Electron stimulated dissociation reaction device with ion isolation functionality in mass spectrometry

Abstract

In one aspect, a method of performing mass spectrometry is disclosed that includes ionizing a sample to generate a plurality of precursor ions, passing the precursor ions through a mass filter to select at least a subset of the ions, introducing the selected ions into a branching radio frequency (RF) ion trap and fragmenting at least some of the selected precursor ions in the ion trap to generate a first plurality of fragment ions. The method can further include isolating at least some of the first plurality of fragment ions in at least one branch of the branching RF ion trap, removing unwanted fragment ions, and releasing remaining ions from the at least one branch and fragmenting at least some of the selected precursor ions to generate a second plurality of fragment ions.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 63 / 051,683, filed July 14, 2020, and entitled "Electron Activation Dissociation Reaction Device with Ion Isolation Functionality in Mass Spectrometry," which is incorporated herein by reference in its entirety. (Technical field)

[0002] The present teachings generally relate to ion dissociation devices that can be incorporated into mass spectrometers to provide tandem MS(n) analysis of analytes. [Background technology]

[0003] Mass spectrometry (MS) is an analytical technique for determining the structure of test chemicals in both qualitative and quantitative applications. MS can be useful for identifying unknown compounds, determining the elemental composition within molecules, determining the structure of a compound by observing its fragmentation, and quantifying the amount of a specific chemical compound in a mixed sample. Mass spectrometers detect chemicals as ions, so that conversion of analytes to charged ions occurs during the sampling process.

[0004] Some mass spectrometers include ion reaction devices, such as collision-induced dissociation devices (CID devices) and electron capture dissociation devices (ECD devices), which It can be employed to cause ion fragmentation to enable obtaining additional structural information about the ion under investigation. The ECD devices with the best performance consist of non-quadrupole RF ion traps or branching RF ion traps (US10014166B2), however, such devices suffer from several drawbacks. For example, ECD devices only allow tandem mass spectrometry (MS / MS) workflows.

[0005] Thus, improved ion reaction devices for use in mass spectrometry and methods for performing multiple tandem mass spectrometry (MS) using such devices are disclosed. n There is a need for a method for performing Summary of the Invention [Means for solving the problem]

[0006] In one aspect, a method of performing mass spectrometry is disclosed that includes ionizing a sample to generate a plurality of precursor ions, passing the precursor ions through a mass filter to select at least a subset of the ions, introducing the selected ions into a branching radio frequency (RF) ion trap, and fragmenting at least some of the selected precursor ions in the ion trap by electron chromatographic deposition (ECD) or chemical electron deposition (CID) to generate a first plurality of fragment ions. The method can further include isolating at least some of the first plurality of fragment ions, releasing the first plurality of fragment ions (or at least some of them) into an interaction region of the ion trap, and fragmenting at least some of them to generate a second plurality of fragment ions. The branching RF ion trap can include an axial section having a trap center and four branches extending laterally from the trap center.

[0007] Isolating at least some of the first plurality of fragment ions can include causing the at least some of the first plurality of fragment ions to enter at least one, typically two, branches of the branched RF ion trap. For example, a DC voltage can be applied to isolation electrodes positioned proximate the lateral branches to cause the at least some of the first plurality of fragment ions to enter at least one of the branches. In some embodiments, the isolation electrodes extend from the proximal end to the distal end of the axial section.

[0008] In some embodiments, a resolving DC voltage can be applied to at least one of the transverse branches in which fragment ions are trapped to remove unwanted fragment ions from the trapped fragment ions (e.g., fragment ions with m / z ratios outside a predefined stability range of the desired fragment ions). The remaining trapped ions in the transverse branches can then be released by adjusting the DC voltage applied to the isolation electrodes. The released ions can undergo a second fragmentation near the trapping center of the RF ion trap.

[0009] In some embodiments, the precursor ions are fragmented using either collision-induced dissociation (CID) or electron capture dissociation (ECD). Additionally, in some embodiments, the first plurality of fragment ions are further fragmented using either CID or ECD.

[0010] In some embodiments, any of the precursor ions and the first plurality of fragment ions are dissociated by electron-induced dissociation (EAD) using an electron beam having an energy in the range of about 0 eV to about 50 eV, including ECD, high-temperature ECD, electron ionization dissociation, electron-induced dissociation, electron impact excitation of ions from organics (EIEIO), negative ion electron capture dissociation (niECD), and electron detachment dissociation (EDD).

[0011] The second plurality of fragment ions can be passed through any type of mass analyzer to generate their mass spectrum. In some embodiments, the mass analyzer can be a time-of-flight (ToF) mass analyzer.

[0012] In a related aspect, a mass spectrometer is disclosed that includes an ion reaction device that includes a branched radio frequency (RF) ion trap that includes eight L-shaped rods positioned axially at a distance from each other to provide an axial section characterized by a central axis for receiving and extracting ions from an ion source and two branch sections extending laterally from a central portion of the axial section and characterized by transverse axes for receiving electrons from an electron source. The mass spectrometer further includes a source for generating electrons such that the electrons enter the ion reaction device along the transverse axis of the ion trap to interact with ions received along the central axis near the central portion of the axial section to cause fragmentation of at least some of the ions to generate a first set of fragment ions. In some embodiments, a magnetic field is applied along the transverse axis to help direct electrons received via the transverse axis to the center of the trap. An isolation electrode is positioned near the branch sections to cause transfer of at least a portion of the first set of fragment ions from the central section to at least one of the branch sections and isolate the ions transferred to the at least one of the branch sections. Furthermore, a DC voltage source is provided to apply a DC voltage to at least one of the L-shaped rods to remove unwanted ions from the set of fragment ions isolated in at least one of the lateral branch sections. The remaining fragment ions can be released from the branch sections by reducing the DC voltage applied to the isolation electrode. The mass spectrometer can further include a gas introduction system (not shown) for introducing a gas into the ion reaction device. In some embodiments, the gas can be helium, nitrogen, or neon. In some embodiments, the gas can include one or more reactive molecules, such as oxygen. A typical pressure of the gas inside the ion reaction device can be a small amount, for example, in the range of about 1 to about 10 mTorr.

[0013] Another DC voltage source can be provided for applying a DC voltage to the isolation electrodes to cause the transfer of at least a portion of the first set of fragment ions into the at least one of the branching lateral sections.

[0014] The mass spectrometer may further include an RF voltage source for applying an RF voltage to the L-shaped rods to radially confine the precursor ions and fragment ions.

[0015] A mass analyzer can be positioned downstream of the ion reaction device to receive the second plurality of fragment ions and generate a mass spectrum thereof. In some embodiments, the mass analyzer can be a time-of-flight (ToF) mass analyzer. The present invention provides, for example, the following items. (Item 1) 1. A method of performing mass spectrometry, said method comprising: ionizing the sample to generate a plurality of precursor ions; mass filtering the precursor ions and selecting at least a subset of the ions; introducing the selected ions into a branching radio frequency (RF) ion trap and causing at least a portion of the selected precursor ions to undergo fragmentation within the ion trap to generate a first plurality of fragment ions; isolating at least a portion of the first plurality of fragment ions; releasing at least some of the isolated ions and causing at least some of the ions to fragment to generate a second plurality of fragment ions; A method comprising: (Item 2) Item 10. The method of item 1, wherein the branched RF ion trap comprises an axial section characterized by a central axis, the axial section having a trap center and four branches extending from the trap center. (Item 3) 3. The method of claim 2, wherein two of the branches are positioned laterally relative to the central axis. (Item 4) 4. The method of claim 3, wherein the step of isolating at least some of the first plurality of fragment ions comprises causing the at least some of the first plurality of fragment ions to enter at least one of the lateral branches. (Item 5) 5. The method of claim 4, wherein the step of causing at least some of the first plurality of fragment ions to enter one of the lateral branches comprises applying a DC voltage to an isolation electrode positioned proximate the branch. (Item 6) Item 6. The method of item 5, wherein the isolation electrode extends from the proximal end to the distal end of the axial section. (Item 7) 6. The method of claim 5, further comprising removing unwanted fragment ions from the fragment ions in the at least one transverse branch by applying a resolving DC voltage to the at least one transverse branch. (Item 8) 8. The method of claim 7, wherein the step of releasing the selected isolated ions comprises adjusting a DC voltage applied to the isolation electrode. (Item 9) 9. The method of claim 8, wherein the released ions undergo the second fragmentation near the trapping center. (Item 10) Item 2. The method according to item 1, wherein the precursor ion and any of the first plurality of fragment ions are dissociated by electron induced dissociation using an electron beam having an energy in a range of about 0 eV to about 50 eV. (Item 11) Item 11. The method of item 10, further comprising passing the second plurality of fragment ions through a mass analyzer to generate a mass spectrum thereof. (Item 12) Item 12. The method of item 11, wherein the mass analyzer comprises a time-of-flight mass analyzer. (Item 13) Item 1, wherein the precursor ions are fragmented using either collision-induced dissociation (CID) or electron-induced dissociation (EAD). (Item 14) Item 14. The method of item 13, wherein the first plurality of fragment ions are fragmented using either CID or EAD. (Item 15) 1. A mass spectrometer, the mass spectrometer comprising an ion reaction device; The ion reaction device a branched radio frequency (RF) ion trap comprising eight L-shaped rods positioned axially at a distance from one another to provide an axial section, the axial section being characterized by a central axis for receiving ions from an ion source, the branched RF ion trap comprising two branch sections extending laterally from a central portion of the axial section, the branch sections being characterized by transverse axes for receiving electrons from an electron source; a source for generating electrons such that the electrons enter the ion reaction device along the transverse axis, the electrons interacting with ions received along the central axis near the central portion of the axial section to cause fragmentation of at least some of the ions and generate a first set of fragment ions; an isolation electrode positioned proximate the branch sections to cause transfer of at least a portion of the first set of fragment ions from the central portion to at least one of the branch sections and to isolate the ions transferred to the at least one of the branch sections; and a DC voltage source for applying a DC voltage to at least one of the L-shaped rods; Equipped with applying the DC voltage induces mass selective instability on at least some of the isolated first set of fragment ions in the at least one branch section and removes unwanted ions from the first set of fragment ions. (Item 16) Item 16. The mass spectrometer of item 15, further comprising an electron source positioned to provide a beam of electrons along the lateral axis. (Item 17) 17. The mass spectrometer of claim 16, further comprising a device for generating a magnetic field parallel to the transverse axis. (Item 18) 16. The mass spectrometer of claim 15, further comprising another DC voltage source for applying a DC voltage to the isolation electrode to cause transfer of the at least some of the first set of ion fragments into the at least one of the branch sections. (Item 19) 16. The mass spectrometer of claim 15, further comprising an RF voltage source for applying an RF voltage to at least one of the L-shaped rods to radially confine the precursor ions and the ion fragments. (Item 20) Item 18. The mass spectrometer of item 17, further comprising a mass analyzer disposed downstream of the ion reaction device for receiving the second set of ion fragments and generating a mass spectrum thereof. (Item 21) Item 19. The mass spectrometer of item 18, wherein the mass analyzer comprises a time-of-flight (ToF) mass analyzer. [Brief explanation of the drawings]

[0016] [Figure 1] 1A and 1B schematically depict an ion reaction device according to an embodiment of the present teachings.

[0017] [Figure 2] FIG. 2 depicts a schematic representation of an ion reaction device operating in a conventional manner.

[0018] [Figure 3] FIG. 3 illustrates a schematic of the isolation of fragment ions in a lateral branch of an ion reaction device according to an embodiment of the present teachings.

[0019] [Figure 4A] FIG. 4A is another schematic diagram of an ion reaction device operating in a conventional manner.

[0020] [Figure 4B] FIG. 4B shows islands of stability and instability for ions with m / z=609 trapped in the ion reaction device depicted in FIG. 4A as a function of applied RF confinement and DC resolving voltages.

[0021] [Figure 5A] FIG. 5A is a schematic diagram of an ion reaction device according to an embodiment of the present teachings.

[0022] [Figure 5B] FIG. 5B shows islands of stability and instability for an ion with m / z=609 trapped in the ion reaction device depicted in FIG. 5A as a function of applied RF confinement and DC resolving voltages illustrating the region of stability for certain values ​​of RF and DC voltages.

[0023] [Figure 6] FIG. 6 illustrates a schematic of a mass spectrometer incorporating ion reactions according to an embodiment of the present teachings.

[0024] [Figure 7A] FIG. 7A shows the mass spectrum of a pair of isomeric glycopeptides separated by liquid chromatography.

[0025] [Figure 7B]FIG. 7B shows the mass spectrum of the isolated glycopeptide without any dissociation.

[0026] [Figure 7C] FIG. 7C shows the mass spectrum of the CID product of the isolated glycopeptide.

[0027] [Figure 7D] FIG. 7D shows the mass spectrum of the isolated CID product with m / z of 557.

[0028] [Figure 7E] Figure 7E shows fragments of an isolated glycopeptide observed by the MS(3) workflow in which CID and EIEIO were employed for ion fragmentation.

[0029] [Figure 8] 8A and 8B show the chemical structures of a pair of isomeric glycopeptides with different sialic acid linkages.

[0030] [Figure 9] FIG. 9 shows the CID-EIEIO spectrum of a standard verified to have alpha(2,3) linkages in a tryptic digest of fetuin, a protein from bovine.

[0031] [Figure 10] FIG. 10 shows another CID-EIEO spectrum of a standard verified to have an alpha(2,6) bond.

[0032] [Figure 11] FIG. 11 shows the identification of unknown linkages of sialic acid in glycopeptides contained in hen's egg yolk.

[0033] [Figure 12A] FIG. 12A depicts a schematic representation of multiple fragment ions at the center of an RF ion trap.

[0034] [Figure 12B] FIG. 12B depicts diagrammatically the application of a DC voltage to the T-bar electrodes that can cause fragment ion transport from the center of the RF ion trap to the lateral branches.

[0035] [Figure 12C] FIG. 12C diagrammatically depicts the removal of unwanted fragment ions from the set of fragment ions isolated in the lateral branches by application of a resolving DC voltage to the L-shaped rods.

[0036] [Figure 12D] FIG. 12D depicts diagrammatically that following removal of unwanted fragment ions, other ions remain trapped in the lateral branches.

[0037] [Figure 12E] FIG. 12E schematically depicts the removal of remaining fragment ions from the lateral branches into the trap center by reducing the DC voltage applied to the T-shaped isolation electrodes. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present teachings generally provide an RF ion reaction device that can be used to cause multiple fragmentation of precursor ions. In other words, the RF ion reaction device according to the present teachings allows for multiple tandem mass spectrometry, i.e., MS(n), to be performed, which can then enable a deeper understanding of the molecular structure under investigation. For example, the systems and methods disclosed herein can be employed to identify sialic acid linkages in glycans.

[0039] Various terms are used herein according to their ordinary meaning in the art. As used herein, the term "about" is intended to indicate a variation of up to 5 percent.

[0040] 1A and 1B, an ion reaction device 100 according to an embodiment of the present teachings includes an axial passage 102 extending along a central axis (CA) from a proximal end 102a, which provides an inlet port through which ions generated by an upstream ion source (not shown in this figure) can enter the ion reaction device, to a distal end 102b, which provides an outlet port through which ions can leave the ion reaction device. Proximate the proximal end 102a and the distal end 102b are mounted electrode gates 103a / 103b, respectively, which enable controlled entry of ions and ejection of ions from the ion reaction device substantially along the central axis (CA).

[0041] The ion reaction device 100 further includes a first set of electrodes 105 that are generally L-shaped and arranged around a central axis (CA). In the figure, only two of the four electrodes of the first set are shown. The other two electrodes are mounted directly behind the depicted L-shaped rod.

[0042] A second set of electrodes 107 (two of which are depicted and two directly behind the depicted electrodes) are mounted at a slight axial distance relative to the first set of electrodes in the direction of the electron beam path (TA) to create a quadrupole configuration.

[0043] The arrangement of the two sets of L-shaped electrodes 105 / 107 relative to each other provides, in addition to the axial path 102, two lateral branches 111 / 113 characterized by a lateral axis (TA). Two electrodes 115 / 116 positioned at each end of the lateral axis (TA) can serve to capture ions within the two lateral branches (e.g., ion fragments generated by fragmentation of multiple precursor ions, as discussed in further detail below). In this embodiment, the two electrodes 115 / 117 include openings 115a / 117a. In this embodiment, a filament 118 is positioned adjacent to the gate electrode 116, where the filament can be heated to generate electrons. An ion lens 115 is positioned downstream of the gate electrode 116. The electrons can enter the ion reaction device via an opening 115a provided in the ion lens 115. In some embodiments, electrons can be employed to cause fragmentation of ions in the vicinity of the trapping center by electron excited dissociation, as discussed in more detail below.

[0044] In addition, the ion reaction device 100 includes a T-shaped electrode 119 (also referred to herein as an isolation electrode) that extends along a central axis (CA) from the proximal end of the ion reaction device to its distal end and can be used to cause fragment ions generated near the center of the ion reaction device to enter at least one of the branches 111 / 113, as discussed in more detail below.

[0045] An RF voltage source 200 operating under the control of a controller 201 applies RF voltages to the quadrupole rods 105 / 107 to radially confine precursors and ion fragments near the central axis (CA) and transverse axis (TA) of the ion reaction device 100. In particular, the polarities of the RF signals applied to the first and second sets of electrodes 105 / 107 are selected so that the generated quadrupole field can confine ions (precursor and fragment ions) near the central axis (CA) and transverse axis (TA). In some embodiments, the frequency of the applied RF signal can be, for example, in the range of about 0.2 MHz to about 2 MHz, and the amplitude of the applied RF signal can be, for example, in the range of about 0 V to about 600 V.

[0046] Two DC voltage sources 300 and 302 apply dc voltages to the two sets of quadrupole rods 105 / 107 such that the dc voltages applied to any two rods in the two sets positioned diagonally relative to each other have the same sign, and the dc voltages applied to any two rods facing each other have opposite signs. For example, with continued reference to Figures 1A and 1B, in this embodiment, voltage source 300 applies a positive dc voltage to L-shaped rods 105a / 107a, and voltage source 302 applies a negative dc voltage to L-shaped rods 105b / 107b.

[0047] A DC voltage source 303 operating under the control of the controller 201 applies a DC voltage to the T-shaped electrode 119. As discussed in more detail below, the controller 201 can control the DC voltage applied to the T-shaped electrode 119 to trap fragment ions generated by fragmentation of multiple precursor ions in one or both of the branches 111 / 113, or to release the trapped fragment ions so that they may undergo further fragmentation near the trap center.

[0048] An ion reaction device according to the present teachings advantageously allows for the isolation of initially formed fragment ions, allowing those fragment ions to undergo further fragmentation. By way of illustration, FIG. 2 schematically depicts an ECD ion reaction device, where ion fragments can be formed near the trapping center. Because the electromagnetic field at the trapping center is not quadrupole (the field at the trapping center is affected by eight rods), it is not feasible to isolate ion fragments and select and release them based on their m / z ratios.

[0049] In contrast, the electromagnetic fields in the lateral branches of the depicted ECD ion reaction device are quadrupole, since they are generated by four side rods. Thus, ion fragments can be isolated in these branches based on their mass by inducing mass-dependent instabilities, as discussed in more detail below.

[0050] As a further illustration, FIG. 4A shows an ion reaction device according to the present teachings, such as the ion reaction device 100 described above, in which a 5-volt DC voltage is applied to the T-shaped electrode to cause ions in the center to be introduced into one or both lateral branches. Simulation results indicate that the applied voltage of 5 volts is not effective, particularly in achieving isolation of fragment ions within the lateral branches. FIG. 4B shows the regions of stability and instability for an ion having m / z=609 as a function of the amplitude of the RF signal and resolving DC voltage applied to the rods, illustrating the difficulty in isolating such an ion using a 5-volt voltage applied to the T-shaped electrode. In contrast, referring to FIGS. 5A and 5B, application of a 20-volt DC voltage to the T-shaped electrode can result in several combinations of RF signal amplitude and DC resolving voltage that can lead to stable isolation of ions in the lateral branches.

[0051] An ion reaction device according to the present teachings can be employed in a variety of mass spectrometers. By way of example, FIG. 6 schematically depicts a mass spectrometer 1000 according to an embodiment incorporating the ion reaction device 100. Without loss of generality, and for illustrative purposes only, FIG. 6 will be discussed in relation to the use of the mass spectrometer 1000 for glycopeptide sugar bond analysis.

[0052] The mass spectrometer 1000 includes a curtain plate 1002 and an orifice plate 1004 having openings 1002a / 1004a through which ions generated by an upstream ion source (not shown) are received. Various ion sources can be employed. Some examples of suitable ion sources include, but are not limited to, electrospray ionization devices, nebulizer-assisted electrospray devices, chemical ionization devices, nebulizer-assisted atomization devices, chemical ionization devices, matrix-assisted laser desorption / ionization (MALDI) ion sources, photoionization devices, laser ionization devices, thermospray ionization devices, inductively coupled plasma (ICP) ion sources, sonic spray ionization devices, glow discharge ion sources, and electron impact ion sources, among others.

[0053] Ion optics QJet, comprising four rods arranged in a quadrupole configuration, forms an ion beam for transmission to downstream components of the mass analyzer. Ion lens IQ0 separates the QJet region from ion guide Q0, which is also formed by a quadrupole arrangement of four rods. Ion guide Q0 can focus ions through a combination of aerodynamics and RF voltages applied to its rods. Ion lens IQ1 and quadrupole stub lens ST1 can focus ions as they pass from ion guide Q0 into mass filter Q1. In this embodiment, Q1 can function as a mass analyzer, allowing selection of ions with a selected m / z ratio (or range of m / z ratios) for passage to reaction-ion device 100 via passage through stub ST2 and entrance electrode 103a.

[0054] Ions exiting the ion reaction device 100 pass through the exit electrode 103b to reach the ion guide Q2, which is formed by a four-rod quadrupole arrangement. Q2 can function as a CID dissociation device and / or an ion guide to introduce ions into a downstream time-of-flight (TOF) mass analyzer. Ions can exit the Q2 ion guide via ion lens IQ3 to reach a downstream mass analyzer, such as a time-of-flight (ToF) mass analyzer.

[0055] In use, multiple precursor ions isolated by Q1 can be introduced into the ion reaction device via its input port. The precursor ions can undergo fragmentation near the trapping center to generate multiple fragment ions (referred to herein as first fragment ions to distinguish them from fragment ions generated by subsequent fragmentation of the first fragment ions, as discussed in more detail below). In some embodiments, fragmentation of the precursor ions at the trapping center can be achieved by collision-induced dissociation (CID). In other embodiments, fragmentation of the precursor ions (or at least some of them) can be achieved by electron-induced dissociation (EAD). For example, in this embodiment, a filament 118 disposed adjacent to a gate electrode 116 positioned at the distal end of the lateral branch 111 generates electrons that pass through the lateral branch 111 into the trapping center and cause at least some of the precursor ions to undergo electron-induced dissociation (EAD), generating the first multiple fragment ions.

[0056] FIG. 12A shows diagrammatically a number of fragment ions 2000 in the centre of the RF trap, including an unwanted portion (shown in grey in this illustration).

[0057] Referring to FIG. 12B, application of a suitable DC voltage to the T-shaped electrode 119 can cause the fragment ions 2000 (or at least a portion of them) to enter one or both of the lateral branches of the ion reaction device 100.

[0058] Referring to FIG. 12C, following the introduction of the first fragment ions into one or both of the transverse branches of the ion reaction device 100, the voltage source 200 can be activated to apply a resolving DC voltage to the ions isolated in those transverse branches to remove unwanted fragment ions (e.g., unwanted ions with m / z ratios outside a predefined m / z range including the target ions) using mass-induced instability, as indicated by the arrows. As shown in FIG. 12D, the remaining ions (i.e., target ions) are still trapped in at least one or both of the branches. Referring to FIG. 12E, these ions can be released from the transverse branches and introduced into the trapping center by lowering the DC voltage applied to the T-shaped electrodes. Due to mass-selective stability, the ions isolated in the branches are released into the trapping center independently of their mass. Before releasing the target ions from the branches, the resolving DC voltage is set to zero.

[0059] The selected first fragment ions introduced into the trapping center can undergo further fragmentation, for example by EAD, using electrons generated by the filament 118 to generate a second plurality of fragment ions. The second plurality of fragment ions can exit the ion reaction device and ion guide Q2 to reach a downstream ToF analyzer, which can generate a mass spectrum of the second plurality of fragment ions.

[0060] In some embodiments, when the selected first fragment ion is released from the EAD device, CID can be applied to the selected first fragment ion by applying a CID activation DC voltage between the EAD device and Q2.

[0061] As described above, an ion reaction device according to the present teachings enables MS(n) mass spectrometry to be performed. As an example, the following MS(3) workflow can be achieved using an ion reaction device according to the present teachings. MS (3): isolation (first MS by Q1) → CID → isolation of CID product (second MS) → CID → mass spectrometry (third MS); MS (3): isolation (first MS by Q1) → ECD → isolation of ECD (second MS) → CID → mass spectrometry (third MS); MS (3): isolation (first MS by Q1) → CID → isolation of CID product (second MS) → ECD → mass spectrometry (third MS), and MS (3): isolation (first MS by Q1) → ECD → isolation of ECD product (second MS) → ECD → mass spectrometry (third MS).

[0062] MS(4) workflows can also be achieved using an ion reaction device according to the present teachings. For example, the following MS(4) workflow can be achieved: MS (4): isolation (1st MS by Q1) → CID → isolation of CID products (2nd MS) → ECD → isolation of ECD products (3rd MS) → CID → mass spectrometry (4th MS).

[0063] The following examples are provided to further elucidate various aspects of the present teachings and are not necessarily intended to represent the best way to practice the invention or the best results that may be obtained. (Example)

[0064] A mass spectrometer similar to that shown in Figure 6 was employed to acquire the data described in this section. A pair of isomeric glycopeptides with structures shown in Figures 8A and 8B with different sialic acid linkages (alpha(2,3) and alpha(2,6)) were used as samples. Sialic acids are represented using diamonds, and linkages were differentiated by the orientation of the bond between the sialic acid (diamond) and the hexose (circle). The goal of the workflow in this example was to distinguish the two linkages in intact glycopeptides. In natural samples, such as digested proteins, the two linkages are mixed.

[0065] A mixed isomeric sample of two glycopeptides with different sialic acid linkages was separated using liquid chromatography (LC). Although such separation can be achieved by LC, it is not possible to distinguish between the types of sialic acid linkages using LC.

[0066] The separated glycopeptides were ionized using electrospray ionization (ESI), and Figure 7A shows the resulting mass spectrum.

[0067] Ions generated by ESI were introduced into the mass spectrometer through curtain plate 1002 and orifice plate 2004, and then into Q1 via QJet, IO0, Q0, IQ1, and ST1. Target glycopeptides with specific m / z ratios were isolated from impurities by the Q1 filter. Figure 7B shows the mass spectrum of the isolated glycopeptide without any dissociation.

[0068] The isolated glycopeptide was introduced into the EAD reaction device through ST2 and the lens electrode 103a. In this example, the first mode of dissociation was via CID. To induce collisional dissociation, the DC bias between the EAD reaction device 100 and the QJet was set to a high value; typically, the QJet bias was set +30 V higher than the EAD reaction device 100. Figure 7C shows the mass spectrum of the CID product of the isolated glycopeptide. As CID acts on the glycans in the glycopeptide, fragments associated with glycan fragments were generated.

[0069] To investigate the sialic acid linkage, a glycan fragment with m / z 557 (see FIG. 7C ) containing one of the linkages shown in FIG. 8A or 8B was selected. For the second dissociation, the fragment with m / z 557 was isolated using the method described herein. The entire CID product was stored in the electron beam branch by applying a voltage (or isolation voltage) of +20 V to the T-shaped electrode. A predetermined resolving DC voltage and a trapping RF signal with a predefined amplitude were applied to isolate the 557 m / z fragment. The resolving DC and trapping RF amplitudes were set to normal conditions, and the isolated CID fragment with m / z 557 was released into the center of the EAD reaction device 100 by setting the voltage applied to the T-shaped electrode to a normal value. FIG. 7D shows the mass spectrum of the isolated CID fragment.

[0070] In this example, the second fragmentation was achieved by electron impact excitation of ions from the organic compound (EIEIO). An electron beam with a kinetic energy of 10 eV was applied to the isolated fragment with m / z 557. EIEIO is a type of electron-induced dissociation that can be applied to singly charged ions. In the case of glycans, EIEIO induces cross-ring cleavage, which can distinguish sialic acid linkages (see Figure 6). As shown in Figure 7E, fragments obtained by CID and EIEIO were observed by the MS(3) workflow.

[0071] Figure 9 shows the CID-E1E10 spectrum of a standard verified to have an alpha(2,3) bond in a tryptic digest of fetuin, a protein from bovine. Figure 10 shows another CID-E1E10 spectrum of a standard verified to have an alpha(2,6) bond. By comparison between the two standard spectra, the m / z of the diagnostic peaks was obtained. For the alpha(2,3) bond diagnostic (Figure 8B), two peaks with m / z of 331.126+H and 348.128+H were found. For the alpha(2,6) bond diagnostic (Figure 8A), a peak with m / z of 305.111+H was found.

[0072] Figure 11 demonstrates the identification of an unknown sialic acid linkage in a glycopeptide contained in hen's egg yolk. The same workflow (CID followed by EIEIO) described above was applied. The spectral appearance is similar to that of the standard with a diagnostic peak associated with the alpha(2,6) linkage. Therefore, it can be concluded that the glycopeptide in the egg yolk has a sialic acid linkage as alpha(2,6).

[0073] Those skilled in the art will appreciate that various modifications can be made to the above-described embodiments without departing from the scope of the present invention, as defined by the following claims.

Claims

1. 1. A method of performing mass spectrometry, said method comprising: ionizing the sample to generate a plurality of precursor ions; passing the precursor ions through a mass filter to select at least a subset of the precursor ions; introducing the at least one subset of the precursor ions into a branching radio frequency (RF) ion trap and causing at least a portion of the at least one subset of the precursor ions to undergo fragmentation within the ion trap to generate a first plurality of fragment ions; isolating at least a portion of the first plurality of fragment ions; releasing at least some of the isolated ions and causing at least some of the isolated ions to undergo fragmentation to generate a second plurality of fragment ions; wherein the branched RF ion trap comprises an axial section characterized by a central axis, the axial section having a trap center and four branches extending from the trap center.

2. The method of claim 1 , wherein two of the branches are positioned transversely to the central axis.

3. 3. The method of claim 2, wherein said step of isolating at least some of said first plurality of fragment ions comprises causing said at least some of said first plurality of fragment ions to enter at least one of said lateral branches.

4. 4. The method of claim 3, wherein the step of causing the at least some of the first plurality of fragment ions to enter one of the lateral branches comprises applying a DC voltage to an isolation electrode positioned proximate the branch.

5. The method of claim 4 , wherein the isolation electrode extends from the proximal end to the distal end of the axial section.

6. 5. The method of claim 4, further comprising removing unwanted fragment ions from the first plurality of fragment ions in the at least one transverse branch by applying a resolving DC voltage to the at least one transverse branch.

7. The method of claim 6 , wherein the step of releasing the selected isolated ions comprises adjusting a DC voltage applied to the isolation electrode.

8. The method of claim 7 , wherein the released ions undergo the fragmentation near the trapping center.

9. 10. The method of claim 1, wherein the precursor ions and any of the first plurality of fragment ions are dissociated by electron induced dissociation using an electron beam having an energy in a range of about 0 eV to about 50 eV.

10. 10. The method of claim 9, further comprising passing the second plurality of fragment ions through a mass analyzer to generate a mass spectrum thereof.

11. The method of claim 10 , wherein the mass analyzer comprises a time-of-flight mass analyzer.

12. The method of claim 1 , wherein the precursor ions are fragmented using one of collision-induced dissociation (CID) and electron-induced dissociation (EAD).

13. The method of claim 12 , wherein the first plurality of fragment ions are fragmented using one of CID and EAD.

14. 1. A mass spectrometer, the mass spectrometer comprising an ion reaction device; The ion reaction device a branched radio frequency (RF) ion trap comprising eight L-shaped rods positioned axially at a distance from each other to provide an axial section and two branch sections, the axial section characterized by a central axis for receiving ions from an ion source, and the two branch sections extending laterally from a central portion of the axial section and characterized by lateral axes for receiving electrons from an electron source; a source for generating electrons such that the electrons enter the ion reaction device along the transverse axis, the electrons interacting with ions received along the central axis near the central portion of the axial section to cause fragmentation of at least some of the ions and generate a first set of fragment ions; an isolation electrode positioned proximate the branch sections to cause transfer of at least a portion of the first set of fragment ions from the central portion to at least one of the branch sections and to isolate the ions transferred to the at least one of the branch sections; and a DC voltage source for applying a DC voltage to at least one of the L-shaped rods; Equipped with applying the DC voltage induces mass selective instability on at least some of the isolated first set of fragment ions in the at least one branch section and removes unwanted ions from the first set of fragment ions.

15. 15. The mass spectrometer of claim 14, further comprising an electron source positioned to provide a beam of electrons along the lateral axis.

16. 16. The mass spectrometer of claim 15, further comprising a device for generating a magnetic field parallel to the transverse axis.

17. 15. The mass spectrometer of claim 14, further comprising another DC voltage source for applying a DC voltage to the isolation electrode to cause transfer of the at least some of the first set of fragment ions into the at least one of the branch sections.

18. 15. The mass spectrometer of claim 14, further comprising an RF voltage source for applying an RF voltage to at least one of the L-shaped rods to radially confine the ions and the first set of fragment ions received from the ion source.

19. A mass spectrometer as described in claim 16, wherein the ion reaction device releases at least a portion of the isolated first set of fragment ions in the at least one branch section, causes at least a portion of the fragment ions to undergo fragmentation, and generates a second set of fragment ions, and the mass spectrometer further comprises a mass analyzer positioned downstream of the ion reaction device for receiving the second set of fragment ions and generating a mass spectrum thereof.

20. 20. The mass spectrometer of claim 19, wherein the mass analyzer comprises a time-of-flight (ToF) mass analyzer.

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