, Figure 8 illustrates the results 800 showing the value of adding CID energy before protein analytes enter the ExD cell 100. In this regard, the results 800 show that the ECD fragments significantly increase in the left side (e.g., at 802 and 804) as CID activation is increased. [0100] Figure 9 illustrates side, isometric front, and back views of a filament cassette (e.g., filament cassette 500), in accordance with an example of the present disclosure. [0101] Referring to Figure 9, different views of the filament cassette 500 with a hole or inlet provided to deliver gas directly into the chamber surrounding the filament are illustrated. For example, the views illustrate side view at 902, isometric front views at 904 and 906, and back view at 908 of a filament cassette. Compared to the isometric front view at 904, the isometric front view at 906 includes a cover removed. The filament cassette 500 may include filament inlet 910 for gas. The filament inlet 910 may provide gas directly to an electron producing filament 914. [0102] The filament cassette 500 may include aperture 912 including the filament 914. Resilient fingers 916 and 918 may be insertable or otherwise slid into corresponding passages (not shown) in the ExD cell 100 to provide engagement of the filament cassette 500 in the ExD cell 100 when enlarged protrusions 920 20230167-02 engage with corresponding enlarged notches (not shown) in the ExD cell 100. In this manner, the filament cassette 500 may be releasably positioned and/or locked into the ExD cell 100. [0103] As shown at 922, posts 924 and 926 may retain the filament 914 there between. [0104] With respect to the aperture 912, a corresponding aperture 928 may be positioned opposite to the aperture 912. The apertures 912 and 928 may be disposed along a central axis to allow analyte ions to pass through the filament cassette 500 and inside the hot filament loop for the filament 914. The apertures 912 and 928 also allow gas to fill the ExD cell 100. [0105] According to examples disclosed herein, gas may be delivered directly into the filament cassette 500. The pressure of the gas may be in the range of 1 to 200 milliTorr for increasing ECD efficiency, and in one example, between 20 to 100 milliTorr. [0106] According to examples disclosed herein, the gas line for gas normally provided to the collision cell may be used to feed into the filament cassette 500. Thus, all of the gas needed for the collision cell operation may flow out of the small central hole in the filament cassette 500. [0107] Figure 10 illustrates a diagrammatic view of venting of gas after leaving the filament cassette 500 to the front and back of an ExD cell to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. 20230167-02 [0108] Referring to Figure 10, additional venting may be provided through magnetic lenses 1000 and 1002, and surrounding lenses 1004, 1006, 1008, and 1010, before and after the filament cassette 500. In this regard, upper and lower vent holes as shown at 1012 and 1014 that are positioned off axis (e.g., at a specified distance from central axis 1016 of the ExD cell 100) allow gas to exit the ExD cell 100 away from the path of ions. The lenses 1004, 1006, 1008, and 1010 may include the vent holes at 1012 and 1014 with various geometric patterns. For example, as shown in Figure 10, the vent holes may include four holes per lens, with two holes placed in an upper area of the lens and two holes placed in a lower area of the lens in the orientation of Figure 10. The vent holes 1012 and 1014 may be of the same size as shown, or of different diameters to adjust the amount of gas exiting to the front collision cell versus the IBC. The lenses 1004, 1006, 1008, and 1010 may also include central holes for ion entrance and exit as shown in Figure 10. [0109] Gas may be vented as shown by the flow path arrows (e.g., at 1018, 1020, etc.) after leaving the filament cassette 500 to the front and back of the ExD cell 100. The vent holes at 1012 and 1014 may allow gas to escape from in between the elements. This reduces the gas flow being present in the ion path (e.g., along the central axis 1016) through the ExD cell 100 as illustrated by alternative paths for gas movement shown by the various flow path arrows (e.g., at 1018, 1020, etc.). 20230167-02 [0110] Figure 11 illustrates how gas from the filament cassette 500 is directed to the collision cell 102 and the IBC 104 to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. [0111] Referring to Figure 11, with the proper choice of lens apertures in the electromagnetostatic cell and suitable venting of gas into the CC 102 and to the IBC 104, the benefits of enhanced electron fragmentation are possible without adding an additional gas load to the mass spectrometer. In this regard, in a similar manner as Figure 1, Figure 11 shows an arrangement where the CC 102 is on the left and the IBC 104 is located on the right relative to the ExD cell 100 (in the orientation of Figure 11). The CC 102 and the IBC 104 may utilize hexapoles with RF to confine ions and utilize gas to cool ions. The conductance of the gas moving forward or to the rear may be modified by small alterations in the diameter of the apertures. For example, changing the diameter from 3 mm to 2 mm restricts gas flow by more than one-half. These changes in diameter can be used to balance the gas flow to the CC 102 and the IBC 104. [0112] In a similar manner as disclosed herein with respect to Figures 1 and 10, gas from the filament cassette 500 may be directed to the CC 102 and the IBC 104, which may utilize 10-20 milliTorr pressure. Based on this configuration, relatively higher gas pressures may be achieved near the filament to maximize ECD while using the escaping gas to supply the CC 102 and the IBC 104. 20230167-02 [0113] Figure 12 illustrates the CC 102, the ExD cell 100, and IBC shielding to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. [0114] Referring to Figure 12, the CC 102, the ExD cell 100, and the use of the exit lens as an RF shield are shown next to IBC. A capacitor allows RF currents induced on the outer electrostatic lens to be transferred to the instrument ground. [0115] The IBC 104 is illustrated as two different RF-dependent hexapoles. The electrical mounting brackets adjacent to the ExD cell 100 result in an asymmetric RF field being induced on the last lens in the ExD cell 100. In this regard, a capacitor may transfer the induced RF current on the exit lens to ground to maintain the RF free region inside the ExD cell 100 used to confine electrons. [0116] Figure 13 illustrates a collision cell, and an ExD cell with an electrically separate RF shield added to the exit lens to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. [0117] Referring to Figure 13, the CC 102, the ExD cell 100 with RF shielding, and IBC shielding are shown. With respect to IBC, RF shielding as shown at 1300 reduces induced voltages in the ExD cell to maintain the RF-free fields used to confine electrons. [0118] Figure 14 illustrates fragment intensity to illustrate operation of the ExD cell 100, in accordance with an example of the present disclosure. 20230167-02 [0119] Referring to Figure 14, top panel 1400 shows the ECD cell operated with gas and looking at the fragmentation of 2% substance P (e.g., a peptide of 11 amino acids). The desired ECD fragments are shown at 1402, and the unwanted unassigned fragments are shown at 1404. Without added gas, the ECD cell would have produced only 2-5% ECD. [0120] The panels at 1406 and 1408 are CID at 25 and 30 V of collision energy. Typical CID fragments are shown at 1410 and 1412, though many other fragments are produced. The CID fragments are lower in intensity than the ECD fragments at 1402. The entire spectrum at 1406 and 1408 is filled with many fragments that may be challenging assign, or can be misinterpreted when looking at unknown samples. [0121] What has been described and illustrated herein is an example along with some of its variations. The terms, descriptions and figures used herein are set forth by way of illustration only and are not meant as limitations. 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- in which all terms are meant in their broadest reasonable sense unless otherwise indicated.

Claims

20230167-02 What is claimed is: 1. An electron-based fragmentation (ExD) cell comprising: a container to enclose a filament cassette; a first inlet that is configured to deliver gas into the container; a second inlet that is configured to deliver ions to the filament cassette; and an ion exit opening. 2. The ExD cell according to claim 1, wherein the first inlet is configured to deliver the gas adjacent to a central axis of the ExD cell. 3. The ExD cell according to claim 1, wherein the first inlet is configured to deliver the gas in regions where electrons are restrained by an electromagnetic field. 4. The ExD cell according to claim 1, wherein the first inlet is configured to deliver the gas into the filament cassette. 5. The ExD cell according to claim 4, wherein the filament cassette comprises: 20230167-02 a filament; and a filament inlet for the gas, wherein the filament inlet is configured to deliver the gas directly to a region next to the filament. 6. The ExD cell according to claim 4, wherein the filament cassette comprises: a filament; posts to provide electrical contacts to the filament; and a filament inlet for the gas, wherein the filament inlet is configured to direct the gas towards the posts to evenly distribute the gas. 7. The ExD cell according to claim 1, further comprising: at least two electrostatic lenses disposed on opposite sides of the filament cassette, wherein the at least two electrostatic lenses include vent holes to allow the gas to exit the ExD cell. 8. The ExD cell according to claim 7, wherein the vent holes are symmetrically disposed in the at least two electrostatic lenses. 20230167-02 9. The ExD cell according to claim 1, further comprising: radio-frequency (RF) shielding to maintain an RF-free environment. 10. The ExD cell according to claim 1, wherein pressure in the container does not substantially drop. 11. An assembly comprising: a collision cell; downstream ion optics; and an electron-based fragmentation (ExD) cell disposed between the collision cell and the downstream ion optics. 12. The assembly according to claim 11, wherein the downstream ion optics is an ion beam compressor. 13. The assembly according to claim 11, wherein the ExD cell comprises: a gas inlet that is configured to deliver gas adjacent to a central axis of the ExD cell; 20230167-02 a gas inlet that is configured to deliver the gas in regions where electrons are restrained by an electromagnetic field; or a gas inlet that is configured to deliver the gas into a filament cassette. 14. The assembly according to claim 13, wherein the filament cassette comprises: a filament; and a filament inlet for the gas, wherein the filament inlet is configured to deliver the gas directly to a region next to the filament. 15. The assembly according to claim 13, wherein the filament cassette comprises: a filament; posts to provide electrical contacts to the filament; and a filament inlet for the gas, wherein the filament inlet is configured to direct the gas towards the posts to evenly distribute the gas. 16. The assembly according to claim 11, wherein the ExD cell comprises a gas container to enclose a filament cassette, and wherein the gas container includes an ion entrance opening and an ion exit opening. 20230167-02 17. The assembly according to claim 11, wherein the ExD cell is disposed between the collision cell and the downstream ion optics by being disposed generally equal distances between the collision cell and the downstream ion optics. 18. The assembly according to claim 11, wherein the ExD cell comprises: at least two electrostatic lenses disposed on opposite sides of a filament cassette, wherein the at least two electrostatic lenses include vent holes to allow gas to exit the ExD cell. 19. An electron-based fragmentation (ExD) cell comprising: a gas container enclosing gas delivered into the ExD cell. 20. An assembly comprising the ExD cell according to claim 19, wherein the ExD cell is disposed prior to an isolating quadrupole.