Plasma EDD in mass spectrometry

Abstract

A method of performing negative electron activated dissociation (negative EAD) in mass spectrometry includes introducing a plurality of negatively charged analyte ions into an ion trap located in a chamber, and trapping the negatively charged analyte ions in a reaction region of the ion trap, introducing a buffer gas into the chamber, electrons are generated using an electron source located in the cavity and external to the ion trap, and the electrons are accelerated to form an electron beam, and the electron beam is introduced into the ion trap such that the accelerated electrons are capable of ionizing at least a portion of the molecules of the buffer gas to generate a plurality of positively charged ions. The accelerated electrons interact with at least a portion of the analyte ions captured in the reaction region to cause its negative EAD, thereby producing a plurality of fragment product ions.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 347,808, filed on June 1, 2022, entitled “Plasma EDD in Mass Spectrometry,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The following relates to mass spectrometers and, more particularly, to a mass spectrometer utilizing electron activated dissociation (EAD), including negative electron induced dissociation (EID) and electron separation dissociation (EDD) applied to negatively charged analyte ions. Background Art

[0004] Mass spectrometry (MS) is an analytical technique used to determine the elemental composition of a substance. Specifically, MS measures the mass-to-charge ratio (m / z) of ions produced by a test substance. MS can be used to identify unknown compounds, to determine the isotopic composition of elements in a molecule, to determine the structure of a specific compound by observing its fragmentation, and to quantify the amount of a specific compound in a sample. A mass spectrometer detects ions, and therefore, during mass analysis, the test sample must be converted into ionic form (i.e., precursor ions or analyte ions). Generally, a mass spectrometer includes at least an ion source, a mass analyzer, and an ion detector. The ion source converts the test sample into gaseous ions, the analyzer separates the gaseous ions based on the m / z ratio of the gaseous ions, and the detector detects the separated ions.

[0005] Mass spectrometer can use EAD to break analyte into smaller fragment ions (for example, fragments comprising independent core bases). Then the m / z ratio based on these smaller fragment ions can be used to carry out mass analysis and quantification to these smaller fragment ions. EDD acts on negatively charged multi-charged analyte ions, such as oligonucleotides, DNA, RNA etc. Negative EID acts on negatively charged singly charged analyte ions, such as acidic peptides, fatty acids and acidic complex lipids. As used herein, negative EAD comprises both EDD and negative EID. Summary of the invention

[0006] Aspects of the present disclosure address the issues disclosed herein and / or elsewhere.

[0007] In one aspect, a method for performing negative EAD in mass spectrometry is disclosed, which includes introducing a plurality of negatively charged analyte ions into an ion trap located in a cavity, and capturing the negatively charged analyte ions in a reaction region of the ion trap. A buffer gas may be introduced into the cavity, and an electron source located in the cavity and outside the ion trap may be used to generate electrons. Electrons may be accelerated to form an electron beam, which may be introduced into the ion trap so that the accelerated electrons can ionize at least a portion of the molecules of the buffer gas to generate a plurality of positively charged ions. In addition, the accelerated electrons interact with at least a portion of the negatively charged analyte ions captured in the reaction region of the ion trap to cause their negative EAD, thereby generating a plurality of fragment product ions. As discussed in more detail below, the positively charged ions may counteract the repulsive force exerted by the electrons on the negatively charged analyte ions to promote the negatively charged analyte ions to remain in the reaction region of the ion trap.

[0008] In some embodiments, the ion trap can be a branched RF ion trap having a longitudinal channel (also referred to herein as a longitudinal branch) extending from an inlet to an outlet, through which negatively charged analyte ions can enter the trap, and through which the fragment product ions can leave the ion trap. Such a branched RF ion trap can also include a transverse channel (also referred to herein as a transverse branch) intersecting the longitudinal channel in the reaction region, wherein the transverse channel has an inlet for receiving an electron beam. For example, the RF ion trap can include two groups of L-shaped rods axially separated from each other, wherein each group of L-shaped rods is arranged according to a multipole configuration, such as a quadrupole, hexapole, or octopole configuration.

[0009] In another aspect, a mass spectrometer includes an ion source for receiving a sample and ionizing one or more analytes in the sample to produce a plurality of negatively charged analyte ions, a cavity including a buffer gas and an ion trap for capturing the negatively charged analyte ions, an electron source located in the cavity and outside the ion trap for generating electrons, and a magnet located in the cavity for forming the electrons into an electron beam introduced into the ion trap. The electron beam is capable of ionizing at least a portion of the molecules of the buffer gas to produce a plurality of positively charged ions, and the electrons interact with at least a portion of the analyte ions to induce negative EAD thereof, thereby producing a plurality of fragment product ions.

[0010] In some embodiments, the ion source includes an electrospray ion source, and the mass spectrometer also includes a plurality of rods arranged in a multipole configuration to form an axial path and a transverse path perpendicular to the axial path, wherein negatively charged analyte ions are introduced into the ion trap via the axial path, and the electron beam is introduced into the ion trap via the transverse path. In some embodiments, the mass spectrometer also includes an RF voltage source for applying an RF voltage to a plurality of rods. A plurality of rods may include a first group of rods and a second group of rods arranged in a quadrupole configuration. In some embodiments, the rods are L-shaped.

[0011] In some embodiments, the positively charged ions include any one of nitrogen molecular ions, helium ions, neon ions, and krypton ions. In some such embodiments, the nitrogen molecular ions include N2 + and N2H + , the helium ions include He + , the neon ions include Ne + , and the krypton ions include Kr + In some embodiments, the electron kinetic energy is in the range of about 20 eV to about 90 eV, depending on the type of gas. The accelerated electrons may have a kinetic energy greater than about 20 eV, for example, in the range of about 20 eV to about 50 eV in the case of nitrogen, neon, or krypton. The accelerated electrons may have a kinetic energy greater than about 50 eV, for example, in the range of about 50 eV to about 90 eV in the case of helium. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Aspects of the disclosure may take form in various components and arrangements of components, and various steps and arrangements of steps.The drawings are only for the purpose of illustrating the preferred embodiments of the disclosure and are not to be construed as limiting.

[0013] Features of embodiments of the present disclosure will be more readily understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0014] Figure 1 schematically illustrates electrons of an electron beam and a negatively charged analyte according to an exemplary embodiment;

[0015] Figure 2 schematically illustrates a mass spectrometer according to an exemplary embodiment;

[0016] Figure 3 schematically illustrates accelerated electrons and positively charged ions according to an exemplary embodiment;

[0017] Figure 4 schematically illustrates negatively charged ions, positively charged ions, and electrons of an electron beam disposed within an ion trap according to an exemplary embodiment;

[0018] Figure 5 is a flow chart of a method for performing electron separation dissociation (EDD) in mass spectrometry according to an exemplary embodiment;

[0019] Figure 6 is an exemplary EDD spectrum generated using the plasma techniques disclosed herein; and

[0020] Figure 7 is an exemplary negative EID spectrum; and

[0021] Figure 8 A computer system according to an exemplary embodiment is schematically depicted.

[0022] Fig.9A is a schematic illustration of a branched RF ion trap MS with permanent magnets.

[0023] Fig. 9B Schematic depiction of the DC bias along the electron beam path in an EAD device.

[0024] Fig.10 Shows K e is the characteristic spectrum at 20 eV.

[0025] Fig.11A shows the generation of K in the charge reduction state e Dependency.

[0026] Fig. 11B Diagram showing the N2 generated in the EAD device + strength.

[0027] Fig. 12A The diagram shows the e Fragments and K values ​​at 18 eV and below e Dependency.

[0028] Fig. 12B It shows that in K e = Spectrum at 23 eV.

[0029] Fig. 12C It shows that in K e = 30eV spectrum.

[0030] Fig.12D It shows that in K e = 35eV spectrum.

[0031] Figures 13A-13D MSMS spectra at 0, 20, 40 and 60 ms are shown.

[0032] Fig.14 Normalized intensity data as a function of reaction time are shown.

[0033] Fig.15 The reaction time as a function of z is shown.

[0034] Fig.16 Shown using (1 / z) 1.3 Normalized strength of dependence versus reaction time.

[0035] Fig.17A is a diagram of the introduction of ions into the EAD cell, which are generated in the grid1-filament1 region.

[0036] Fig. 17B is a graphical representation of the negative ETD of DNA16-PO in the absence of mutual precursor-reagent capture in conventional ETD operation.

[0037] Fig. 17C The reaction rate of beam-type negative ETD is shown.

[0038] Figures 18A-18D is a graphical representation of the validation of the EDD model.

[0039] Fig.19 It is shown that a high energy electron beam can induce strong EDD.

[0040] Figures 20A-20H Shown are the EDD spectra of DNA20-PS in various charge states z.

[0041] Fig.21 and Figure 22A-22J The EDD spectra with fragment density plots for all fragments are shown.

[0042] Figure 23A-23C It was shown that when the PS moieties were substituted around the termini in DNA-PO (DNA20-POPS), the fragments cleaved at the PS moieties were 100 times stronger than those cleaved at PO.

[0043] Figure 24A-Figure 24E It was shown that when the PS moieties were substituted around the termini in DNA-PO (DNA20-POPS), the fragments cleaved at the PS moieties were 100 times stronger than those cleaved at PO.

[0044] Figure 25A-25B It is shown that even with weak strength at the PO portion, a . The spectrum was sequenced with w fragments.

[0045] Figure 26A-26C The graph shows that CID shows no significant PO / PS differences in dissociation efficiency.

[0046] Figure 27A-Figure 27E and Figure 28A-28H Shown is a fragment intensity plot of an oligonucleotide with a locked ribosulect near the terminus.

[0047] Fig.29 The plasma EDD spectrum is shown.

[0048] Figure 30A-Figure 30B and Figure 31A-Figure 31C The b, c, d, x, and y fragments generated via CID are illustrated.

[0049] Figure 32A-Figure 32C and Fig.33Shown is a survey of impurities in a decomposed GAP sample. DETAILED DESCRIPTION

[0050] It will be understood that, for the sake of clarity, the following discussion will explain various aspects of the embodiments of the present disclosure, while omitting certain specific details wherever it is convenient or appropriate to do so. For example, the discussion of the same or similar features in alternative embodiments may be omitted. For the sake of brevity, known ideas or concepts may also not be discussed in detail. It will be appreciated by those of ordinary skill in the art that some embodiments of the present disclosure may not require certain details of the details specifically described in each implementation, and these details are only set forth herein to provide a thorough understanding of the embodiments. Similarly, it will be clear that the described embodiments may be easily modified or varied according to common knowledge without departing from the scope of the present disclosure. The following detailed description of the embodiments should not be construed as limiting the scope of the applicant's teachings in any way.

[0051] As used herein, the terms "approximately" and "substantially" and "substantially equal" refer to changes in numerical quantities and / or complete states or conditions that may occur, for example, through measurements or processing processes in the real world; through unconscious errors in these processes; through differences in the manufacture, source or purity of compositions or reagents, etc. Typically, the terms "approximately" and "substantially" as used herein mean 10% larger or smaller than the value or range of values ​​or complete conditions or states. For example, a concentration value of about 30% or substantially equal to 30% may mean a concentration between 27% and 33%. These terms also refer to equivalent changes that will be recognized by those skilled in the art, as long as these changes do not cover known values ​​practiced by the prior art.

[0052] 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 " / ".

[0053] The present disclosure generally relates to mass spectrometers. As described above, mass spectrometers can use negative EAD to fragment analytes (e.g., oligonucleotides, DNA, RNA, acidic peptides, fatty acids, acidic complex lipids, etc.) into smaller fragment ions. Mass spectrometers using negative EAD retain negatively charged analyte ions in an ion trap. These mass spectrometers also use an electron beam that collides with the negatively charged analyte ions in the ion trap. The collision fragments the negatively charged analyte ions. Figure 1As depicted, an ion trap potential (or a pseudo potential generated by an RF field) 102 can be used to confine a plurality of negatively charged analyte ions 104 in a radial direction, and an electron beam can be introduced into the ion trap to interact with the trapped negatively charged analyte ions so as to fragment the negatively charged analyte ions via negative EAD. Electrons 106 in such an electron beam generate a potential 108 that can repel the negatively charged analyte ions 104, thereby reducing the number of collisions between the negatively charged analyte ions 104 and the electrons 106, and thus reducing the EDD efficiency of the negatively charged analyte ions. Therefore, there is a need for a negative EAD mass spectrometer with increased collision efficiency between the negatively charged analyte ions and the electron beam.

[0054] In one aspect, the present disclosure relates to methods for performing mass spectrometry and mass spectrometers that can be used to practice these methods. These methods include capturing multiple negatively charged analyte ions in an ion trap, and introducing an electron beam and positively charged ions into the ion trap. In some embodiments, introducing both the negatively charged electron beam and the positively charged ions can produce a substantially electrically neutral environment, i.e., a neutral plasma, in the trap. This neutral environment can promote the negatively charged analyte ions to remain in the reaction area (e.g., the center) of the ion trap, which can increase the number of collisions between the electrons of the electron beam and the negatively charged analyte ions.

[0055] Figure 2 A mass spectrometer 200 according to an exemplary embodiment is schematically depicted.

[0056] In this embodiment, the mass spectrometer 200 includes an electrospray ion source 202 that produces a plurality of negatively charged analyte ions 204. The ion source 202 is in communication with a sample holder (not shown) that provides analytes (e.g., oligonucleotides, etc.) to the ion source 202. The mass spectrometer 200 also includes a vacuum chamber 206 in communication with the ion source 202. The charged analyte ions 204 travel in the direction of arrow 208 and pass through the holes of the curtain plate 210 to enter the vacuum chamber 206.

[0057] Once within the cavity 206, the charged analyte ions 204 pass through a differential pumping region 212 disposed within the vacuum cavity 206. The differential pumping region 212 includes a plurality of rods 214, which in this embodiment are arranged in a quadrupole configuration.

[0058] The mass spectrometer 200 also includes an RF voltage source 216, a DC voltage source 218, and an AC voltage source 220, each of which is under the operation of a controller 222. The RF voltage source 216 can apply an RF voltage to the rods 214 to generate an RF electric field. The RF electric field, combined with gas dynamics, can focus the charged analyte ions 204 into an ion beam for transmission to downstream components of the mass spectrometer.

[0059] The charged analyte ions 204 pass through the ion guide (QJet) region 212 and are further focused by the IQ0 lens 224 and enter the vacuum chamber 226. The charged analyte ions 204 continue in the direction of arrow 208 and pass through the ion guide (Q0) in another differential pump region 228. In this embodiment, the ion guide includes four rods 230 ( Figure 2 The RF voltage source 216 is electrically connected to the rod 230 and provides an RF voltage to the rod 230 to generate an RF electric field for providing radial confinement of ions near the central axis of the rod 230.

[0060] The charged analyte ions 204 continue to propagate in the direction of arrow 208 and enter the vacuum chamber 232 via the IQ1 ion lens 234. Once within the vacuum chamber 232, the charged analyte ions 204 pass through a Q1 region 236 disposed within the vacuum chamber 232. The Q1 region 236 includes a Brubaker lens (or stubby lens) 238, a quadrupole mass filter 240, and a stubby lens 242. The stubby lens 238 is located upstream of the mass filter 240, while the stubby lens 242 is located downstream of the mass filter 240. The quadrupole mass filter 240 includes a plurality of rods 244 ( Figure 2 Only two of them are shown in the figure). The short thick lens 238 focuses the charged analyte ions 204 leaving the vacuum chamber 226 into the mass filter 240.

[0061] Application of RF voltages to the rods of the quadrupole mass filter 240 and a resolving DC voltage provides radial confinement of the ions and also allows selection of analyte ions having a target m / z range of interest to pass through the quadrupole mass filter 240 and be focused via a stub lens 242 into a dissociation device 246 located downstream of the mass filter 240. The charged analyte ions 204 enter the dissociation device 246 via an IQ2 lens 248, which further focuses the charged analyte ions 204.

[0062] The dissociation device 246 includes a chamber 250 in which an ion trap 252 is disposed. The ion trap 252 is defined by a first L-shaped electrode 254 and a second L-shaped electrode 256 (also referred to as L-shaped rods 254 and 256, respectively) axially separated from each other, an electrode 258 (e.g., a lens electrode), and an optional electrode 260. In the center of the ion trap 252 is a reaction region 262. Although Figure 2 Mass spectrometer 200 is shown including electrode 260, but in other embodiments, electrode 260 may be omitted.

[0063] In this embodiment, the first L-shaped electrode 254 and the second L-shaped electrode 256 include four electrodes ( Figure 1 208 ), they are arranged in a quadrupole configuration and axially separated from each other to provide an ion reaction region 262 therebetween. The first L-shaped electrode 254 and the second L-shaped electrode 256 form an axial path (in the direction of arrow 208) through which the charged analyte ions 204 can pass. In addition, the arrangement of the first L-shaped electrode 254 and the second L-shaped electrode 256 forms a lateral path perpendicular to the axial path. The ion trap 252 formed by the first L-shaped electrode 254 and the second L-shaped electrode 256 can be referred to as a "branch ion trap".

[0064] The RF voltage source 216 and the DC voltage source 218 operating under the control of the controller 222 provide voltages to the L-shaped electrodes 254 and 256, which trap the negatively charged analyte ions 204 in the ion trap 252. In this embodiment, since the first L-shaped electrode 254 and the second L-shaped electrode 256 are provided with RF voltages, the ion trap 252 can be referred to as a "branched RF ion trap."

[0065] Electrode 258 and electrode 274 are located near the opening of the transverse path defined by first L-shaped electrode 254 and second L-shaped electrode 256. DC voltage source 218 can be used to apply a DC voltage to electrodes 258 and 274 so as to maintain electrodes 258 and 274 at a potential that will prevent negatively charged analyte ions 204 (e.g., oligonucleotides) from leaking out of ion trap 252 via the transverse path. Thus, electrode 274 further defines ion trap 252.

[0066] The mass spectrometer 200 includes a gas reservoir 264 in communication with the chamber 250. The gas reservoir 264 supplies a neutral buffer gas (eg, neon, krypton, helium, nitrogen, argon, etc.) to the chamber 250 via an input port 266.

[0067] Reference Figure 2 and Figure 3, the mass spectrometer 200 also includes a thermal electron source 268 (e.g., a filament) that generates a plurality of electrons 270, a grid electrode 272 and a pole electrode 274 located between the electron source 268 and an inlet 276. In this embodiment, the mass spectrometer 200 may also include a magnet 278 configured to generate a magnetic field extending from the electron source 268 across the trap center 262 to the pole electrode 258 to confine the electrons and form an electron beam.

[0068] The DC voltage source 218 may also apply a DC voltage to the gate electrode 256 and the pole electrode 272 so that the gate electrode 256 is positively biased relative to the electron source 268. The bias of the electron beam source 268 is set in the range of about -20 to -50 volts relative to the branch ion trap. In these cases, the accelerated electrons may have a kinetic energy greater than at least 20 eV in the ion trap 252, and may specifically have a kinetic energy in the range of about 20 eV to about 50 eV (e.g., 30 eV, 35 eV, 40, etc.). In some embodiments, the controller 222 controls the temperature of the electron source 268 to increase or decrease the current associated with the emitted electrons 270. For example, the current generated by the electrons 270 may be in the range of about 10 to about 100 microamperes.

[0069] Electrons 270 are introduced into the ion trap 252 as an electron beam via an inlet 276 of the transverse path. For example, the electron beam may have a diameter of about 1 mm. The electrons 270 ionize the molecules disposed in the ion trap 252 via electron impact ionization (EI), thereby generating a plurality of positively charged ions 280 (e.g., N2 + 、He + 、Ne + , Kr + The positive charge of the ions 280 can neutralize the negative charge of the electron beam 270, thereby providing a substantially electrically neutral plasma, thereby reducing and preferably eliminating the repulsive forces experienced by the negatively charged ions 204 via interaction with the electron beam.

[0070] Typically, when electrons are introduced into an ion trap alone, the electrons can generate a negatively charged environment in the reaction region of the ion trap into which the electrons are introduced. The negatively charged environment can repel negatively charged analyte ions. This repulsion can expel the negatively charged analyte ions from the reaction region of the ion trap before the analyte ions can interact with the electrons. Therefore, in many cases, the fragmentation of the negatively charged analyte ions via the negative EAD may be minimized. The present teaching overcomes this difficulty by ionizing multiple gas molecules introduced into the ion trap via the same electron beam used for the negative EAD, so as to produce multiple positively charged ions, which can substantially neutralize the electric field generated by the electrons, thereby promoting the negatively charged analyte ions to remain in the reaction region of the ion trap, and thus improve the efficiency of the negative EAD of the negatively charged analyte ions. In some embodiments, the electron beam has kinetic energy sufficient to provide an electrostatic potential well that confines at least a portion of the positively charged ions in the reaction region.

[0071] Without being limited to any particular theory, better retention of analyte ions within the reaction region can result in an increase in the number of collisions between electrons and negatively charged ions, and thus increase the probability of negatively charged analyte ions being fragmented via negative EAD. In addition, the faster reaction rates associated with negative EADs relative to the reaction rates associated with electron transfer reactions can promote fragmentation of negatively charged analyte ions before they leave the reaction region of the ion trap.

[0072] By way of further illustration, in Figure 4 The above process is schematically depicted in FIG. 4. In this example, the RF potential 402 confines the negatively charged analyte ions 404 within the ion trap 400, and the electrons 406 of the electron beam generate a potential 408 that would normally repel the negatively charged analyte ions 404. In this example, positively charged ions 410 generated via ionization of the buffer gas molecules introduced into the ion trap 400 can counteract and preferably neutralize the potential 408 (e.g., causing the potential to vanish), thereby allowing the negatively charged analyte ions 404 to enter and be retained within the path of the electron beam.

[0073] Refer again Figure 2 and Figure 3 , the pole electrodes 274 and 258 are negatively biased relative to the center 262 of the ion trap 252. That is, the pole electrodes 274 and 258 are negatively biased relative to the first L-shaped electrode 254 and the second L-shaped electrode 256. This negative bias of the pole electrodes prevents the negatively charged analyte ions 204 from escaping the ion trap 252 through the entrance 276, while allowing the negatively charged electrons 270 to enter the ion trap 252.

[0074] The electrode 260 is located near the axial exit 282 of the ion trap 252. The AC voltage source 220 provides an AC voltage to the electrode 260, which generates a pseudo-potential barrier that contains the negatively charged analyte ions 204 in the trap 252. However, the fragment ions of interest (e.g., fragment ions with a certain m / z ratio) can overcome the AC pseudo-potential barrier to enter the downstream Q2 collision cell 284 via the hole of the IQ2 lens 286. In the collision cell, the fragment ions 288 collide with the buffer gas molecules supplied by the gas reservoir 264. These collisions result in cooling of the fragment ions 288. The fragment ions 288 continue to propagate in the direction of the arrow 208 and leave the collision cell 284 via the hole through the lens 290. In some embodiments in which the electrode 260 is omitted, after applying the negative EAD, the lens 286 is opened to extract the fragment ions 288 from the reaction device 250 to the mass analyzer 292.

[0075] The mass spectrometer 200 also includes a mass analyzer 292 (e.g., a time-of-flight (TOF) analyzer or another type of mass analyzer) located downstream of the collision cell 284, which receives the fragment ions 288 and provides mass spectral data associated with the fragment ions 288. An analysis module 294 receives the mass spectral data generated by the mass analyzer 292 and processes the data to generate a mass spectrum of the fragment ions 288 and to associate the mass spectrum of the fragment ions 288 with the negatively charged analyte ions 204 from which the fragment ions 288 were generated.

[0076] Reference now Figure 5 , according to an exemplary embodiment, a method 500 of performing electron separation dissociation (EDD) and negative electron induced dissociation (EID) in mass spectrometry is shown.

[0077] At 502, a buffer gas is introduced into a chamber of a reaction device as discussed previously herein.

[0078] At 504 , the analyte (eg, oligonucleotide) is ionized to produce a plurality of negatively charged analyte ions as discussed previously herein.

[0079] At 506, negatively charged analyte ions (of isolated single m / z species, roughly separated over a wide range of m / z values, or unseparated) are introduced and captured into an ion trap located in a cavity of a reaction device as discussed previously herein.

[0080] At 508, an electron source (eg, a hot filament) external to the ion trap generates electrons as discussed previously herein.

[0081] At 510, electrons are accelerated to form an electron beam, and the electron beam is introduced into an ion trap. While in the trap, the electron beam ionizes at least a portion of the buffer gas molecules to produce positively charged ions within the trap, and also interacts with at least a portion of the negatively charged analyte ions trapped in the ion trap to fragment at least a portion thereof via EDD or negative EID, thereby producing a plurality of fragment product ions.

[0082] At 514, the mass analyzer receives the fragment product ions and generates mass spectral data corresponding to the m / z ratios of the ions, as discussed previously herein. Also, at 514, the analysis module receives the mass spectral data generated by the mass analyzer and processes the data to generate mass spectra of the fragment ions and to correlate the mass spectra of the fragment ions with the negatively charged analyte ions from which the fragment ions were generated, as discussed previously herein.

[0083] Now refer to Figure 6 , shows an example of an EDD spectrum using the disclosed plasma technique. In this example, K e =35eV electron beam irradiates the deprotonated DNA sample for 20ms. • The w and w fragments are dominant in the EDD spectrum. The sequence coverage obtained is 100%.

[0084] Now refer to Figure 7 , shows an example of a negative EID spectrum, where negative EID is applied to a phosphoester. In this example, K e =35eV electron beam irradiates a deprotonated phosphatidylglycerol (PG) sample. The complete structural information of the lipid is displayed in a single spectrum.

[0085] The data associated with the following examples were acquired using a research-grade Sciex mass spectrometer configured in accordance with the present teachings.

[0086] Example

[0087] The following examples further illustrate some embodiments of the present disclosure.

[0088] In these examples, the EAD unit used to collect the data discussed below is a branched RF ion trap with permanent magnets, such as Fig.9A As shown schematically in FIG.

[0089] In one set of experiments, a research-grade EAD-TOF instrument was employed, while in the other experiments a commercial system was used. The branched RF ion trap is a six-way crossed RF ion trap, which includes eight L-shaped electrodes. Six negatively biased DC lenses are installed to confine negative ions in six axial directions.

[0090] The RF frequency at which the EAD unit is operated in commercial grade systems is 800 KHz. The RF frequency at which the EAD unit is operated in research grade TOF instruments is set to 600 kHz in order to confine ions of higher mass to charge ratio (m / z).

[0091] Two N52 grade neodymium magnets provide a magnetic field of about 300 mT along the electron beam axis. The buffer gas is introduced into the Q2 and EAD cells, where the pressure in the EAD cell is almost the same as that in the Q2 cell because four holes with a diameter of 5 mm are set on the IQ2B lens to achieve high gas conductivity. Nitrogen is used as the buffer gas (CAD) in commercial instruments.

[0092] At a constant voltage V f A Y2O3-coated iridium disk (Kimball Physics, NH) was Joule-heated to generate a thermal electron beam. The electron beam energy (K e ) is defined by the bias of the iridium disk relative to the DC bias of the branch ion trap electrodes. An ammeter (A1) that measures the voltage drop across the resistor is inserted into the Grid 1 line to monitor electron beam generation used in commercial systems. Another ammeter (A2) is inserted into the shorted Filament 2-Grid 2 lines to measure the current through the EAD device, which can be used in research-grade instruments.

[0093] The EAD unit was installed between Q1 and Q2 of the Q-TOF mass spectrometer. In all experiments using research-grade instruments, the Zeno trap pulse was turned off to avoid strong species saturating the ion detection system. The EAD unit in the Zeno trap system was operated in simultaneous capture mode (or quasi-flow-through mode). The K was acquired using proprietary OS software. e Data with values ​​greater than 20 eV. Zeno trapping was activated in experiments performed using a commercial system.

[0094] Fig. 9B Schematic depiction of the DC bias along the electron beam path in an EAD device.

[0095] I. Production of positive ions

[0096] Irradiation with electron beam in synchronized capture mode z = 9 - Charged state of DNA20-PS:[M-9H] 9- At A1, the nominal electron beam intensity is set at 3000 nA. Fig.11A shows the generated K of the charge reduced state (CRS) e Dependence, while in K e = 18, 23, 30 and 35 eV at the characteristic spectra such as Fig.10 as shown in .

[0097] CRS:[M-nH](n-1)- It can be generated by electron separation or electron transfer as shown below:

[0098] [M-nH] n- +e- [M-nH] .(n-1)- +2e-

[0099] [M-nH] n- +m + [M-nH] .(n-1)- +m

[0100] like Fig.11A As shown in , the charge reduction of n = 9 starts at 10 eV, and the generation of CRS gradually increases, where K e Lower than the ionization energy (IE) of N2: 15.58eV. Fig. 11B As shown in e When the IE is higher than N2, N2 is generated in the EAD device. + .

[0101] In K e At values ​​of 18 eV and below, the spectrum mainly contains precursor ions, and the fragments are still weak, such as Fig. 12A The precursor is strongly consumed at Ke = 18 eV, and the generation of CRS (z = 8 - ) increases sharply. This abrupt change in CRS generation for electron energies above IE is consistent with the + There is agreement that ions play an important role in precursor consumption.

[0102] When the consumption of precursor ions is 62%, the first CRS (z=8 - ) shows that K e =23eV. e =23eV spectrum is as follows Fig. 12B These fragments are visible in the same proportion as the remaining precursor ions. The intensity of the first CRS is higher at higher K e The product intensity decreases at K because the second charge reduction consumes CRS. e is maximized at about 30 eV (e.g., Fig. 12C ), in K e The third CRS (z=-6) reaches its maximum value at about 30eV. Fig. 12C \ Fig.12D It shows that in K e = 35 eV, where the peak intensity is lower than that at K e =30 eV, but have similar profiles.

[0103] II. Measurement of charge reduction rate

[0104] The progress of charge reduction is observed as a function of electron irradiation time to obtain the charge reduction rate for each charge state. (dT) 15 K e =40 eV electron beam irradiation. Conventional acquisition mode was programmed with a fixed ion loading time of 50 ms and a variable reaction time from 0 to 60 ms. A blank period was inserted after ion extraction to keep the total duration of the scan function constant. Figures 13A-13D The MSMS spectra at 0, 20, 40 and 60 ms are shown, and Fig. 13C The intensity variation of each CRS normalized to the vertical scale is shown, illustrating the observed cascade charge reduction.

[0105] use Fig.14 The data in Fig.15 As shown, calculate the reaction rate associated with each charge state. The observed charge reduction rate has (1 / z) 1.3 , where higher charge states exhibit lower charge reduction rates, which is consistent with the known dependence of reaction rates on charge state z for multiplicative ECD (electron impact dissociation) of protonated peptides and proteins. 2 The dependence is opposite. Use (1 / z) 1.3 Dependency Reproduction Fig.14 ,like Fig.16 shown.

[0106] If charge reduction is caused by electron transfer, the higher charge state should react faster due to attractive cation-anion interactions. Perform experiments to examine negative ETD (electron transfer dissociation). Specifically, refer to Fig.17A , N2 + ions are introduced into the EAD cell generated in the grid 1-filament 1 region. The bias voltage applied to the filament is set so as to suppress electrons from entering the trap. Although N2 + Ions are unstable in the RF field, but the nitrogen ion beam passed through the oligonucleotide precursor. A positive current of 0.1 µA was measured at the second filament operating as a Faraday cup. The negative ETD of DNA16-PO (e.g. Fig. 17B ), without the mutual precursor-reagent capture in conventional ETD operation.

[0107] Fig. 17C The reaction rates of beam-type negative ETD are shown. The reaction rate of the highly charged precursor is faster, while the reaction rate of the low-charge state is slower, which is consistent with z 2 Dependent anion-cation interactions ( Fig.13D ), which is consistent with the result of electron beam irradiation ( Fig.15)on the contrary. Fig.15 The results presented in suggest that the enhanced dissociation mechanism is based on EDD.

[0108] III. Neutralization of the electron beam potential

[0109] Reference Figures 18A-18D , validating the EDD model. In other words, it has been demonstrated that positive ions contribute to neutralizing the electron beam potential. The potential height generated by the electron beam is measured using a probe anion. Probe anions (m / z = -166) generated by electron spray ionization (ESI) are injected into the electron beam after being isolated by Q1. A ToF analyzer monitors the anions transmitted through the electron beam. The kinetic energy of the probe anions is ramped in a manner similar to the ramping of the collision energy (CE) in CID operation. Fig.18A The blue curve in corresponds to electron beam off. The tail on the negative CE side shows the kinetic energy distribution of the probe anion. e When set to 26 eV (orange curve), the electron beam is at (dT) 15 In the absence of induced dissociation, the transport of the probe anion is disturbed by the electron beam potential, requiring a higher kinetic energy (+2 eV) for transport. e When the voltage is set to 35 eV (black curve), the transport of the probe anion is restored and the distribution becomes almost the same as that in the electron-free case. This indicates that K e =35eV is equivalent to no electrons, that is, N2 + The positive ions should counteract the electron beam potential. Fig.19 Schematically depicted, a high energy electron beam can induce strong EDD.

[0110] Observed N2H + It is made of N2 + Produced by neutral reaction with ions of residual vacuum gas. N2 + and N2H + (m / z 28 and 29, respectively) were trapped under plasma EDD conditions, although these ions were outside the stability of the RF ion trap due to the frequent application of high RF amplitudes, which is equivalent to LMCO at 200 m / z. However, due to the typical electron beam intensity of 50 µA at A2, electron beam trapping of nitrogen ions was still achieved, resulting in a calculated depth of the electron beam potential of about 1 V for positive ions.

[0111] Without being limited to any particular theory, in the presence of N2 + In positive ion plasmas, the advantage of EDD over ETD can be explained by assuming that 10 10ions, when pushed out by the electron beam, it can immediately fill the electron beam well with the overflow of positive ions swept out by LMCO. On the other hand, a 50µA electron beam provides 10 12 electrons, which is 1 million times the number of cations, which may make EDD the dominant process.

[0112] IV. Chemistry of Plasma EDD

[0113] In various embodiments, the dissociation efficiency of the intermediate state produced by electron desorption is a parameter that can be used to evaluate the efficiency of EDD. This can understand the choice of charge state from the various precursor charge states produced by ESI to achieve good sequence coverage in oligonucleotide dissociation. In CID, the lower charge precursor state provides better coverage because the generation of internal fragments is less than the higher charge precursor state, however, the sensitivity may be low because the abundance of such lower charge precursors in the ESI product is generally low.

[0114] The EDD applied to various precursor charge states was examined. Figures 20A-20H It is shown that K e =25eV electron beam irradiation charge state is z=4 - , 6 - , 8 - and 10 - EDD spectrum of DNA20-PS. Figures 20A-20H It also shows that K e = EDD of DNA20-PS for various precursor charge states at 35 eV and 45 eV. Specifically, Figure 20G Shows K e =25ev when z=10 - Spectra annotated with single charge transitions of the precursor charge states. Figures 20A-20H It is shown that highly charged ions produce abundant fragments, while lower charged ions produce almost no fragments, which is similar to the ECD in peptides and proteins. Again, without being limited to any particular theory, strong intermolecular tension due to dense charges favors dissociation. No significant K was observed in the dissociation pattern. e Dependency.

[0115] V. Dissociation of Modified Oligonucleotides

[0116] The dissociation rules and efficiencies associated with EDD of ONTs undergoing typical modifications were examined. e =35 eV and a nominal electron beam intensity of 3000 nA at A1. The sample was injected into the LC to obtain an EDD spectrum in a targeted MSMS operation. Fig.21 and Figure 22A-22J EDD spectra and fragment intensity plots for all fragmentation types are shown.

[0117] For DNA20-PS, fragment types a and w were dominant, which were the same as for DNA-PO, but the spectrum of the PS form showed significantly lower CRS and very high dissociation efficiency. When PS was partially substituted around the termini in DNA-PO (DNA20-POPS), the fragments cleaved at the PS moiety were 100 times stronger than those cleaved at PO ( Figure 23A-23C and Figure 24A-Figure 24E Although the signal intensity at the PO part is weak, a . and w fragments to sequence the spectrum ( Fig.23A and Figure 25A-25B On the other hand, CID did not show such a significant PO / PS difference in dissociation efficiency (see Figure 26A-26C ).

[0118] Figure 27A-Figure 27E and Figure 28A-28H The plasma EDD spectrum shows Fig.29 ) extracted from an oligonucleotide with a locked ribose (GAP) near the end, i.e., a GCT and CAA at the end. The data show that dissociation at the locked portion is strongly inhibited in any fragmentation type. Although the intensity is weak, w fragments (e.g. Figure 28A-28J ). EDD did not dissociate the locked moiety well, but CID produced fragments. The main fragment types observed via CID were not the standard aB and w fragments. Instead, b, c, d, x, and y fragments were observed (see Figure 30A-Figure 30B and Figure 31A-Figure 31C ).

[0119] Since the locking ribose is introduced near the end of the ONT, the CID and the EDD may be complementary, ie, the CID sequence near the end and the EDD sequence in the middle portion of the ONT may be determined.

[0120] Investigation of impurities in degraded GAP samples by LC-MSMS (see Figure 32A-32C and Fig.33 ). One observed change was the loss of adenine (-A) from the precursors, which showed distinct patterns of -135, -133, and -117 Da. These precursors were not separated by the Q1` mass filter, which was operated in low resolution mode with a 3 m / z window, nor did LC separate the -135 precursor or the -133 precursor well. EDD spectra were obtained by targeted acquisition ( Figure 32A-32C ). The EDD spectrum of each precursor was enhanced by summing the different parts of the LC elution peak (see Fig.32A , Fig.32B ).

[0121] The sulfur loss from PS in GAP samples was also investigated (e.g. Fig.33 ). The experimental loss I of the precursor ion is 15.981, which matches the mass difference between sulfur and oxygen atoms, confirming the existence of PO replacing PS. Fig.33 a. Fig.33 c shows the sequencing results of a and w fragments of the unmodified GAP molecular structure. Fig.33 b. Fig.33 d shows the sequencing results of fragments a and w, but one PS in the fragment is changed to PO. The modified positions are distributed between positions 6 and 13, and positions 7 and 8 are heavily modified.

[0122] Now refer to Figure 8 , shows a computer system 800 according to an exemplary embodiment. In some embodiments, the computer system 800 is used as the controller 222.

[0123] As used herein, a computer system (or device) is any system / device capable of receiving, processing, and / or sending data. Examples of computer systems include, but are not limited to, personal computers, servers, handheld computing devices, tablet computers, smartphones, multiprocessor-based systems, mainframe computer systems, and distributed cloud computing environments including any of the above systems.

[0124] like Figure 8 As shown, computer system 800 includes one or more processors or processing units 802 , a system memory 804 , and a bus 806 that couples various components of computer system 800 , including system memory 804 , to processor 802 .

[0125] The system memory 804 includes a computer-readable storage medium 808 and a volatile memory 810 (e.g., random access memory, cache, etc.). As used herein, a computer-readable storage medium includes any medium capable of storing computer-readable program instructions and capable of being accessed by a computer system. The computer-readable storage medium 808 includes non-volatile and non-transient storage media (e.g., flash memory, read-only memory (ROM), hard drive, etc.). The computer-readable program instructions as described herein include program modules (e.g., routines, programs, objects, components, logic, data structures, etc.) that can be executed by a processor. In addition, when executed by a processor, the computer-readable program instructions can direct a computer system (e.g., computer system 800) to operate in a specific manner so that the computer-readable storage medium (e.g., computer-readable storage medium 808) includes an article of manufacture. Specifically, when the processor 802 executes the computer-readable program instructions stored in the computer-readable storage medium 808, they create a device for implementing the various functions described herein.

[0126] The bus 806 may be one or more of any type of bus structures capable of transferring data between components of the computer system 800 (eg, a memory bus, memory controller, peripheral bus, accelerated graphics port, etc.).

[0127] In some embodiments, Figure 8 As depicted, computer system 800 may include one or more external devices 812 and a display 814. As used herein, an external device includes any device that allows a user to interact with a computer system (e.g., a mouse, keyboard, touch screen, etc.). External devices 812 and display 814 communicate with processor 802 and system memory 804 via input / output (I / O) interface 816.

[0128] The display 814 can display a graphical user interface (GUI) that can include a plurality of selectable icons and / or editable fields. A user can use an external device 812 (e.g., a mouse) to select one or more icons and / or edit one or more editable fields. Selecting an icon and / or editing a field can cause the processor 802 to execute computer-readable program instructions stored in the computer-readable storage medium 808. In one example, a user can use the external device 812 to interact with the computer system 800 and cause the processor 802 to execute computer-readable program instructions associated with the various functions described herein.

[0129] The computer system 800 may also include a network adapter 818 that allows the computer system 800 to communicate with one or more other computer systems / devices via one or more networks (eg, a local area network (LAN), a wide area network (WAN), a public network (Internet), etc.).

[0130] Although various embodiments are described and illustrated in detail in the drawings and the foregoing description, these illustrations and descriptions should be regarded as illustrative or exemplary rather than restrictive; the embodiments of the present disclosure are not limited to the disclosed embodiments. By studying the drawings, the present disclosure, and the appended claims, those skilled in the art can understand and implement other variations of the disclosed embodiments when practicing the embodiments of the present disclosure.

[0131] 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. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

Claims

1. A method for performing electron separation dissociation (EDD) and negative electron induced dissociation (EID) in mass spectrometry, the method comprising: introducing a plurality of negatively charged analyte ions into an ion trap located in a cavity, and trapping the negatively charged analyte ions in a reaction region of the ion trap, introducing a buffer gas into the chamber, generating electrons using an electron source located in the cavity and external to the ion trap, and accelerating the electrons to form an electron beam, and introducing the electron beam into the ion trap so that the accelerated electrons can ionize at least a portion of the molecules of the buffer gas to generate a plurality of positively charged ions, The accelerated electrons interact with at least a portion of the analyte ions trapped in the reaction region to cause electron separation dissociation (EDD) and negative electron induced dissociation (EID) thereof, thereby generating a plurality of fragment product ions.

2. The method of claim 1, wherein the accelerated electrons have a kinetic energy greater than about 25 eV when nitrogen, neon, and krypton are used as the buffer gas.

3. The method of claim 1, wherein the accelerated electrons have a kinetic energy greater than about 50 eV when helium is used as the buffer gas.

4. The method according to claim 1, wherein the positively charged ions include any one of nitrogen molecular ions, helium ions, neon ions, and krypton ions. 5 . The method of claim 4 , wherein when nitrogen, neon, and krypton are used as the buffer gas, the electron kinetic energy is in the range of about 30 eV to about 50 eV.

6. The method of claim 4, wherein when helium is used as the buffer gas, the electron kinetic energy is in the range of about 50 eV to about 90 eV.

7. The method according to claim 6, wherein the nitrogen molecular ions include N2 + and N2H + , the helium ions include He + , the neon ions include Ne + , and the krypton ions include Kr + .

8. A method according to any preceding claim, wherein the ion trap comprises: a branched RF ion trap having a longitudinal channel extending from an inlet to an outlet, wherein the negatively charged analyte ions can enter the trap through the inlet and the fragment product ions can leave the trap through the outlet; and A transverse channel intersects the longitudinal channel at the reaction zone, the transverse channel having an inlet for receiving the electron beam.

9. The method of claim 8, wherein the branched RF ion trap comprises two sets of L-shaped rods axially separated from each other, wherein the sets of L-shaped rods are arranged according to a multipole configuration, and the method further comprises: An RF voltage is applied to the rods to generate an RF electric field for confining the analyte ions within the reaction region.

10. A method according to any preceding claim, further comprising confining the electron beam using at least one magnet located in the cavity.

11. A method according to any preceding claim, wherein the electron beam provides an electrostatic potential well which confines the positively charged ions within the reaction region.

12. A mass spectrometer comprising: an ion source for receiving the sample and ionizing one or more analytes to produce a plurality of negatively charged analyte ions, a chamber comprising a buffer gas and an ion trap for capturing the negatively charged analyte ions, an electron source located in the cavity and outside the ion trap for generating electrons, and at least one magnet, located in the cavity, for forming the electrons into an electron beam, the electron beam being introduced into the ion trap, wherein the electron beam is capable of ionizing at least a portion of the molecules of the buffer gas to generate a plurality of positively charged ions, and The electrons interact with at least a portion of the analyte ions to cause electron separation dissociation (EDD) and negative electron induced dissociation (EID) thereof, thereby generating a plurality of fragment product ions.

13. The mass spectrometer of claim 12, wherein the ion source comprises an electrospray ion source.

14. The mass spectrometer according to claim 12 or 13, further comprising: a plurality of rods arranged in a multipole configuration to form an axial path and a transverse path perpendicular to the axial path, wherein the negatively charged analyte ions are introduced into the ion trap via the axial path and the electron beam is introduced into the ion trap via the transverse path; as well as An RF voltage source is provided for applying an RF voltage to the plurality of rods.

15. The mass spectrometer of claim 14, wherein the plurality of rods comprises a first set of L-shaped rods and a second set of L-shaped rods arranged in a quadrupole configuration.

16. The mass spectrometer of claim 15, wherein the rod is L-shaped.

17. The mass spectrometer according to any one of claims 12 to 16, wherein the positively charged ions include any one of nitrogen molecular ions, helium ions, neon ions, and krypton ions.

18. The mass spectrometer of claim 17, wherein the nitrogen molecular ions include N2 + and N2H + , the helium ions include He + , the neon ions include Ne + , and the krypton ions include Kr + .

19. The mass spectrometer of claim 12, wherein when nitrogen, neon, and krypton are used as the buffer gas, the electron kinetic energy is in the range of about 25 eV to about 50 eV.

20. The mass spectrometer of claim 12, wherein when helium is used as the buffer gas, the electron kinetic energy is in the range of about 50 eV to about 90 eV.

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