Linear RFQ ion trap in magnetic field

Abstract

In one aspect, an apparatus for use in a mass spectrometer is disclosed, which comprises an ion reaction device, which includes an ion dissociation unit (e.g., an ion-electron interaction unit) that can receive a plurality of precursor ions and cause their dissociation to generate a plurality of product ions, and a linear ion trap that can receive the product ions and isolate a target product ion of interest for mass analysis. The linear ion trap is positioned in a magnetic field and includes a set of quadrupole rods formed of a paramagnetic material to substantially shield the space between the quadrupole rods from the magnetic field, thereby reducing and preferably eliminating the interference of the magnetic field with the process of isolating the target product ion of interest.

Description

LINEAR REQ ION TRAP IN MAGNETIC FIELDRelated Applications

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 698,684 filed on September 25, 2024, the contents of which are incorporated herein by reference in their entirety.Technical Field

[0002] The present teachings are generally directed to systems and methods for mass spectrometry and more particularly to systems and methods for performing tandem mass spectrometry.Background

[0003] Mass spectrometry (MS) is an analytical technique for determining the structure of test chemical substances with both qualitative and quantitative applications. MS can be useful for identifying unknown compounds, determining the composition of atomic elements in a molecule, determining the structure of a compound by observing its fragmentation, and quantifying the amount of a particular chemical compound in a mixed sample. Mass spectrometers detect chemical entities as ions such that a conversion of the analytes to charged ions must occur.

[0004] An advanced type of mass spectrometry, generally known as tandem mass spectrometry, employs multiple stages of mass analysis for providing detailed structural information about molecules. In one example of a two-stage mass spectrometry, one or more analytes within a sample under analysis are ionized to generate a plurality of precursor ions and a mass filter is utilized to select those precursor ions having mass-to-charge ratios within a bandpass of the mass filter. Subsequently, the selected precursor ions are fragmented to generate fragment ions and a mass spectrum of the fragment ions is acquired and analyzed to obtain information about the analytes.

[0005] Tandem mass spectrometry offers a practical way for deeper molecular structure elucidation in mass spectrometry. Collision-induced dissociation (CID) and electron-based dissociation (ExD), which includes activation by a free electron (i.e., electron capture dissociation: ECD and its derivatives) or an electron transferred from a reagent anion (electron transfer14907-7108-5162, v. 1dissociation: ETD) provide orthogonal molecular structural information because these popular techniques are based on alternative dissociation mechanisms. CID is based on thermal dissociation or molecular vibrational state excitation mechanism. ExD, on the other hand, is based on a radical- induced dissociation mechanism that introduces an unpaired electron or radical electron. ExD can include multiple types of dissociation principles, such as electron capture dissociation (ECD), hot ECD, electron transfer dissociation (ETD), electronically excited dissociation (EED), and electron detachment dissociation (EDD). Ultra-violet photodissociation (UVPD) is another orthogonal dissociation method, including electronic excitation of molecular internal states.

[0006] Notwithstanding great progress that has been made in tandem mass spectrometry, there is still a need for improved systems and methods for performing mass spectrometry.Summary

[0007] In one aspect, an apparatus for use in a mass spectrometer is disclosed, which comprises an ion reaction device, where the ion reaction device includes a longitudinal pathway and a transverse pathway intersecting the longitudinal pathway at an ion reaction region, said longitudinal pathway having an input port (herein also referred to as an inlet) for receiving precursor ions and an output port (herein also referred to as an outlet) through which ions can exit the longitudinal pathway and said transverse pathway further having a transverse input port (herein also referred to as a transverse inlet) for receiving an electron beam for interacting with the precursor ions in said ion reaction region to generate product ions and a transverse output port through which the product ions can exit the transverse pathway. The ion reaction device further includes at least one magnet for generating a magnetic field for guiding the electron beam along the transverse pathway, said magnetic field extending along the transverse pathway and through said output port thereof to a region external to said transverse pathway. A linear ion trap is positioned at least partially within the magnetic field in said external region and includes a plurality of electrodes arranged in a quadrupole configuration to provide a passageway for receiving the product ions exiting the transverse pathway through the transverse output port, wherein said plurality of electrodes of the linear ion trap includes a paramagnetic material, thereby substantially shielding said passageway from the magnetic field.

[0008] By way of example, and without limitation, the paramagnetic material can be any of paramagnetic stainless steel and soft iron. In some embodiments, the quadrupole rods of the linear24907-7108-5162, v. 1ion trap can have a length in a range of about 10 mm to about 50 mm and a quadrupole radius (i.e., the distance between each quadrupole rod to the center of the linear ion trap) in a range of about 2 mm to about 5 mm.

[0009] In various embodiments, the ion reaction device further comprises a first set of L- shaped electrodes arranged in a multipole configuration and a second set of L-shaped electrodes arranged in a multipole configuration, where said first and said second set of L-shaped electrodes are arranged relative to one another so as to provide said longitudinal and said transverse pathways. By way of example, and without limitation, any of the first set and the second set of the L-shaped electrodes is arranged according to a quadrupole configuration.

[0010] In various embodiments, the apparatus further includes at least an RF voltage source for application of a set of RF voltages to the quadrupole electrodes of the linear ion trap and at least a first DC voltage source for applying a resolving DC voltage (herein also referred to as a “discriminating DC voltage”) to at least one of said quadrupole electrodes.

[0011] In various embodiments, the apparatus further includes a controller in communication with the RF voltage source and the first DC voltage source for configuring the set of RF voltages and the resolving DC voltage for trapping a product ion with an isolation target mass-to-charge ratio (m / z) within said passageway. By way of example, the controller can be configured to control the RF voltage source and the first DC voltage source to generate the set of RF voltages and the resolving DC voltage such that the isolation target m / z corresponds to the apex of an a-q stability diagram of the Mathieu equation.

[0012] In various embodiments, the apparatus can further include an AC voltage source for applying an AC voltage to at least one of the quadrupole rods of the linear ion trap for causing resonant excitation of unwanted product ions so as to discard said unwanted product ions from the linear ion trap. This AC voltage can also be used for resonant excitation of ions to induce collisional dissociation.

[0013] In various embodiments, the apparatus can further include an electron source that is positioned in proximity of said transverse input port and provides an electron beam. A first pole electrode can be positioned between the electron source and the transverse input port, where the first pole electrode is configured for application of a first DC voltage thereto for confining ions in34907-7108-5162, v. 1the reaction device. A second pole electrode is positioned between the transverse output port and a proximal, ion-receiving end of the linear ion trap and is configured for application of a second DC voltage thereto for controlling propagation of the ions along said transverse pathway, delivery of the product ions into the passageway and retention of at least a portion of the product ions therein.

[0014] In various embodiments, the apparatus further includes a first gate electrode that is positioned between the first pole electrode and the electron source, where the first gate electrode is maintained at a DC potential configured to extract electrons from the electron source.

[0015] In various embodiments, the apparatus further includes a second gate electrode that is positioned between the second pole electrode and the plurality of rods of the linear ion trap, where the second gate electrode is maintained at a DC potential configured to inhibit electrons and / or positive ions exiting the ion reaction device from colliding with said plurality of rods of the linear ion trap.

[0016] In various embodiments, the apparatus further includes an end electrode that is positioned at a distal end of the passageway and is configured for application of a third DC voltage thereto such that a potential difference between said end electrode and said second pole electrode and a potential difference between said end electrode and the quadrupole rods of the linear ion trap facilitate axial confinement of the product ions within the linear ion trap.

[0017] In various embodiments, the at least one magnet includes two magnets that are positioned, respectively, in proximity of the input port and the output port of the transverse pathway.

[0018] In a related aspect, an apparatus for use in a mass spectrometer is disclosed, which includes an ion reaction device that is configured to receive precursor ions and having an ion reaction region in which the received precursor ions interact with an electron beam to generate product ions, said ion reaction device further having at least one magnet for generating a magnetic field for guiding the electron beam, wherein said magnetic field extends to a region external to the ion reaction device, and a linear ion trap positioned at least partially in the magnetic field in said region external to the reaction device, said linear ion trap having a plurality of rods arranged in a quadrupole configuration to provide a passageway for receiving the product ions from the ion44907-7108-5162, v. 1reaction device, wherein said plurality of rods of the linear ion trap comprises a paramagnetic material, e.g., formed entirely of a paramagnetic material, for substantially shielding the passageway from the magnetic field.

[0019] In various embodiments, the paramagnetic material includes any of paramagnetic stainless steel and soft iron.

[0020] In various embodiments, the ion reaction device can further include a first set of L- shaped electrodes arranged in a multipole configuration and a second set of L-shaped electrodes arranged in a multipole configuration, said first and said second set of L-shaped electrodes arranged relative to one another so as to provide a longitudinal pathway and a transverse pathway, wherein said longitudinal pathway includes an input port for receiving precursor ions and an output port through which ions can exit the longitudinal pathway and said transverse pathway includes a transverse input port for receiving the electron beam and a transverse output port through which the product ions can exit the transverse pathway to reach the linear ion trap.

[0021] In various embodiments, the apparatus further includes at least an RF voltage source for application of a set of RF voltages to the quadrupole electrodes of the linear ion trap and at least a first DC voltage source for applying a resolving DC voltage to at least one of said quadrupole electrodes. The apparatus can further include a controller in communication with the RF voltage source and the first DC voltage source for configuring the set of RF voltages and the resolving DC voltage for trapping a species of said product ions with an isolation target mass-to- charge ratio (m / z) within said passageway. The controller can be configured to control the second RF voltage source and the first DC voltage source to generate the set of RF voltages and the resolving DC voltage such that the isolation target m / z corresponds to the apex of an a-q stability diagram of the Mathieu equation.

[0022] In various embodiments, the apparatus can further include an AC voltage source for applying an AC voltage to at least one of the quadrupole rods of the linear ion trap for causing resonant excitation of unwanted product ions so as to discard said unwanted product ions from the linear ion trap.54907-7108-5162, v. 1

[0023] In various embodiments, the apparatus can further include an AC voltage source for applying an AC voltage to at least one of the quadrupole rods of the linear ion trap for causing resonant excitation of at least one ion species so as to induce collisional dissociation.

[0024] In various embodiments, the apparatus can further include an electron source that is positioned in proximity of the transverse input port and provides the electron beam.

[0025] In various embodiments, a first pole electrode can be positioned between the electron source and the transverse input port, where the first pole electrode is configured for application of a first DC voltage thereto for controlling delivery of the product ions into the passageway and retention of at least a portion of the product ions therein.

[0026] In various embodiments, a gate electrode is positioned between the second pole electrode and the quadrupole rods of the linear ion trap, where the gate electrode is maintained at a DC potential configured to prevent electrons and / or positive ions exiting the transverse channel from entering the linear ion trap. Further, an end electrode can be positioned at a distal end of the passageway of the linear ion trap and be configured for application of a third DC voltage thereto such that a potential difference between said end electrode and said second pole electrode and a potential difference between the end electrode and the quadrupole rods facilitate axial confinement of the product ions within the linear ion trap.

[0027] In a related aspect, a mass spectrometer is disclosed, which includes an ion source for receiving a sample and ionizing one or more analytes within the sample to generate a plurality of precursor ions, and an apparatus for receiving the precursor ions and generating a plurality of product ions, where the apparatus includes an ion reaction device. The ion reaction device can in turn include a longitudinal pathway and a transverse pathway intersecting the longitudinal pathway at an ion reaction region, said longitudinal pathway having an input port for receiving precursor ions and an output port through which ions can exit the longitudinal pathway and said transverse pathway having an input port for receiving an electron beam for interacting with the precursor ions in said ion reaction region to generate product ions and a transverse output port through which the product ions can exit the transverse pathway, and at least one magnet for generating a magnetic field for guiding the electron beam along the transverse pathway, said magnetic field extending along the transverse pathway and through said output port thereof to a region external to said transverse pathway. A linear ion trap is positioned at least partially within the magnetic field in64907-7108-5162, v. 1said external region and includes a plurality of electrodes arranged in a quadrupole configuration to provide a passageway for receiving the product ions exiting the transverse pathway through the output port thereof, where the plurality of electrodes of the linear ion trap includes a paramagnetic material (e.g., formed entirely of the paramagnetic material), thereby substantially shielding said passageway from the magnetic field.

[0028] In various embodiments, the mass spectrometer can further include a mass analyzer that is positioned downstream of said apparatus and is configured to receive the product ions exiting the output port of the longitudinal pathway and to generate ion detection signals. The mass spectrometer can further include an analysis module that is configured to receive and process the ion detection signals so as to generate a mass spectrum of the product ions.

[0029] In various embodiments, the mass spectrometer further includes an ion fragmentation device that is positioned downstream of the apparatus for receiving at least a portion of the product ions and fragmenting the received product ions to generate a second set of product ions. A mass analyzer positioned downstream of the ion fragmentation device can receive the second set of product ions and generate mass detection signals for generating a mass spectrum of the second set of the product ions.

[0030] Further understanding of various aspects of the present teachings can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.Brief Description of the Drawings

[0031] FIG. 1A schematically depicts an embodiment of an apparatus according to the present teachings,

[0032] FIG. IB is a perspective schematic view of two sets of L-shaped quadrupole rods employed in an ion dissociation unit of the apparatus shown in FIG. 1A,

[0033] FIG. 1C is a perspective schematic view of a linear ion trap employed in the apparatus of FIG. 1A,

[0034] FIG. ID schematically depicts an apparatus according to another embodiment of the present teachings,74907-7108-5162, v. 1

[0035] FIG. 2 schematically illustrates application of RF and DC voltages to various components of the linear ion trap,

[0036] FIG. 3A schematically depicts the apparatus shown in FIG. 1 A further identifying the application of DC and RF voltages to various components of the apparatus during ion loading,

[0037] FIG. 3B shows the relative values of DC bias voltages applied to various elements of the apparatus and those applied to an upstream mass filer and an ion guide during a normal ion loading mode,

[0038] FIG. 3C shows the relative values of DC bias voltages applied to various elements of the apparatus and those applied to an upstream mass filter and an upstream ion guide during an ion loading mode in which ions undergo collision induced dissociation due to collisions with molecular of a gas (such as nitrogen) with which the apparatus is filled,

[0039] FIG. 4A shows application of DC and / or RF voltages to various elements of the apparatus while electron activated dissociation of ions received by the ion dissociation unit of the apparatus is performed,

[0040] FIG. 4B shows the relative values of DC bias voltages applied to various elements of the apparatus and those applied to an upstream mass filter and an upstream ion guide during performance of electron activated dissociation of the ions received by the apparatus,

[0041] FIG. 5A shows application of DC and / or RF voltages to various elements of the apparatus depicted in FIG. ID while electron activated dissociation of ions received by the ion dissociation unit of the apparatus is performed,

[0042] FIG. 5B shows the relative values of DC bias voltages applied to various elements of the apparatus during performance of electron activated dissociation of ions received by the apparatus,

[0043] FIG. 6A shows application of DC and / or RF voltages to various elements of the apparatus shown in FIG. 1A during transfer of product ions generated via dissociation of precursor ions from the ion dissociation unit of the apparatus to the linear ion trap,84907-7108-5162, v. 1

[0044] FIG. 6B shows the relative values of DC bias voltages applied to various elements of the apparatus shown in FIG. 6A during transfer of the product ions from the ion dissociation unit to the linear ion trap,

[0045] FIG. 7A shows application of DC and / or RF voltages to various elements of the apparatus shown in FIG. 1A during isolation of a target product ion of interest in the linear ion trap using a-q instability,

[0046] FIG. 7B shows the relative values of DC bias voltages applied to various elements of the apparatus shown in FIG. 7A during isolation of a target product ion of interest in the linear ion trap,

[0047] FIG. 8A shows application of DC and / or RF voltages to various elements of the apparatus shown in FIG. 1A during isolation of a target product ion of interest in the linear ion trap using AC resonant ejection, e.g., subsequent to the use of a-q instability,

[0048] FIG. 8B shows the relative values of DC bias voltages applied to various elements of the apparatus shown in FIG. 8A during isolation of a target product ion of interest in the linear ion trap using AC resonant ejection, e.g., subsequent to the use of a-q instability,

[0049] FIG. 9A shows application of DC and / or RF voltages to various elements of the apparatus shown in FIG. 1A during isolation of a target product ion of interest in the linear ion trap using notched white noise, e.g., subsequent to the use of a-q instability,

[0050] FIG. 9B shows the relative values of DC bias voltages applied to various elements of the apparatus shown in FIG. 9A during isolation of a target product ion of interest in the linear ion trap using notched white noise, e.g., subsequent to the use of a-q instability,

[0051] FIG. 10A shows application of DC and / or RF voltages to various elements of the apparatus shown in FIG. 1A during isolation of a target product ion of interest in the linear ion trap using notched white noise, e.g., subsequent to the use of a-q instability,

[0052] FIG. 10B shows the relative values of DC bias voltages applied to various elements of the apparatus shown in FIG. 10A during isolation of a target product ion of interest in the linear ion trap using notched white noise, e.g., subsequent to the use of a-q instability,94907-7108-5162, v. 1

[0053] FIG. 11 A shows application of DC and / or RF voltages to various elements of the apparatus shown in FIG. 1A during transfer of an isolated product ion from the linear ion trap to the ion dissociation unit of the apparatus depicted in FIG. 1A,

[0054] FIG. 11B shows relative values of DC bias voltages applied to various elements of the apparatus shown in FIG. 11A during transfer of the isolated product ion from the linear ion trap to the ion dissociation unit of the apparatus,

[0055] FIG. 12 A shows application of DC and / or RF voltages to various elements of the apparatus shown in FIG. 1A during extraction of an isolated product ion from the apparatus depicted in FIG. 1A,

[0056] FIG. 12B shows relative values of DC bias voltages applied to various elements of the apparatus shown in FIG. 12A during normal extraction of the isolated product ion from the apparatus,

[0057] FIG. 12C shows relative values of DC bias voltages applied to various elements of the apparatus shown n FIG. 12A during CID extraction of the isolated product ion from the apparatus,

[0058] FIG. 13A is a schematic view of a mass spectrometer according to an embodiment in which an apparatus according to an embodiment of the present teachings is incorporated,

[0059] FIG. 13B is a schematic view of a mass spectrometer according to another embodiment in which an apparatus according to another embodiment of the present teachings is incorporated,

[0060] FIG. 14A shows a full range display of an a-q stability diagram for a linear ion trap having non-magnetic rods utilized in an apparatus according to the present teachings, where the m / z of a test ion was 830.5 and a supplemental dipole excitation voltage at 0 = 0.7276 was applied,

[0061] FIG. 14B shows an expanded display at the apex of a-q stability diagram of FIG. 14A,

[0062] FIG. 14C shows an expanded display of the a-q stability diagram of FIG. 14A near the low-mass cut-off, showing that the magnetic field splits the dipole resonant line,

[0063] FIG. 14D shows a full range display of an a-q stability diagram for a linear ion trap having paramagnetic rods, where the m / z of a test ion was 830.5 and a supplemental dipole excitation voltage at 0 = 0.7276 was applied,104907-7108-5162, v. 1

[0064] FIG. 14E shows an expanded display of the a-q stability shown in FIG. 14D at the apex,

[0065] FIG. 14F shows an expanded display of the a-q stability shown in FIG. 14D near the low-mass cut-off,

[0066] FIG. 15 shows mass spectra of a tuning solution obtained by the TOF mass analyzer (solid peaks) and the linear ion trap using the a-q apex (the dotted peaks),

[0067] FIG. 16 shows dipolar resonant ejection profiles of ions with m / z = 829.5, 830.5, and 831 / 5, where a small resolving DC voltage of -4V was applied and 0 value of the supplemental AC was 0.7276, equivalent to 276.491 kHz. On both sides of the resonant ejection edges, a resolution of 2 was demonstrated, which can isolate a molecule from a molecule with a doublebond difference,

[0068] FIG. 17A shows an MS spectrum of cardiolipin: CL18: 1 standard in the ammonium acetate solvent. Protonated, mono-sodiated, di-sodiated, and tri-sodiated precursors were observed. Mono-lyso CL was contaminated,

[0069] FIG. 17B shows a beam-type CID (70 eV) spectrum of the protonated CL18:1 precursor ions, wherein the m / z at 603.54 corresponds to a subunit of two acyls and a glycerol backbone,

[0070] FIG. 17C shows a beam-type CID (70 eV) spectrum of the mono-sodiated CL18: 1 precursor ions, where the CID produced two major species (m / z = 877.49 is a subunit similar to a glycerophospholipid with a PGP (phospho-glycerol-phospho) headgroup),

[0071] FIG. 17D shows a beam-type CID (70 eV) spectrum of the di-sodiated CL18:1 precursor ions, where the ion at m / z = 899.47 is a glycerophospholipid-like product,

[0072] FIG. 17E shows a beam-type CID (70 eV) spectrum of the tri-sodiated CL18: 1 precursor ions, where the ion at m / z = 903.448 is a glycerophospholipid-like product,

[0073] FIG. 18A shows EED of isolated CID fragment ion of [CL18: 1+Na]+at m / z = 603.5 seen in FIG. 17C,114907-7108-5162, v. 1

[0074] FIG. 18B shows EED of isolated CID fragment ion of [CL18:1+Na]+at m / z = 877.5 seem in FIG. 17C,

[0075] FIG. 18C shows an expanded version of the spectrum shown in FIG. 18B around the backbone-acyl chain region,

[0076] FIG. 19A shows a MS spectrum of mono-sodiated CLs expressed in E. Coli,

[0077] FIGS. 19B - 19K illustrate CID spectra of each mono-sodiated precursor ions seen in FIG. 19A,

[0078] FIG. 20A shows EED spectrum of the CID product (m / z = 823.45 [32: 1]) of precursor m / z = 1385.8=CL(65:2),

[0079] FIG. 20B shows EED spectrum of the CID product (m / z =837.47 [33:1]) of precursor m / z = 1385.8=CL(65:2),

[0080] FIG. 20C shows EED fragment intensity of the acyl region, where the transition between zero-double bond ( bars showing with hatching 0) and one-double bond (bars shown with hatching 1) indicates the position of the double bond (n-7 = co-7), or 9,

[0081] FIG. 20D shows enhancement of fragment intensities at CH3(CH2)n+H and CH3(CH2)n+i+H (clear bars (ring)), indicating the existence of triangular-ring structure in the acyl chain. This CID product has a triangular-ring structure at 9-10 position, a 9, 10-methylene- hexadecanoyl acyl group,

[0082] FIG. 20E shows reconstructed CL structure of m / z = 1385.8,

[0083] FIG. 21 A shows an expanded spectrum around the glycerol backbone and the acyl chains of 18: 1 (1 IE), or trans-vaccenoyl acyl group,

[0084] FIG. 21B shows fragment intensity profile of the acyl chains associated with the expanded spectrum of FIG. 21 A,

[0085] FIG. 21C shows an expanded spectrum around the glycerol backbone and the acyl chains of 11,12-methylene-octadecanoyl acyl group or lactobacilloyl acyl group, and

[0086] FIG. 21D shows fragment intensity profile of the acyl chains associated with the expanded spectrum of FIG. 21C, and124907-7108-5162, v. 1

[0087] FIG. 22 is a block diagram of a computer system suitable for use in various embodiments of the present teachings, e.g., for controlling various voltage sources and / or analysis of mass spectra data.Detailed Description

[0088] It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also, for brevity, not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of the specifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

[0089] As used herein, the terms "about" and "substantially equal" refer to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the terms "about" and "substantially" as used herein mean 10% greater or less than the value or range of values stated or the complete condition or state. For instance, a concentration value of about 30% or substantially equal to 30% can mean a concentration between 27% and 33%. The terms also refer to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art.

[0090] As used herein the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ". Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent description of a corresponding block or item or feature of a corresponding apparatus. Some or all134907-7108-5162, v. 1of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some embodiments, one or more of the most important method steps may be executed by such an apparatus.

[0091] The phrase “substantially free of a magnetic field,” as used herein, indicates that a magnetic field, if any, has a magnitude less than about 0.1 Tesla, and has preferably a vanishing magnitude.

[0092] The phrase “paramagnetic material” is used in accordance with its ordinary meaning in the art to refer to a substance that becomes weakly magnetized when placed in an external magnetic field, aligning its magnetic moments parallel to the external magnetic field direction.

[0093] This disclosure generally relates to an ion reaction apparatus for use in a mass spectrometer, where the ion reaction apparatus can include an ion dissociation unit that can receive a plurality of precursor ions and cause their dissociation to generate a plurality of product ions and a linear ion trap positioned in a magnetic field generated by at least one magnet, where the linear ion trap can receive the product ions and isolate a product ion of interest for further analysis. In various embodiments, the linear ion trap includes a set of quadrupole rods that are formed of a paramagnetic material to substantially shield the interior of the linear ion trap (i.e., the space between the rods) from the magnetic field.

[0094] In some embodiments, the dissociation of the precursor ions within the dissociation unit can be achieved via exposure of the precursor ions to an electron beam. In other embodiments, the precursor ions can undergo collision-induced dissociation (CID) within the ion dissociation unit to generate the product ions. The product ion isolated within the linear ion trap can be transferred back to the ion dissociation unit and subsequently extracted from the apparatus for introduction into downstream components of a mass spectrometer in which the ion reaction apparatus is incorporated.

[0095] Without any loss of generality, in the following description of various embodiments of the present teachings, it is assumed that the precursor ions are positively charged. But the present teachings apply equally to negatively-charged ions, e.g., via inverting the polarities of the DC bias voltages, with the exception of the DC bias voltages associated with electron source bias and e-144907-7108-5162, v. 1gate electrode. The electron source bias can be configured relative to the L-shaped electrodes in the same manner as that for positively-charged ions so as to obtain a desired electron kinetic energy. The ON and OFF states of e-gate electrode are also biased the same as that for the case of positively-charged ions.

[0096] FIG. 1A schematically depicts an apparatus 100 according to an embodiment for use in a mass spectrometer. As discussed in more detail below, in various embodiments, the apparatus 100 can be used to cause dissociation of precursor ions into product ions. More specifically, in this embodiment, the apparatus 100 includes an ion dissociation device 102, which can be ionelectron reaction device (e.g., an electron activated dissociation (EAD)), which includes two sets of L-shaped rods 104 and 106, which are separated axially relative to one another to form an ion dissociation volume (region) 108, such as an electron-ion reaction region, therebetween.

[0097] With reference to FIGS. 1A and IB, in this embodiment, the rod set 104 includes four rods 104a, 104b, 104c, and 104d, each of which has a generally L-shaped configuration characterized by an axial segment and a transverse segment (e.g., the axial segment 104aL and the transverse segment 104aT). Similarly, the rod set 106 includes four rods 106a, 106b, 106c, and 106d, each of which has a generally L-shaped configuration characterized by an axial segment and a transverse segment (e.g., the axial segment 106aL and the transverse segment 106aT). In this embodiment, the longitudinal and transverse segments of each rod have a convex surface.

[0098] Both rod sets 104 / 106 are arranged relative to one another according to a quadrupolar configuration and provide a longitudinal channel 110, formed by the axial segments of the rods, and a transverse channel 112, formed by the transverse segments of the rods. The longitudinal channel 110 extends between an inlet 110a and an outlet 110b along an axial axis (LA) (i.e., an axis extending from the inlet to the outlet of the longitudinal channel at the center thereof), where a plurality of precursor ions can be introduced into the longitudinal channel 110 via the inlet 110a. The outlet 110b can, in turn, allow ions, e.g., a target product ion (i.e., a product ion of interest) and any remaining precursor ions, to exit the apparatus. An ion lens IQ2A positioned in proximity of the inlet of the longitudinal channel facilitates the introduction of the precursor ions into the longitudinal channel and an ion lens IQ2B positioned in proximity of the outlet of the longitudinal channel facilitates the extraction of the product ions from the longitudinal channel.154907-7108-5162, v. 1

[0099] An RF voltage source (not shown in this figure) can apply RF signals to the rod sets 104 and 106 so as to generate a quadrupolar electromagnetic field for providing radial confinement of the ions within the longitudinal channel 110 as well as the transverse channel 112. A DC voltage source (not shown in this figure) can apply a DC bias voltage to the two sets of the L-shaped quadrupole rods.

[0100] With continued reference to FIG. 1A as well as FIG. IB, in this embodiment, the apparatus 100 further includes a pair of T-shaped electrodes 121 and 122 that are positioned, respectively, within the upstream and the downstream segments of the longitudinal channel 110, where each T-shaped electrode includes a base (121b and 122b) and two stems (121s and 122s) that extend from the base and penetrate at least partially into the longitudinal channel towards the longitudinal axis (i.e., an axis extending at the center of the longitudinal channel from the inlet of the channel to its outlet). In some embodiments, the tip of the stems can be positioned at a distance between about 3-8 mm, from the longitudinal axis (LA).

[0101] With particular reference to FIG. 1A, the transverse channel 112 includes an inlet 112a through which an electron beam can be introduced into the transverse channel to propagate to the ion-electron reaction region to interact with the precursor ions to generate product ions. More specifically, an electron- emitting device 124 includes a cathode 124a that can emit electrons in response to application of a voltage thereto. A gate electrode (herein also referred to as e-gate, or first gate) is positioned in front of the cathode and is maintained at a positive DC potential relative to the cathode to facilitate the extraction of the electrons from the cathode. A first magnet 126 can focus the electrons into an electron beam 128 for entry into the transverse channel 112. In this embodiment, the magnet 126 and 208 is a permanent magnet, though in other embodiments it can be an electromagnet. By way of example, and without limitation, each of the magnets 126 and 208 can be a neodymium magnet. In other embodiments, rather than utilizing an electron beam for causing the dissociation of the precursor ions, the precursor ions can be dissociated via collision induced dissociation.

[0102] In particular, in this embodiment, the electron beam passes through openings provided by the gate electrode (e-gate) and a pole electrode (pole 1) to enter the transverse channel 112. The application of voltages to the gate electrode and the pole electrode can facilitate the introduction of the electron beam into the transverse channel 112. In this embodiment, the pole164907-7108-5162, v. 1electrode (pole 1) is DC-biased at a voltage greater than that applied to the set of the L-shaped electrodes so as to confine the precursor ions and product ions when they reach the pole electrodes. In this embodiment, T bar electrode (121) is DC-biased at a voltage greater than that applied to the set of the L-shaped electrodes so as to confine the precursor ions and product ions when they reach the trap center. A DC bias voltage applied to the gate electrode (e-gate) is set higher than that of the cathode bias to extract electrons from the electron source (= electron beam ON) or set lower than that of the cathode bias to prevent electron emission (= electron beam OFF).

[0103] The product ions generated within the ion-electron reaction region via electron-ion interactions or generated via collision- induced dissociation can exit outlet 112b of the transverse channel 112 to be received by a linear ion trap 200. A pole electrode (pole 2) is positioned between the outlet 112b of the transverse channel and an inlet of the linear ion trap 200. The application of a DC voltage to the pole electrode (pole 2) via a DC voltage source (not shown in the figure) can bias the pole electrode (pole 2) negative (i.e., lower than the bias of the electron source) so as to reflect the electron beam back to the electron-ion reaction region. Such negative bias applied to Pole 2 can be used to collect positively charged ions produced by ionization of a gas (such as nitrogen gas) introduced into an enclosure in which the apparatus is positioned and any residual gas by the electron beam. This prevents the entry of positively-charged product ions into the linear ion trap 200. Further, the DC bias of the linear ion trap may be more positive than the electron beam path to prevent the positively charged unwanted ions from entering the linear ion trap.

[0104] With reference to FIG. 1A as well as FIG. 1C, in this embodiment, the linear ion trap 200 includes four electrodes 204a, 204b, 204C, and 204D (herein collectively referred to as electrodes or rods 204), which extend from a proximal end (in proximity of the inlet of the linear ion trap) to a distal end (in proximity of the outlet of the linear ion trap) and are arranged relative to one another in a quadrupole configuration to provide a passageway therebetween for receiving the product ions via the outlet 112b of the transverse channel 112. The product ions can enter the passageway between the four electrodes 204 via an opening provided at the proximal end of the electrodes.

[0105] A terminal electrode 206 (herein also referred to with the acronym “D wall”) is positioned in proximity of the distal end of the electrodes 204. The application of a DC voltage to the electrode 206 can facilitate axial trapping of the product ions within the passageway.174907-7108-5162, v. 1

[0106] A second magnet 208 at least partially surrounds the quadrupole rods 204 of the linear ion trap. Similar to the first magnet 126, the second magnet 208 can be a permanent magnet or an electromagnet. By way of example, and without limitation, the second magnet 208 can be a neodymium magnet.

[0107] The combination of the first and the second magnets can cooperatively generate a magnetic field that is oriented substantially along the longitudinal axis of the transverse channel (a central axis extending between the inlet and the outlet of the transverse channel). In various embodiments, the resultant magnetic field generated via the superposition of the magnetic fields provided by the two magnets can have a strength in a range of about 0.2 Tesla to about 0.5 Tesla. Further, the use of both magnets can enhance the uniformity of the magnetic field along the transverse channel, which can in turn improve the confinement of the electron beam within the transverse channel.

[0108] As discussed in more detail below, the magnetic field within the passageway formed between the quadrupole rods 204 of the linear ion trap can, however, adversely affect the functioning of the linear ion trap. Accordingly, in this embodiment, the quadrupole rods 204 are formed of a paramagnetic material so as to substantially shield the passageway between the quadrupole rods 204 from the magnetic fields generated by first and the second magnets. In other words, the paramagnetic quadrupole rods of the linear ion trap ensure that the space between the quadrupole rods is substantially free of a magnetic field. By way of example, the paramagnetic rods 204 can shield the space between the quadrupole rods 204 such that any residual magnetic field remaining in the space between the quadrupole rods would be less than about 0.1 Tesla, and preferably zero.

[0109] A variety of paramagnetic materials can be utilized for forming the quadrupole rods 204. Some examples of such paramagnetic materials include, without limitation, paramagnetic stainless steel and soft iron.

[0110] An RF voltage source (such as the RF voltage source 206 schematically illustrated in FIG. 2) can apply RF voltages to the quadrupole rods 204 to ensure radial confinement of the product ions received by the linear ion trap. Further, at least two DC voltage sources (such as the DC voltage sources 216 / 218 schematically depicted in FIG. 2) can apply a DC resolving voltage across two of the quadrupole rods to help isolate a target product ion (i.e., a product ion having a184907-7108-5162, v. 1target m / z value) from the set of the product ions that are received by the linear ion trap, as discussed in more detail below. The same or a different DC voltage source can also apply a DC bias voltage to the quadrupole rods to adjust a DC bias voltage between the linear ion trap and the two sets of the L-shaped quadrupole rods. Further, an AC voltage source can apply an AC voltage to at least one of the quadrupole rods 204 to facilitate the isolation of the target product ion.

[0111] FIG. ID schematically depicts another embodiment of an ion-electron reaction apparatus 100’ according to the present teachings, which is similar to the above apparatus 100 except that it further includes a gate electrode (gate-2) that is positioned between the pole electrode (pole 2) and the inlet of the quadrupole rod set 204 of the linear ion trap.

[0112] In some embodiments, a DC bias voltage applied to the pole electrode (pole 2) can be selected to ensure that positive precursor and product ions are axially trapped in the electron beam path by positive DC biases applied to pole 1 and pole 2. Meanwhile, the gate electrode (gate 2) can be biased to cause reflection of the electron beam back to the ion-electron reaction region. Positively charged ions generated as a result of ionization of gas molecules (such as nitrogen molecules utilized in the apparatus for cooling of the ions and vacuum residual gas) are prevented from entering the linear ion trap because the negative bias on the gate 2 collects such positive ions. Further, the DC bias of the linear ion trap may be more positive than the electron beam path to prevent the positively charged unwanted ions from entering the linear ion trap.

[0113] FIG. 2 schematically depicts the application of RF, DC and AC voltages to the pole electrode (pole 2), the D wall as well as the quadrupole rods 204 of the linear ion trap 200. More specifically, in this example, an RF voltage source 206 can generate an RF voltage that is amplified by an RF amplifier 206a to generate an amplified RF voltage. The amplified RF voltage is in turn applied to a transformer 210, which generates two RF voltages having the same amplitudes but opposite phases for application to quadrupole rods 204 such that the phase of the RF voltages applied to one pair of radially opposed rods is 180 degrees different from the phase of the RF voltages applied to the other pair of radially opposed rods.

[0114] A DC voltage source 212 generates a DC bias voltage that is amplified by an amplifier 212a for application to the pole electrode (pole 2) and a DC voltage source 214 generates a DC bias voltage that is amplified by an amplifier 214a for application to the D wall. By way of example, and without limitation, the DC bias voltage applied to the pole electrode (pole 2) can be194907-7108-5162, v. 1in a range of about -10 volts (V) to about 60 volts relative to the L-shaped electrodes and the DC voltage applied to the D wall can be in a range of about 1 to about 30V relative to the linear quadrupole electrodes 204.

[0115] Further, DC voltage sources 216 / 218 can generate DC voltages that can be amplified by amplifiers 216a / 218a, respectively, for application to a pair of radially opposed rods of the quadrupole rods 204 to provide a DC voltage difference between those rods, which can act as a resolving DC voltage for facilitating the isolation of a target product ion, as discussed in more detail below.

[0116] Finally, an AC voltage source 220 can generate an AC voltage that can be amplified by an AC amplifier 220 to be applied to the one of the quadrupole rods 204. By way of example, and without limitation, the frequency of the AC voltage can be in a range of about 5 kHz to about 500 kHz and the amplitude of the AC voltage can be in a range of about 0 V to about 10 V.

[0117] As discussed in more detail below, the AC voltage can be utilized to obtain a high resolution isolation of a target product ion. For example, in various embodiments, the use of the RF voltage and the DC resolving voltage may result in an isolation window in a range of about 10 - 20 m / z. In such embodiments, the AC voltage can be utilized to reduce the size of the isolation window to about 1 m / z, thereby providing a much higher resolution for the isolation of the target product ion. Without being limited to any particular theory, the lower resolution achieved in the linear ion trap without the use of the AC voltage may be due to space charge interference of the trapped ions, the effect of a cooling gas that fills the trap, and / or the axial DC field used for ion confinement.

[0118] A controller 230 is in electrical communication with the RF and DC voltage source to control their operation, e.g., to activate and / or deactivate them for performing various operational steps, and / or set the amplitudes and / or frequencies of the voltages they generate.

[0119] With reference to FIGS. 3A, 3B, in use, in one example of an ion loading step (which is herein referred to as “normal loading”), a plurality of precursor ions can be introduced into the ion-electron reaction apparatus to be trapped within the ion-electron reaction region thereof. With particular reference to FIG. 3B, in this mode of ion loading, the application of a DC voltage to the electron gate (e-gate) prevents the entry of electrons into the transverse channel. Further, the204907-7108-5162, v. 1application of DC bias voltages to the IQ2A and IQ2B lenses and T-bar electrodes generates a DC potential minimum in the ion-electron reaction region for trapping a plurality of precursor ions entering the longitudinal axis of the apparatus via its inlet. More specifically, in this embodiment, the IQ2A electrode is biased at a DC voltage less than the DC voltage applied to the rods of an upstream mass filter QI (not shown in this figure), which may be present in certain embodiments of a mass spectrometer in which an ion-electron reaction apparatus according to the present teachings is incorporated, as discussed in more detail below.

[0120] In this example, the DC bias voltage applied to the QI mass filter is in turn lower than a DC bias voltage applied to an ion guide (e.g., DC bias voltage applied to a set of quadrupole rods of such an ion guide) positioned upstream of the QI mass filter. In this manner, the DC voltages applied to the Q0, QI, and IQ2A electrodes provide a DC potential ramp that causes acceleration of positive precursor ions and hence facilitate their entry into the longitudinal channel of the ionelectron reaction apparatus. In this mode of loading the apparatus with precursor ions, the DC bias of the L-shaped quadrupole rod sets relative to the DC voltage applied to the IQ2A lens is not sufficient to accelerate the ions to a kinetic energy that would result in collisional dissociation of those ions as they collide with molecules of a gas, such as nitrogen, within the apparatus, though such a gas can cause cooling of the ions received by the apparatus. By way of example, and without limitation, the energy of ions entering the apparatus during the normal mode of loading can be in a range of about 1 eV to about 5 eV for majority of molecular species.

[0121] Upon entry of ions into the ion-electron reaction apparatus, the DC voltages applied to the IQ2A and IQ2B electrodes provide axial confinement of the precursor ions within the longitudinal channel and a DC voltage applied to the T-bar electrodes helps retain the precursor ions within the ion-electron reaction region. Further, RF voltages applied to the two sets of the quadrupole rods can provide radial confinement of the precursor ions in a manner well known in the art.

[0122] With reference to FIG. 3C, in another mode of loading ions into the electron-ion apparatus, sufficient kinetic energy is imparted to the ions entering the apparatus so as to cause their dissociation via collisions with the gas molecules (typically nitrogen) within a housing in which the ion-electron reaction apparatus is positioned. In particular, in this example, the difference between the DC bias voltage applied to the L-shaped quadrupole rods and the DC214907-7108-5162, v. 1voltage applied to the IQ2A lens is greater than the respective DC voltage difference in the normal mode of loading discussed above in connection with FIG. 3B. In fact, the DC voltage difference in this example is sufficiently high (e.g., in a range of about 5 eV to about 100 eV depending on the molecular species) to accelerate the precursor ions to an energy at which the collision of the accelerated ions with the background gas molecules can cause dissociation of the accelerated ions to generate a plurality of product ions. In other words, in such an embodiment, rather than utilizing an electron beam to cause dissociation of the precursor ions to form product ions, the collision of the ions with the background gas molecules leads to the dissociation of the ions to generate product ions as the first dissociation in tandem mass spectrometry.

[0123] In the normal mode of ion loading, subsequent to loading of the precursor ions into the electron-ion reaction device, the trapped precursor ions can be exposed to an electron beam, e.g., to cause electron activated dissociation (EAD) of at least a portion of the precursor ions. FIGS. 4A and 4B schematically show an example of application of RF and DC voltages to various components of the electron-ion reaction apparatus to allow exposure of the trapped precursor ions to the electron beam. More specifically, in this example, the DC voltage applied to the e-gate electrode is set at a positive voltage relative to the electron source to extract electrons from the electron source, which is a thermalized electron emitter cathode. The DC voltage applied to the pole electrode (pole 1) is adjusted to confine the precursor and the product ions in the reaction device.

[0124] In this example, the interaction of the electron beam with the trapped precursor ions can cause electron activated dissociation (e.g., electron capture dissociation) of at least a portion of the precursor ions to generate a plurality of product (fragment) ions. The T-bar DC volage is adjusted to ensure that the product ions and any remaining precursor ions stay within the electronion reaction region. The DC voltage applied to pole electrode 2 is adjusted to a negative DC voltage to cause the repulsion of the electrons and hence their back propagation to the electron-ion interaction region. Further, the DC bias voltage applied to the quadrupole rods of the linear ion trap is greater than the DC bias voltage applied to any portion of the electron beam path to ensure that the electron ionization products from the nitrogen gas and the residual gas are not introduced into the linear ion trap.224907-7108-5162, v. 1

[0125] FIGS. 5A and 5B show an example of exposure of the precursor ions trapped within the electron-ion interaction region to an electron beam in an embodiment of an apparatus having a gate electrode between the outlet of the transverse channel and the pole electrode (pole 2). Similar adjustment of bias voltages as those discussed above in connection with FIGS. 4A and 4B can be performed to allow the introduction of the electron beam into the electron-ion interaction region of the apparatus except that in this case the pole electrode (pole 2) is maintained at a higher positive potential to keep the precursor and product ions in the reaction region. The DC voltage at e-gate electrode is adjusted so that the e-gate electrode is maintained at a negative potential so as to reflect the electrons back to the electron-ion reaction region. Further, the DC bias voltage applied to the quadrupole rods of the linear ion trap is greater than the DC bias voltage applied to any portions of the electron beam path to ensure that the electron ionization products from the nitrogen gas and the residual gas are not introduced into the linear ion trap.

[0126] Subsequent to the interaction of the precursor ions with the electron beam or beam type CID described in FIG. 3C, the product ions are transferred to the linear ion trap. The e-gate is closed via application of an appropriate bias voltage thereto so as to stop the flow of electrons into the transverse channel. By way of example, FIGS. 6A and 6B schematically depict one way of transferring the product ions into the linear ion trap. More specifically, in this example, the voltage applied to the pole electrode (pole 2) is adjusted such that pole electrode 2 is at a lower potential than the potential associated with the L-shaped electrodes and the T-bar electrodes and the DC bias voltage applied to the quadruple rods of linear ion trap is adjusted such that these quadrupole rods are at a lower potential than the pole electrode 2. This provides a DC potential ramp that causes the transfer of the product ions from the ion-electron reaction region to the linear ion trap. The DC bias voltage applied to the D wall electrode inhibits the leakage of the product ions out of the linear ion trap. Further, the RF voltages applied to the quadrupole rods of the linear ion trap provide radial confinement of the received product ions.

[0127] Subsequent to the completion of the transfer of the product ions into the linear ion trap, a target ion species of interest is isolated and the other ions are discarded. By way of example, FIGS. 7A and 7B schematically depict one example of isolating a target ion of interest by using the Mathieu stability parameters. For example, the RF voltage and the DC resolving voltage applied to the rods of the linear ion trap can be adjusted, e.g., by keeping the RF voltage fixed at234907-7108-5162, v. 1q=0.706 while scanning the DC voltage, such that the target ion of interest can be stably maintained within the ion trap while other ions will experience unstable trajectories that result in their removal from the trap, for example, via collisions with the quadruple rods of the linear ion trap. For example, the RF voltage and the resolving DC voltage can be selected such that the a-q parameters associated with the target ion of interest would correspond to the apex of the stability region.

[0128] By way of another example, FIGS. 8A and 8B depict the use of the AC voltage applied to one of the rods of the linear ion trap for isolating a target ion of interest within the linear ion trap. By way of example, subsequent to the use of a-q parameters to discard unwanted product ions, in some cases, some unwanted product ions may still remain within the ion trap. In some such cases, the AC voltage can be tuned to the resonant frequency of an unwanted ion remaining in the linear ion trap to cause its radial excitation so as to discard the ion, for example, via its impact with the quadrupole rods of the linear ion trap. If multiple unwanted ions remain to be discarded, the AC voltage can be tuned successively to the resonant frequencies of those ions to cause their removal from the linear ion trap.

[0129] Alternatively, with respect to FIGS. 9A and 9B, a plurality of AC voltages at different frequencies (herein also referred to as white noise) can be applied concurrently to the rods of the quadrupole rod set of the linear ion trap to cause excitation of a plurality of unwanted product ions so as to discard those ions from the linear ion trap via the radial excitation thereof and their impact with the rods of the quadruple rod set. A notch corresponding to the resonant frequency of the target product ion is, however, provided in the white noise to ensure that the target product ion remains stably trapped within the linear ion trap.

[0130] By way of another example, FIGS. 10A and 10B schematically depict the use of collision induced dissociation (CID) using resonant excitation. The frequency of the AC field is selected to excite the oscillation motion of the target ions. The amplitude of the AC field is set lower than the amplitude needed for resonant ejection of ions so as not to eject the ions but resonantly excite them via gradual absorption of kinetic energy due to collisions with the gas molecules (nitrogen gas in many application) into their vibrational excited states. When the vibrational states become highly excited, the ions are dissociated.

[0131] Subsequent to isolation of a target product ion of interest within the linear ion trap that product ion is transferred to the ion electron reaction region between the two L-shaped quadruple244907-7108-5162, v. 1rod sets while the electron beam is prevented from entering the transverse channel. By way of example, with reference to FIGS. 11 A and 11B, such a transfer of the isolated product ion from the linear ion trap to the electron-ion reaction region can be achieved by lowering the DC potential of the pole electrode 2 such that it is lower than the bias voltage applied to the quadruple rods of the linear ion trap and higher than the bias voltage applied to the L-shaped quadrupole rods so as to cause the product ions to exit the linear ion trap and enter the transverse channel of the dissociation device. The pole 1 electrode is maintained at a higher potential than the L-shaped quadrupole rods to inhibit the leakage of the product ions out of the dissociation device via the inlet of the transverse channel.

[0132] Following the transfer of the product ions from the linear ion trap to the reaction device, the product ions can be extracted from the reaction device to be transferred to downstream components of a mass spectrometer in which the apparatus is incorporated. With reference to FIGS, 12A and 12B, for extraction of the product ions from the apparatus, the voltage applied to the exit electrode IQ2B is adjusted to be lower than the DC potential of the exit electrode relative to the DC potential of the L-shaped electrodes to allow the product ions to exit the reaction device while maintaining the DC potential of the exit electrode IQ2B higher than the DC bias voltage of a subsequent component, e.g., the DC bias voltage applied to the rods of a collision cell Q2 in this example. The step-wise reduction in the DC potential between the L-shaped quadrupole rods and the collision cells causes the extraction of the product ions from the reaction device and their transfer to the collision cell in which the extracted product ions can undergo collisional cooling.

[0133] In some embodiments, during the extraction of the product ions from the reaction device, a voltage differential between the exit electrode and a downstream collision cell is sufficiently high to impart enough kinetic energy to the product ions as they are transferred to the collision cell so as to cause their collisional dissociation within the collision cell. By way of illustration, FIG. 12C schematically depicts such an embodiment in which the DC voltage applied to the rods of the collision cell Q2 positioned downstream of the ion-electron reaction apparatus is adjusted to be negative relative to the DC voltage applied to the exit electrode IQ2B so as to cause ion extraction to Q2. The ions accelerated by the bias difference between the ion-electron reaction device and Q2 that they may have sufficient kinetic energy to undergo CID within the collision cell Q2. By way of example, the difference between the voltages applied to the ion-254907-7108-5162, v. 1electron reaction device and the collision cell (more specifically the rods of an ion guide within the collision cell) can be in a range of about 5 eV to about 60 eV to induce collisional dissociation. In various embodiments, the voltage difference can be optimized based on the molecular species.

[0134] An ion-electron apparatus according to various embodiments of the present teachings can be incorporated into a variety of mass spectrometers. By way of illustration, FIG. 14A schematically depicts such a mass spectrometer 1400 that includes an ion source 1402, e.g., an electrospray ion source, which can receive a sample and ionize one or more analytes within the sample to generate a plurality of precursor ions that pass through the orifices of a skimmer plate 1404 and a curtain plate 1406 to reach an ion guide QJet, where the QJet ion guide includes a set of rods 1408 arranged in a quadrupole configuration, two of which 1408a / 1408b are visible in the figure and employs a combination of gas dynamics and radio frequency fields to cause focusing of the ions. The ions exiting the QJet ion guide are received by an ion guide Q0 that includes a set of quadrupole rods 1410, two of which 1410a / 1410b are visible in the figure, to which RF voltages can be applied for causing radial confinement of the ions and generate an ion beam that is in turn received by an ion mass filter QI. The ion guides QJet, Q0, and the mass filter QI are disposed in differentially-pumped chambers that are maintained at progressively lower pressures.

[0135] An ion lens IQ0 focuses the ions exiting the Q0 ion guide into the mass filter QI. The mass filter QI includes a stubby lens 1412 that includes a set of quadrupole rods (two of which 1412a / 1412b are visible in the figure) to which an RF field can be applied to cause focusing of the ions. The mass filter QI further includes a set of quadrupole rods 1414, two of which 1414a / 1414b are visible in the figure, to which a combination of RF and DC voltages can be applied to allow the selection of a precursor ion having a particular m / z ratio for transmission to a downstream electron-ion reaction apparatus 100 according to an embodiment of the present teachings, via passage through a stubby lens 1416 having a set of quadrupole rods (two of which 1416a / 1416b are visible in the figure) to which RF voltages can be applied. The product ion of interest exiting the electron-ion reaction apparatus 100 is received by a downstream collision cell Q2, which includes two sets of quadrupole rods 1418 / 1420 to which RF voltages can be applied for providing radial confinement of the ions. In this example, both the ion-electron reaction device 100 and the collision cell Q2 are positioned within an enclosure provided by a housing 1421 that is pressurized264907-7108-5162, v. 1via introduction of nitrogen gas to allow collisional cooling of ions, such as the product ions received by the collision cell Q2.

[0136] The ions exiting the cell Q2 are received by a time-of-flight (ToF) mass analyzer 1422, which can provide mass analysis of those ions. An analysis module 1424 receives the ion detection signals generated by the TOF mass analyzer and processes those signals to generate a mass spectrum.

[0137] FIG. 13B schematically depicts another mass spectrometer 1500 according to another embodiment, which is identical in all respects to the mass spectrometer 1400 discussed above, except that this embodiment includes an ion reaction apparatus (such as the ion reaction apparatus 100’ discussed in connection with FIG. ID) in which a gate electrode is positioned between pole electrode 2 and the quadrupole rods of the linear ion trap.

[0138] A controller and an analysis module for use in the practice of the present teachings (such as the controller 230 depicted in FIG. 2 or the analysis module 1424 depicted in FIGS. 13A and 13B) can be implemented using hardware, firmware and / or software. By way of example, in various embodiments, the controller and the analysis module can be implemented as a computer system described below.

[0139] By way of example, FIG. 22 is a block diagram that illustrates such a computer system 1000, upon which embodiments of the present teachings may be implemented. Computer system 1000 includes a bus 1022 or other communication mechanism for communicating information, and a processor 1042 coupled with bus 1020 for processing information. Computer system 1000 also includes a memory 1060, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus 1020 for determining base calls, and instructions to be executed by processor 1040. Memory 1060 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 1040. Computer system 1000 further includes a read only memory (ROM) 1080 or other static storage device coupled to bus 1020 for storing static information and instructions for processor 1040. A storage device 1100, such as a magnetic disk or optical disk, is provided and coupled to bus 1020 for storing information and instructions.274907-7108-5162, v. 1

[0140] Computer system 1000 may be coupled via bus 1020 to a display 1120, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 1140, including alphanumeric and other keys, is coupled to bus 1020 for communicating information and command selections to processor 1040. Another type of user input device is cursor control 1160, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 1040 and for controlling cursor movement on display 1120. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

[0141] Consistent with certain implementations of the present teachings, results are provided by computer system 1000 in response to processor 1040 executing one or more sequences of one or more instructions contained in memory 1060. Such instructions may be read into memory 1060 from another computer-readable medium, such as storage device 1100. Execution of the sequences of instructions contained in memory 1060 causes processor 1040 to perform the process described herein. Alternatively hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0142] The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to processor 1040 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device 1100. Volatile media includes dynamic memory, such as memory 1060. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 1020.

[0143] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, papertape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.284907-7108-5162, v. 1

[0144] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 1040 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 1000 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 1020 can receive the data carried in the infra-red signal and place the data on bus 1020. Bus 1020 carries the data to memory 1060, from which processor 1040 retrieves and executes the instructions. The instructions received by memory 1060 may optionally be stored on storage device 1100 either before or after execution by processor 1040.

[0145] In accordance with various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0146] The following examples are provided to further elucidate various aspects of the present teachings and may not necessarily indicate an optimal way of practicing the present teachings and / or the optimal results that may be obtained.

[0147] Examples

[0148] A free-electron-based dissociation device, or an EAD device, according to an embodiment of the present teachings was developed and installed in a commercially available TOF MS instrument, 7600 ZenoTOF (SCIEX). The EAD device is a six-way-cross branched radiofrequency ion trap with magnets. The first branch was used for ion introduction from a quadrupole mass filter, and the second branch was used for product extraction to the TOF mass spectrometer. The third and fourth branches were used for electron beam introduction. The fifth and sixth branches were closed by DC potentials applied to T-bar electrodes.294907-7108-5162, v. 1

[0149] For further molecular structural analysis, multi-stage tandem mass spectrometry combined with multiple dissociation techniques, MS(n), can be useful. Isolation of a dissociation product is often applied between two consecutive dissociations.

[0150] In this example, an electron beam source installed in a permanent magnet was removed to install a linear RFQ ion trap for product isolation. The linear RFQ trap is herein referred to as a "Dilated" trap, or its abbreviation “D trap.” Using this EAD-D trap combination, an efficient nearcomplete structural identification of cardiolipins (CLs) was demonstrated. Cardiolipins (l',3'-Bis- (l,2-diacyl-sn-glycero-3-phospho)-sn-glycerol) have four acyl groups. Thus, it is challenging to analyze detailed chain structure of these compounds by a single application of CID or EED. In a conventional MS(3) workflow, CID followed by UVPD in the positive ionization mode and UVPD in the negative ionization mode have been utilized to characterize native CLs, determining each chain length and double bond positions (or cyclic structure) and regioisomerism (i.e., sn isomerism). (Anal. Chem. 2019, 91, 12509-12516). Minor CID products with a sodiated glycerol backbone and two acyl chains were selected to identify sn isomerism, but their intensities can be 1 / 300 of major CID products. To enhance such CID products, two phosphate groups were derivatized. Cis / trans isomerism at the double bond and chirality at the center carbon in the center glycerol group were still missing.

[0151] In this example, an EED methodology is described that can identify the structure of glycerophospholipids nearly completely, including cis / trans identification in a double bond. This method is fully applicable to major sodiated CID products with Phospho-Glycerol-Phospho "head group." Applying EED on isolated CID products with a PGP head group demonstrated a near complete structural identification of cardiolipins in a single MS(3) experiment in positive mode in this work as follows.

[0152] Samples and ionization

[0153] CL18:1 standard and E. Coli cardiolipin extract from Avanti Polar lipid (Alabaster,Alabama) were obtained. Both samples were provided in triply sodiated form. No further purification or desalting process was applied to the samples.

[0154] A solvent of methanol (50%v): dichloromethane (50%v) that contained 2 mM of ammonium acetate was used as the working solution. The concentration of the working solution304907-7108-5162, v. 1was 10 pg / mL for the CL18: 1 standard and 50 pg / mL for the E. Coli extract. The working solution was ionized by a Turbo V ESI source (SCIEX) in infusion. The infusion flow rate was 5-10 pL / min.

[0155] ESI condition was carefully tuned to reduce contamination of dimerized precursor ions and ammonium acetate adducts. ESI voltage (5500 V), Gas 1 and Gas 2 (25 psi), and curtain gas (35 psi) provided the optimized condition, which were standard values for many compounds, but the temperature of Gas 2 (400 °C) and declustering potential (DP, 300 V) were essential, which were the maximum value allowed by 7600 ZenoTOF (SCIEX).

[0156] D trap: linear RFQ ion trap stored in a magnet

[0157] A linear ion trap was installed in a magnet in the EAD device for isolation purposes, as shown in FIG. 1A. As noted above this ion trap is herein referred to as a "Dilated trap" or "D trap." This trap was installed in a commercial-grade 7600 ZenoTOF. The electron gate (e- gate) in the D trap side was removed.

[0158] The quadrupole rods of the D trap had a length of 27 mm. The radius of the linear quadrupole (distance of the rod from the center axis), ro, was 2.14 mm. A wall electrode, which has a conical shape, was placed at another side of the EAD device. Pole 2 was shared with the EAD trap as another wall electrode. The D trap was filled with Nitrogen gas. The D trap was stored in a gas-tight holder except for the hole on pole 2, so the nitrogen gas pressure was almost the same as the EAD device. RF voltage with a frequency of 760 kHz was applied to the D trap. Its amplitude was adjustable from 0 V to 600 V peak-to-peak for each RF phase. Applicable quadrupole DC voltage or resolving DC was 0 to ±60 V for each quadrupole rod pair. The RF amplitude was calibrated to represent the apex of the stability diagram for a m / z value. The quadrupole DC or resolving DC is in voltage [V] to show the difference from the zero-resolving for each quadrupole rod pair.

[0159] An AC voltage source was used to apply a dipole supplemental AC voltage to the D trap for resonant ejection. This resonant ejection was used to select a CID product from a pair with one double bond difference in an E. Coli CL identification experiment.

[0160] The D trap and the EAD were controlled using a LabView-based driver software programmed in-house. For this purpose, the EAD electronics initially controlled by SCIEX OS were detached from the EAD trap. The 7600 ZenoTOF was driven in CID mode using SCIEX OS314907-7108-5162, v. 1software for precursor ionization, biasing of the front end (Turbo V source - QI), precursor isolation using QI, Q2 cell (nitrogen gas-filled CID device), and mass analysis by the TOF analyzer. A Lab View-based driver allows a flexible combination of CID, EAD, and product isolation. Nitrogen gas flow (or CAD gas flow) was controlled by a mass flow controller.

[0161] Stability diagram of the D trap

[0162] In the experiments, ions were isolated using the apex of the a-q stability diagram of the Mathieu equation. For good isolation resolution, the apex should be as sharp as possible. A weak magnetic field (2 ml) can help increase the Q filter's isolation resolution, but a field that is too strong decreases the resolution. Because the linear ion trap was installed in a strong magnetic field of 0.4 T, the magnetic field may have an influence on isolation resolution, if not shieled. A conventional choice for the rod material is non- magnetic stainless steel, such as SS304, but to reduce the magnetic field inside the D trap, paramagnetic rods were also made from SS410. It was confirmed that the paramagnetic rods function as a magnetic shield.

[0163] Isolated positive ions with m / z of 830.5 (1st13C species of ALILTLVS peptide) in the tuning solution (product number: 5049910, SCIEX) were used for the test. Ions were loaded into the D trap at the fixed RF amplitude with zero resolving DC; then, the RF amplitude was changed to a target value. A target resolving DC was applied for 2 ms, then the resolving DC was set at zero. Finally, the RF amplitude was set at the original value for the ion loading to release the remaining ions to the TOF mass analyzer. The number of remaining ions was monitored by the TOF analyzer. The target RF amplitude was ramped from zero to 1500 volts at a fixed resolving DC value. The resolving DC value was changed step by step. A supplemental dipole excitation voltage at P=0.7276 (276.491 kHz) was also applied to obtain the stability diagrams depicted in FIGS. 14A- 14F

[0164] FIGS. 14A-14C show the stability diagram when non-magnetic rods (made of SS304 stainless steel) were used in the D trap. The overall boundary shape is similar to the standard a-q diagram of a linear radio-frequency quadrupole. The test ions were removed from the trap on the dipole resonant line; however, the resonant line was split into two and curled around a=0 or the resolving DC zero (See, FIG. 14C).324907-7108-5162, v. 1

[0165] The split and curled dipole resonant line was reproduced by ion motion simulation. When the resolving DC voltage was zero and the magnetic field was also zero, the secular motions in the x direction and y direction had the same frequency; however, the non-zero magnetic field degenerates the secular frequencies and provides different frequencies. From the distance of the two resonant profiles, the magnetic field in the D trap was calculated as 0.33 T, which showed a good agreement with the nominal magnetic field strength of the magnet used in the EAD device.

[0166] FIGS. 14D - 14F show the stability diagram when the paramagnetic rods (made of S410 stainless steel) were used. The overall shape was similar to the standard a-q diagram of a linear radio-frequency quadrupole. The resonant line was continuous across a=0, though a small distortion was observed (FIG. 14F). From the distance of the two resonant frequencies at a=0, the magnetic field strength was calculated to be 0.14 T.

[0167] Hexapole instability observed in the paramagnetic rod assembly near the apex in parameter a was positive in FIG. 14D, and on the line: (m / z: 900, -2V)-(m / z: 960, 4V) in FIG. 14E. To avoid this hexapole instability, the negative side of the stability diagram was used in the a-q apex isolation described next. A significant difference in the shape of the apexes on the negative side in FIG. 14B and FIG. 14E.

[0168] Isolation using the a-q apex

[0169] The resolving DC was scanned simultaneously with the RF amplitude near the a-q stability apex to obtain mass spectra. This method is equivalent to isolation using a quadrupole filter (FIG. 15). For the solid peaks, the TOF spectrum was obtained without resolving DC voltage, i.e., the test ions were trapped and released from the D trap. For the pink curves (peaks shown in cross lines), the trapped ions were mass analyzed (or isolated) at the a-q apex in the D trap. For a direct comparison of the two methods, each peak height in the TOF spectrum was normalized to show the total intensity of the molecular species, which is the sum of the13C series. The resolution of the D trap was tuned to obtain nearly half transmission to the non-resolved intensities that are shown in the heights of the solid peaks. FW-HM of peaks were 18 in m / z for m / z = 609 and 21 for m / z = 829.

[0170] The resolution was strongly affected by buffer nitrogen gas pressure, the total number of trapped ions before isolation, and molecular species and their charge states. The resolution was334907-7108-5162, v. 1improved when the gas introduction flow was set lower (0.1 standard-cubic centimeter or seem) than 0.3 seem. The peak widths of 732(2+) and 1026(2+) were wider than other singly charged ions (FIG. 15). This suggests that the motion of the ions is strongly affected by the collision crosssection between the ions and buffer gas, and a large collision cross-section makes the resolution worse. Another observation is that m / z: 829 species (ALILTLVS peptides) were lost easily for a long isolation duration. This shows that the peptide ions were dissociated by a large micro motion near the bl boundary in the CID manner and lost from the trap.

[0171] This method isolated one carbon difference with nearly half ion transmission in the following cardiolipin analysis.

[0172] Isolation using resonant excitation ejection

[0173] The ejection of unwanted ions after a-q apex isolation was used, if needed. The 0 value of the supplemental AC was 0.7276, equivalent to 276.491 kHz. A small resolving DC of -4V made the resonant peak width narrower. FIG. 16 shows dipolar resonant ejection profiles of m / z = 829.5, 830.5, and 831.5 at the lower m / z edge and the higher m / z edge.

[0174] When m / z: 829.5 was depleted, m / z: 831.5 was still nearly intact in the lower m / z edge. When m / z: 831.5 was depleted, m / z: 829.5 was still nearly intact in the high m / z edge. On both sides of the resonant ejection edges, a resolution of 2 was demonstrated, which can isolate a molecule from a molecule with a double- bond difference. In this example, this resonant ejection was applied only to Cardiolipin 65:3 to isolate a target species from one double bond different species.

[0175] Electronic excited dissociation (EED) or Electron Impact Excitation of Ions from Organics (EIEIO)

[0176] EED or EIEIO workflows are known. Many complex lipids can be ionized as singly charged species in the protonated or metal-adducted forms, such as sodiated and potassiated forms. EED works well in the EAD device utilized in this example for the structural identification of complex lipids. The EAD device in 7600 ZenoTOF was used without modification except for the installment of a D trap in the second electron emitter side.

[0177] MS(3)344907-7108-5162, v. 1

[0178] A beam-type CID was used before EAD for cardiolipin analysis. Isolated precursor ions by QI mass filter were introduced into the EAD device with high kinetic energy to induce CID. For this purpose, the DC bias of the front end (composed of the ion source, ion guides, and QI filter) was set at high bias (collision energy: CE = 50 eV in SCIEXOS), and the bias of the EAD cell was set at a low voltage (0V). In this setting, collisional activation energy was 70 eV. The CID products were accumulated inside the EAD device. The CID products were transferred to the D trap for isolation of a CID product. After isolation, the isolated CID products were brought back to the EAD device to irradiate them with the electron beam. The electron beam energy, Ke, was 10 eV. The EAD products were released in Q2 for TOF mass analysis.

[0179] Results and discussion (Near-complete structural identification of authentic CL standard)

[0180] FIG. 17A shows an MS spectrum of intact CL18:1 standard. In the MS spectrum, protonated, mono-sodiated, di-sodiated, and tri-sodiated precursors were confirmed. Mono-lyso CLs impurities were detected in the sample.

[0181] EED was applied to intact CL18: 1 standard with different sodiation levels. However, EED applied to intact CLs was not suitable for detailed structural analysis, i.e., four acyl chains were too complicated to obtain full information about regioisomerism and the number of double bonds, locations, and cis / trans isomerism. CID was tried to see if it would produce useful subunits for EED analysis. Further, the number of sodiation was evaluated to determine the optimal number for the following EED experiments to analyze the subunits.

[0182] CID applied to CL18: 1 standard

[0183] The mono-isotopic peak of each sodiation level of the precursor was isolated before applying beam-type CID (70 eV). FIGS. 17B-17E show the CID spectra of each sodiation level of the CL18: 1 standard.

[0184] In the protonated case (FIG. 17B), the precursor produced only one major CID product with a glycerol backbone and two acyl groups. The mono-sodiated precursor (FIG. 17C) produced two CID products. One has the glycerol backbone and two acyl groups are the same as (FIG. 17B), and another has a PGP head group, glycerol backbone, and two acyl groups. The di-sodiatated (FIG. 17D) and tri-sodiated precursor (FIG. 17E) provided more complicated peak profiles. If354907-7108-5162, v. 1EED provides detailed information when the 603 -type fragment is used, the protonated precursor is the best to obtain high efficiency and high confidence in the subunit structure. The mono- sodiated precursor is also promising when the 603-type fragment is not useful. The 877-type fragment can also be promising because it is major peak without accompanying peaks. Di-sodiated and tri-sodiated may introduce misidentification because their CID spectra contain many peaks with different intensity levels.

[0185] MS(3) of CL18: 1 standard

[0186] EED was applied to both 603-type and 877-type fragments to determine whether EED on these fragments would provide complete structural information of the subunits (See, FIGS. 18A, 18B and 18C). The CID products were transferred to the D trap to apply the a-q apex isolation of each target. The isolated CID products were brought back to the EAD device; then, the electron beam was applied. Electron beam energy (Ke) was 10 eV, which is the default value for complex lipid analysis. The reaction time was 10 ms, and the electron beam current was adjusted to consume 50-80% of the initial precursors. The EED products were released to the TOF mass analyzer through Q2. In the TOF mass analyzer, Zeno pulsing was ON to enhance signals. The TOF signal was accumulated for 1 minute. It is over-accumulation, but it provided good s / n and signal intensity statistics to allow confident data analysis for the proof-of-concept of MS(3) identification. In offline data processing, the electron impact (El) background produced from residual vacuum gas was subtracted. This subtraction did not cause the loss of real signal from CLs, because the El background is composed of hydrocarbons, but the produced fragments contain sodium and many oxygen atoms. TOF was capable of resolving the difference satisfactorily.

[0187] FIGS. 18A, 18B and 18C show EED spectra applied to the 603 fragments and the 899 fragments. The appearance of the EED spectrum of the 603 fragment (FIG. 18A) is different from regular glycerophospholipids. It is because this portion has a glycerol backbone and two acyl chains, but it does not have an electrically polar head group. Actually, the charge can be located in the acyl groups, as an intense fragment with m / z=265.25 was observed. The double bond position was indicated, but it was not easy to identify regioisomerism (assignment of acyl chains to sn-1 or sn-2 in the glycerol backbone.)

[0188] The appearance of the EED spectrum of the 877 fragment (FIG. 18B), on the other hand, is very similar to regular glycerophospholipids. The headgroup signal was (m / z=274.97).364907-7108-5162, v. 1The thin solid arrow is consistent with PGP (phospho-glycerol-phospho combination). Glycerol backbone diagnostic peaks (315.00 and 317.98, the dotted arrows) clearly appeared. This confirms it is not a sphingosine backbone, which should appear between the two arrows with 316 as a single peak. In the glycerol region (FIG. 18C), a single peak of the C-C cleavage at 582.237 (homolytic cleavage, radical), paired peaks of the C-0 cleavage at 595.246 (one hydrogen lost) and 596.254 (homolytic, radical), triplet peaks of the C-(C=O) at 611.241 (strong, one hydrogen lost), 612.249 (weak: homolytic, radical ), and 613.257 (strong, one hydrogen gained) were clearly observed. The C-C cleavage (the red arrow) is the regioisomer diagnostic peak, which provides information regarding the length and the number of double bonds in the sn-1 acyl chain. Precursor miz - the m / z of this diagnostic peak gives the length and the number of double bonds in the sn-1 acyl chain. In the acyl chain region (FIG. 18C), the double bond position is clearly indicated. At a glance, a V-shaped intensity profile has appeared at the double bond. For more detailed analysis, a 2H mass shift in the fragment m / z series appeared across the double bond. This feature is used intensively in the following E. Coli CL analysis, which is applicable to more complicated spectra.

[0189] A conventional structural identification strategy for glycerophospholipids is fully applicable when a mono-sodiated CL is selected and CID is applied to produce glycerophospholipid-like subunits with a sodiated PGP head group. The ammonium acetate solvent is promising for producing intense mono-sodiated precursors.

[0190] E. Coli cardiolipin extract

[0191] MS(3) was applied to mono-sodiated CLs in the E. Coli extract to show the usefulness of this strategy for analysis of native samples. A high temperature of gas 2 and high declustering potential help reduce dimerized precursor and acetate adduct ions, but still some contamination was observed. Additional collisional activation (i.e., CID) of 50 eV was applied to kill the remaining dimerized precursors and ammonium acetate and sodium acetate adduct ions (FIG. 19A). The relative intensity ratio of mono-isotopic peaks of each CL precursor in this MS spectrum was used for the calculation of the abundance of CLs in the extract.

[0192] CID as the 1st dissociation in the MS(3) experiments374907-7108-5162, v. 1

[0193] Next, CID was applied to each isolated precursor ion to form subunits. QI was used to isolate the mono-isotopic peak of each precursor ion with the unit m / z isolation in QI. Beam-type CID is applied in Q2 filled with nitrogen gas. Collision energy (CE) was 70 eV

[0194] FIGS. 19B-19K show the m / z region of the "877.49"-type fragment or the CID fragments with a sodiated PGP head group. One to three pairs have appeared in each precursor. The brute compositions in the subunit acyls are included in FIGS. 19B-19K, which were calculated from the m / z of the CID product, where it was assumed the PGP head group. Using the CID products, the brute acyl composition of each intact precursor was successfully reconstructed. Such peak pairs are shown in the green line connections. Interestingly, paired intensities are very similar. This suggests that the peak intensity is proportional to the abundance of different types of isomeric CL precursors. The intensity ratios of the different peak pairs were used for the calculation of the abundance of isomeric CLs.

[0195] EED as the 2nd dissociation in the MS(3) experiments

[0196] EED was applied to each subunit. The same workflow as the CL18:1 standard was applied. The CID products were transferred to the D trap. CID products with different numbers of carbons were isolated by the a-q apex isolation method. The difference in the number of double bonds (FIG. 19F) was isolated by the resonant ejection using the supplemental AC after rough a- q apex isolation. EED (Ke = 10 eV) was applied to an isolated CID product after transferring it to the EAD device. Electron beam current and duration of electron application were adjusted to consume about 60% of the precursor ions. Reaction time was 10-20 ms.

[0197] In TOF spectrum accumulation, Zeno pulsing was activated because some EED fragments were very weak, less than 1 count per ten seconds for the lowest abundant precursor ions. MS(3) TOF spectra were accumulated for 2 minutes to 20 minutes. Again, this is overaccumulation, but good s / n and good signal statistics were obtained for confident structural identification.

[0198] By way of illustration, FIGS. 20A-20E show the case of (g) CL65:2 precursor that appeared in FIG. 18G.

[0199] FIGS. 20A and 20B show the EED spectra of the two subunits, 823.46 [32: 1] and 837.47 [33:1], The regioisomer diagnostic peak has appeared in each spectrum. In FIG. 20A, the384907-7108-5162, v. 1sn diagnostic peak is 554.21, m / z difference from the precursor m / z (823.46) is 269.25, which indicates the sn-1 acyl is 16:0. The sn-2 is calculated using the sn-1 acyl as total (32: 1) - sn-l(16:0) = sn-2(16: l). In FIG. 20B, the sn diagnostic peak is 568.22, m / z difference from the precursor m / z (837.47) is 269.25, which indicates the sn-1 acyl is also 16:1. The sn-2 is calculated using the sn- 1 acyl as total (33: 1) - sn-l(16:0) = n-2(17:l).

[0200] FIGS. 20C and 20D show intensity profiles of the acyl chain fragments that appeared in FIGS. 20A and 20B, which provide the detailed structures of the acyl chains. Using known diagnostic rules, the double bond position can be determined to be at n-7, or 9 in FIG. 20C. This double bond is cis because the hydrogen lost fragment: m / z: 737.34 is much weaker than the homolytic cleaved radical fragment: 738.353. This subunit is identified as PGP [16:0 / 16: 1 (9Z)].

[0201] The 837.43 subunit was 17:1, but the intensity profile was quite different from the case of double bonds. One hydrogen is obtained by the neutral loss fragment side (the terminal side of the chain), which is in great contrast with the double bond case, i.e., two hydrogen loss. In FIG. 21D, the intensity of such one hydrogen gain product is shown in bars with hatching designated as 2, indicating a significant enhancement. This is the diagnostic rule of a cyclic structure (triangle) in fatty acids and acyl chains. This subunit is identified as PGP[16:0 / 17-cyclo], where 17-cyclo represents a 9,10-methylene-hexadecanoyl acyl group.

[0202] Using the above subunit analysis, the structure of CL65:2 in the E. Coli extract was reconstructed as shown in FIG. 20E, i.e., CL([16:0 / 16: l(9Z)] > [16:0 / 17-cyclo]). represents the remaining ambiguity of the chirality or permutation at the center glycerol group, T- or 3'-.

[0203] In the E. Coli extract, other types of acyl groups have appeared. For example, FIGS. 21A - 21D show unsaturated acyl groups that appeared in the E. Coli CL extract, other than the acyls that appeared in FIGS. 20A- 20E.

[0204] In FIG. 21A, the subunit is identified as PGP[16:0 / 18:l] using the sn diagnostic rules. The double bond position in 18: 1 was determined to be n-7 or the 11 position. This double bond was identified as trans because the hydrogen-lost peak intensity is similar to the 766.37 peak by homolytic cleavage. So, the full structure of this subunit is PGP[16:O / 18: 1(11E)]. The same analysis method was applied to the 35:1 subunit in FIG. 21B. It has a triangular structure similar394907-7108-5162, v. 1to 17:1, but the chain is longer. So, the complete structure of this subunit is PGP[16:0 / 19-cyclo], where 19-cyclo represents 11,12-methylene-octadecanoyl acyl group or lactobacilloyl acyl group.

[0205] Table 1 below summarizes the identified CLs in this example.Table 117-cyclo = 940-niethylene-bexadecanoyl29-cyc!o = 12,12-methyiene-octadecanoyl = iactobacilloyl

[0206] Interestingly, the detected acyls with the same length and the same number of double bonds have the same structure. For 17:1, for example, only cyclic-type acyls (17-cyclo) were found, but acyls with a double bond (17: 1) were not found.

[0207] In this example, near-complete structural identification of CLs was achieved, including conformation of the backbone section, i.e., phosphate-glycerol-phosphate "head group," each acyl group structure including the chain length, the double bond position and its cis / trans isomerism, triangular cyclic structure, and sn-regioisomers of each subunit.

[0208] In summary, an ion-electron reaction device, including an EAD device and a linear RFQ ion trap were constructed, where the linear ion trap was used for fragment isolation purposes. As discussed in detail above, in order to prevent interference of a magnetic field generated by a magnet, within which the linear ion trap was positioned, with ion motion within the linear ion trap,404907-7108-5162, v. 1the quadrupole rods of the linear ion trap were made from paramagnetic stainless steel to substantially shield the space between the quadrupole rods from the magnetic field.

[0209] The device was utilized to demonstrate a near-complete structural analysis of native cardiolipins. A mono-sodiated precursor is the most promising candidate for CL analysis, e.g., via application of EED (10 eV) after beam-type CID (70 eV) to a mono-sodiated CL precursor as MS(3) workflow.

[0210] The above descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.

[0211] Depending on certain implementation requirements, embodiments of the present teachings, the controller can be implemented in hardware, firmware and / or in software.

[0212] In some embodiments, the instructions for operating the optical system can be stored using a non-transitory storage medium such as a digital storage medium, for example a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable.

[0213] While various embodiments have been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; embodiments of the present disclosure are not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing embodiments of the present disclosure, from a study of the drawings, the disclosure, and the appended claims.

[0214] In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single processor or other processing414907-7108-5162, v. 1unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0215] Those having ordinary skill in the art will appreciate that various changes can be made to the above embodiments without departing from the scope of the present teachings.4907-7108-5162, v. 1

Claims

What is claimed is:

1. An apparatus for use in a mass spectrometer, comprising: an ion reaction device including: a longitudinal pathway and a transverse pathway intersecting the longitudinal pathway at an ion reaction region, said longitudinal pathway having an input port for receiving precursor ions and an output port through which ions can exit the longitudinal pathway and said transverse pathway having a transverse input port for receiving an electron beam for interacting with the precursor ions in said ion reaction region to generate product ions and a transverse output port through which the product ions can exit the transverse pathway, at least one magnet for generating a magnetic field for guiding the electron beam along the transverse pathway, said magnetic field extending along the transverse pathway and through said output port thereof to a region external to said transverse pathway, and a linear ion trap positioned at least partially within the magnetic field in said external region and having a plurality of electrodes arranged in a quadrupole configuration to provide a passageway for receiving the product ions exiting the transverse pathway through the transverse output port, wherein said plurality of electrodes of the linear ion trap includes a paramagnetic material, thereby substantially shielding said passageway from the magnetic field.

2. The apparatus of Claim 1, wherein said paramagnetic material comprises any of paramagnetic stainless steel and soft ion.

3. The apparatus of Claim 1, wherein said ion reaction device further comprises a first set of L-shaped electrodes arranged in a multipole configuration and a second set of L-shaped electrodes arranged in a multipole configuration, said first and said second set of L- shaped electrodes arranged relative to one another so as to provide said longitudinal and said transverse pathways, wherein optionally any of said first set and said second set of the L-shaped electrodes are arranged according to a quadrupole configuration.434907-7108-5162, v.

14. The apparatus of any one of Claims 1 - 3, further comprising at least an RF voltage source for application of a set of RF voltages to the quadrupole electrodes of the linear ion trap and at least a first DC voltage source for applying a resolving DC voltage to at least one of said quadrupole electrodes.

5. The apparatus of Claim 4, further comprising a controller in communication with said RF voltage source and said first DC voltage source for configuring the set of RF voltages and the resolving DC voltage for trapping a product ion with an isolation target mass-to- charge ratio (m / z) within said passageway.

6. The apparatus of Claim 5, wherein the controller is configured to control said RF voltage source and said first DC voltage source to generate the set of RF voltages and the resolving DC voltage such that the isolation target m / z corresponds to the apex of an a-q stability diagram of the Mathieu equation.

7. The apparatus of Claim 5, further comprising an AC voltage source for applying an AC voltage to at least one of the quadrupole rods of the linear ion trap for causing resonant excitation of unwanted product ions so as to discard said unwanted product ions from the linear ion trap.

8. The apparatus of Claim 5, further comprising an AC voltage source for applying an AC voltage to at least one of the quadrupole rods of the linear ion trap for causing resonant excitation of at least a portion of the ions received by the linear ion trap so as to cause collisional dissociation thereof.

9. The apparatus of any one of Claims 1-3, and 5-8, further comprising an electron source positioned in proximity of said transverse input port and providing the electron beam.

10. The apparatus of Claim 9, further comprising a first pole electrode positioned between the electron source and the transverse input port, said first pole electrode configured for444907-7108-5162, v. 1application of a first DC voltage thereto for controlling delivery of the electron beam into the transverse pathway.

11. The apparatus of Claim 10, further comprising a second pole electrode positioned between the transverse output port and a proximal, ion-receiving end of the linear ion trap and configured for application of a second DC voltage thereto for controlling propagation of the electron beam along said transverse pathway, delivery of the product ions into the passageway and retention of at least a portion of the product ions therein.

12. The apparatus of Claim 11, further comprising a gate electrode positioned between the second pole electrode and the plurality of rods of the linear ion trap, wherein the gate electrode is maintained at a DC potential configured to inhibit electrons and positive ions exiting the ion reaction device from colliding with said plurality of rods of the linear ion trap.

13. The apparatus of Claim 11, further comprising an end electrode positioned at a distal end of the passageway and configured for application of a third DC voltage thereto such that a potential difference between said end electrode and said second pole electrode and a potential difference between said end electrode and the quadrupole rods of the linear ion trap facilitate axial confinement of the product ions within the linear ion trap.

14. The apparatus of Claim 1, wherein said at least one magnet comprises two magnets positioned, respectively, in proximity of the input port and the output port of the transverse pathway.

15. An apparatus for use in a mass spectrometer, comprising: an ion reaction device configured to receive precursor ions and having an ion reaction region in which the received precursor ions interact with an electron beam to generate product ions, said ion reaction device further having at least one magnet for generating a magnetic field for guiding the electron beam, wherein said magnetic field extends to a region external to the ion reaction device; and454907-7108-5162, v. 1a linear ion trap positioned at least partially in the magnetic field in said region external to the reaction device, said linear ion trap having a plurality of rods arranged in a quadrupole configuration to provide a passageway for receiving the product ions from the ion reaction device, wherein said plurality of rods of the linear ion trap comprises a paramagnetic material for substantially shielding the passageway from the magnetic afield.

16. The apparatus of Claim 15, wherein said paramagnetic material comprises any of paramagnetic stainless steel and soft iron.

17. The apparatus of Claim 15, wherein the ion reaction device further comprises a first set of L-shaped electrodes arranged in a multipole configuration and a second set of L-shaped electrodes arranged in a multipole configuration, said first and said second set of L- shaped electrodes arranged relative to one another so as to provide a longitudinal pathway and a transverse pathway, wherein said longitudinal pathway includes an input port for receiving precursor ions and an output port through which ions can exit the longitudinal pathway and said transverse pathway includes a transverse input port for receiving the electron beam and a transverse output port through the product ions can exit the transverse pathway to reach the linear ion trap.

18. The apparatus of any one of Claims 15 - 17, further comprising at least an RF voltage source for application of a set of RF voltages to the quadrupole electrodes of the linear ion trap and at least a first DC voltage source for applying a resolving DC voltage to at least one of said quadrupole electrodes.

19. The apparatus of Claim 18, further comprising a controller in communication with said RF voltage source and said first DC voltage source for configuring the set of RF voltages and the resolving DC voltage for trapping a species of said product ions with an isolation target mass-to-charge ratio (m / z) within said passageway.

20. The apparatus of Claim 19, wherein the controller is configured to control said second RF voltage source and said first DC voltage source to generate the set of RF voltages and the464907-7108-5162, v. 1resolving DC voltage such that the isolation target m / z corresponds to the apex of an a-q stability diagram of the Mathieu equation.

21. The apparatus of Claim 20, further comprising an AC voltage source for applying an AC voltage to at least one of the quadrupole rods of the linear ion trap for causing resonant excitation of unwanted product ions so as to discard said unwanted product ions from the linear ion trap.

22. The apparatus of Claim 20, further comprising an AC voltage source for applying an AC voltage to at least one of the quadrupole rods of the linear ion trap for causing resonant excitation of at least a portion of the ions received by the linear ion trap so as to cause collisional dissociation thereof.

23. The apparatus of any one of Claims 16, 17, and 19 - 22, further comprising an electron source positioned in proximity of said transverse input port and providing the electron beam.

24. The apparatus of Claim 20, further comprising a first pole electrode positioned between the electron source and the transverse input port, said first pole electrode configured for application of a first DC voltage thereto for controlling delivery of the product ions into the passageway and retention of at least a portion of the product ions therein.

25. The apparatus of Claim 24, further comprising a gate electrode positioned between the second pole electrode and the quadrupole rods of the linear ion trap, wherein the gate electrode is maintained at a DC potential configured to inhibit electrons and positive ions exiting the ion reaction device from colliding with the quadrupole rods of the linear ion trap.

26. The apparatus of Claim 25, further comprising an end electrode positioned at a distal end of the passageway and configured for application of a third DC voltage thereto such that a potential difference between said end electrode and said second pole electrode and a potential difference between the end electrode and the quadrupole rods facilitate axial confinement of the product ions within the linear ion trap.474907-7108-5162, v.

127. A mass spectrometer, comprising: an ion source for receiving a sample and ionizing one or more analytes within the sample to generate a plurality of precursor ions; and an apparatus for receiving the precursor ions and generating a plurality of product ions, wherein said apparatus comprises: an ion reaction device including: a longitudinal pathway and a transverse pathway intersecting the longitudinal pathway at an ion reaction region, said longitudinal pathway having an input port for receiving precursor ions and an output port through which ions can exit the longitudinal pathway and said transverse pathway having an input port for receiving an electron beam for interacting with the precursor ions in said ion reaction region to generate product ions and a transverse output port through which the product ions can exit the transverse pathway, at least one magnet for generating a magnetic field for guiding the electron beam along the transverse pathway, said magnetic field extending along the transverse pathway and through said output port thereof to a region external to said transverse pathway, and a linear ion trap positioned at least partially within the magnetic field in said external region and having a plurality of electrodes arranged in a quadrupole configuration to provide a passageway for receiving the product ions exiting the transverse pathway through the output port thereof, wherein said plurality of electrodes of the linear ion trap includes a paramagnetic material, thereby substantially shielding said passageway from the magnetic field.

28. The mass spectrometer of Claim 27, further comprising a mass analyzer positioned downstream of said apparatus and configured to receive the product ions exiting the output port of the longitudinal pathway and generating ion detection signals.484907-7108-5162, v.

129. The mass spectrometer of Claim 28, further comprising an analysis module configured to receive and process the ion detection signals so as to generate a mass spectrum of the product ions.

30. The mass spectrometer of any one of Claims 28 and 29, wherein said mass analyzer comprises a time-of-flight mass analyzer.

31. The mass spectrometer of Claim 27, further comprising an ion fragmentation device positioned downstream of said apparatus for receiving at least a portion of the product ions and fragmenting said received product ions to generate a second set of product ions.

32. The mass spectrometer of Claim 31, further comprising a mass analyzer positioned downstream of said ion fragmentation device to receive said second set of product ions and generate mass detection signals for generating a mass spectrum of said second set of the product ions.4907-7108-5162, v. 1

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