Dissociation method of DNA in mass spectrometry

Abstract

A method for dissociating an oligonucleotide is disclosed. A plurality of precursor ions of one or more oligonucleotides is loaded into an ion trap. Negative radical-induced dissociation is applied to the plurality of precursor ions in the ion trap during a first time period, producing charge-reduced ions. Resonant CID is applied in the ion trap during a second time period to dissociate the charge-reduced ions. A pause without any dissociation in the ion trap is performed during a third time period to cool ions produced from the previous resonant CID or again negative radical-induced dissociation is applied in the ion trap during the third time period to again produce charge-reduced ions from the plurality of precursor ions while at a same time allowing ions produced from the previous resonant CID to be cooled. The last two steps are repeated one or more times.

Description

DISSOCIATION METHOD OF DNA IN MASS SPECTROMETRYRELATED APPLICATION(S)

[0001] This application claims priority to U.S. Provisional Application No. 63 / 600,857 filed on November 20, 2023, the contents of which are incorporated herein by reference in their entirety.INTRODUCTION

[0002] The teachings herein relate to a method for dissociating an oligonucleotide. More particularly the teachings herein relate to systems and methods for applying negative radical-induced dissociation (negRID) followed by pulsed resonant collision-induced dissociation (CID) to dissociate an oligonucleotide.

[0003] The systems and methods herein can be performed in conjunction with a processor, controller, or computer system, such as the computer system of Figure 1.PO-DNA and PO-RNA Dissociation

[0004] Collision-induced dissociation (CID), electron detachment dissociation (EDD) and negative electron-transfer dissociation (ETD) have been used to dissociate oligonucleotides. CID is the most popular method to sequence oligonucleotides, but EDD and ETD may provide better sequence coverage in the cases the precursor ions have a highly charged state. More specifically, these electron-based methods have been shown to effectively dissociate modified DNAs and RNAs, such as phosphorothioate deoxyribonucleotides (PS-DNAs) and phosphorothioate ribonucleotides (PS-RNAs). However, when these methods are applied to regular DNAs and RNAs, or phosphodiester deoxyribonucleotides (PO-DNAs) andphosphodiester ribonucleotides (PO-RNAs), the dissociation efficiency is significantly reduced.

[0005] As a result, additional systems and methods are needed for dissociating PO-DNAs and PO-DNAs using radical-induced dissociation methods, such as EDD and ETD.LC-MS and LC-MS / MS Background

[0006] Mass spectrometry (MS) is an analytical technique for the detection and quantitation of chemical compounds based on the analysis of mass-to-charge ratios (m / z) of ions formed from those compounds. The combination of mass spectrometry (MS) and liquid chromatography (LC) is an important analytical tool for the identification and quantitation of compounds within a mixture. Generally, in liquid chromatography, a fluid sample under analysis is passed through a column filled with a chemically-treated solid adsorbent material (typically in the form of small solid particles, e.g., silica). Due to slightly different interactions of components of the mixture with the solid adsorbent material (typically referred to as the stationary phase), the different components can have different transit (elution) times through the packed column, resulting in separation of the various components.

[0007] Note that for singly charged species, the terms “mass” and “m / z” are used interchangeably herein. One of ordinary skill in the art understands that a mass can be found from an m / z by multiplying the m / z by the charge. Similarly, the m / z can be found from a mass by dividing the mass by the charge.

[0008] In LC-MS, the effluent exiting the LC column can be continuously subjected to MS analysis. The data from this analysis can be processed to generate anextracted ion chromatogram (XIC), which can depict detected ion intensity (a measure of the number of detected ions of one or more particular analytes) as a function of retention time.

[0009] In MS analysis, an MS or precursor ion scan is performed at each interval of the separation for a mass range that includes the precursor ion. An MS scan includes the selection of a precursor ion or precursor ion range and mass analysis of the precursor ion or precursor ion range.

[0010] In some cases, the LC effluent can be subjected to tandem mass spectrometry (or mass spectrometry / mass spectrometry MS / MS) for the identification of product ions corresponding to the peaks in the XIC. For example, the precursor ions can be selected based on their mass / charge ratio to be subjected to subsequent stages of mass analysis. For example, the selected precursor ions can be fragmented (e.g., via collision-induced dissociation), and the fragmented ions (product ions) can be analyzed via a subsequent stage of mass spectrometry.Fragmentation Techniques Background

[0011] Electron-based dissociation (ExD), ultraviolet photodissociation (UVPD), infrared photodissociation (IRMPD), and collision-induced dissociation (CID) are often used as fragmentation techniques for tandem mass spectrometry (MS / MS). CID is the most conventional technique for dissociation in tandem mass spectrometers. CID, in-source fragmentation, blackbody infrared radiative dissociation and IRMPD are examples of thermal-dissociation methods in this description. Thermal-dissociation methods included herein are non-radical dissociation methods that do not involve the use of radical formation in the dissociation process.

[0012] ExD can include, but is not limited to, electron-induced dissociation (EID), electron impact excitation in organics (EIEIO), electron capture dissociation (ECD), electron detachment dissociation (EDD), plasma electron detachment dissociation (pEDD), or electron transfer dissociation (ETD). Radical-induced dissociation methods, mentioned herein, include ExD, UVPD, and electron photodetachment dissociation (EPD).Tandem Mass Spectrometry or MS / MS Background

[0013] Tandem mass spectrometry or MS / MS involves ionization of one or more compounds of interest from a sample, selection of one or more precursor ions of the one or more compounds, fragmentation of the one or more precursor ions into product ions, and mass analysis of the product ions.

[0014] Tandem mass spectrometry can provide both qualitative and quantitative information. The product ion spectrum can be used to identify a molecule of interest. The intensity of one or more product ions can be used to quantitate the amount of the compound present in a sample.

[0015] A large number of different types of experimental methods or workflows can be performed using a tandem mass spectrometer. These workflows can include, but are not limited to, targeted acquisition, information dependent acquisition (IDA) or data dependent acquisition (DDA), and data independent acquisition (DIA).

[0016] In a targeted acquisition method, one or more transitions of a precursor ion to a product ion are predefined for a compound of interest. As a sample is being introduced into the tandem mass spectrometer, the one or more transitions are interrogated during each time period or cycle of a plurality of time periods or cycles. In other words, the mass spectrometer selects and fragments the precursorion of each transition and performs a targeted mass analysis for the product ion of the transition. As a result, a chromatogram (the variation of the intensity with retention time) is produced for each transition. Targeted acquisition methods include, but are not limited to, multiple reaction monitoring (MRM) and selected reaction monitoring (SRM).

[0017] MRM experiments are typically performed using “low resolution” instruments that include, but are not limited to, triple quadrupole (QqQ) or quadrupole linear ion trap (QqLIT) devices. With the advent of “high resolution” instruments, there was a desire to collect MS and MS / MS using workflows that are similar to QqQ / QqLIT systems. High-resolution instruments include, but are not limited to, quadrupole time-of-flight (QqTOF) or orbitrap devices. These high-resolution instruments also provide new functionality.

[0018] MRM on QqQ / QqLIT systems is the standard mass spectrometric technique of choice for targeted quantification in all application areas, due to its ability to provide the highest specificity and sensitivity for the detection of specific components in complex mixtures. However, the speed and sensitivity of today’s accurate mass systems have enabled a new quantification strategy with similar performance characteristics. In this strategy (termed MRM high resolution (MRM-HR) or parallel reaction monitoring (PRM)), looped MS / MS spectra are collected at high-resolution with short accumulation times, and then fragment ions (product ions) are extracted post-acquisition to generate MRM-like peaks for integration and quantification. With instrumentation like the TRIPLETOF® Systems of AB SCIEX™, this targeted technique is sensitive and fast enough to enable quantitative performance similar to higher-end triple quadrupoleinstruments, with full fragmentation data measured at high resolution and high mass accuracy.

[0019] In other words, in methods such as MRM-HR, a high-resolution precursor ion mass spectrum is obtained, one or more precursor ions are selected and fragmented, and a high-resolution full product ion spectrum is obtained for each selected precursor ion. A full product ion spectrum is collected for each selected precursor ion but a product ion mass of interest can be specified and everything other than the mass window of the product ion mass of interest can be discarded.

[0020] In an IDA (or DDA) method, a user can specify criteria for collecting mass spectra of product ions while a sample is being introduced into the tandem mass spectrometer. For example, in an IDA method a precursor ion or mass spectrometry (MS) survey scan is performed to generate a precursor ion peak list. The user can select criteria to filter the peak list for a subset of the precursor ions on the peak list. The survey scan and peak list are periodically refreshed or updated, and MS / MS is then performed on each precursor ion of the subset of precursor ions. A product ion spectrum is produced for each precursor ion. MS / MS is repeatedly performed on the precursor ions of the subset of precursor ions as the sample is being introduced into the tandem mass spectrometer.

[0021] In proteomics and many other applications, however, the complexity and dynamic range of compounds is very large. This poses challenges for traditional targeted and IDA methods, requiring very high-speed MS / MS acquisition to deeply interrogate the sample in order to both identify and quantify a broad range of analytes.

[0022] As a result, DIA methods, the third broad category of tandem mass spectrometry, were developed. These DIA methods have been used to increase thereproducibility and comprehensiveness of data collection from complex samples. DIA methods can also be called non-specific fragmentation methods. In a DIA method the actions of the tandem mass spectrometer are not varied among MS / MS scans based on data acquired in a previous precursor or survey scan. Instead, a precursor ion mass range is selected. A precursor ion mass selection window is then stepped across the precursor ion mass range. All precursor ions in the precursor ion mass selection window are fragmented and all of the product ions of all of the precursor ions in the precursor ion mass selection window are mass analyzed.

[0023] The precursor ion mass selection window used to scan the mass range can be narrow so that the likelihood of multiple precursors within the window is small. This type of DIA method is called, for example, MS / MSALL. In an MS / MSALLmethod, a precursor ion mass selection window of about 1 Da is scanned or stepped across an entire mass range. A product ion spectrum is produced for each 1 Da precursor mass window. The time it takes to analyze or scan the entire mass range once is referred to as one scan cycle. Scanning a narrow precursor ion mass selection window across a wide precursor ion mass range during each cycle, however, can take a long time and is not practical for some instruments and experiments.

[0024] As a result, a larger precursor ion mass selection window, or selection window with a greater width, is stepped across the entire precursor mass range. This type of DIA method is called, for example, SWATH acquisition. In a SWATH acquisition, the precursor ion mass selection window stepped across the precursor mass range in each cycle may have a width of 5-25 Da, or even larger. Like the MS / MSALLmethod, all of the precursor ions in each precursor ion mass selectionwindow are fragmented, and all of the product ions of all of the precursor ions in each mass selection window are mass analyzed.

[0025] However, identifying compounds of interest in a sample analyzed using SWATH acquisition, for example, can be difficult. It can be difficult because either there is no precursor ion information provided with a precursor ion mass selection window to help determine the precursor ion that produces each product ion, or the precursor ion information provided is from a mass spectrometry (MS) observation that has a low sensitivity. In addition, because there is little or no specific precursor ion information provided with a precursor ion mass selection window, it is also difficult to determine if a product ion is convolved with or includes contributions from multiple precursor ions within the precursor ion mass selection window.

[0026] As a result, a method of scanning the precursor ion mass selection windows in SWATH acquisition, called scanning SWATH, was developed. Essentially, in scanning SWATH, a precursor ion mass selection window is scanned across a mass range so that successive windows have large areas of overlap and small areas of non-overlap. This scanning makes the resulting product ions a function of the scanned precursor ion mass selection windows. This additional information, in turn, can be used to identify the one or more precursor ions responsible for each product ion.SUMMARY

[0027] A system, method, and computer program product are disclosed for dissociating an oligonucleotide. In a first step, a plurality of precursor ions of one or more oligonucleotides is loaded into an ion trap. In a second step, negRID is applied tothe plurality of precursor ions in the ion trap during a first time period, producing charge-reduced ions from the plurality of precursor ions. In a third step, resonant CID is applied in the ion trap during a second time period to dissociate the charge- reduced ions. In a fourth step, a pause without any dissociation in the ion trap is performed during a third time period to cool ions produced from the previous resonant CID, or again negRID is applied in the ion trap during the third time period to again produce charge -reduced ions from the plurality of precursor ions while at the same time allowing ions produced from the previous resonant CID to be cooled. In a fifth step, the third and fourth steps are repeated one or more times. In a sixth step, product ions produced from the second step to the fifth step are transferred to a mass analyzer for mass analysis to obtain a mass spectrum.

[0028] In one general aspect, a method for dissociating an oligonucleotide can include: (a) loading a plurality of precursor ions of one or more oligonucleotides into an ion trap; (b) applying negative radical-induced dissociation (negRID) to the plurality of precursor ions in the ion trap during a first time period, thereby producing charge-reduced ions from the plurality of precursor ions; (c) applying resonant collision-induced dissociation (CID) in the ion trap during a second time period to dissociate the charge-reduced ions; (d) pausing without any dissociation in the ion trap during a third time period to cool ions produced from the previous resonant CID or applying again negRID in the ion trap during the third time period to again produce charge -reduced ions from the plurality of precursor ions while at a same time allowing ions produced from the previous resonant CID to be cooled; (e) repeating steps (c) and (d) one or more times; and (f) transferring product ions produced from steps (b)-(e) to a mass analyzer for mass analysis to obtain a mass spectrum.

[0029] In some embodiments, the one or more oligonucleotides can include deoxyribonucleic acid (DNA) with one to all phosphodiester (PO).

[0030] In some embodiments, the one or more oligonucleotides can include ribonucleic acid (RNA) with one to all phosphodiester (PO).

[0031] In some embodiments, the negRID can include electron detachment dissociation (EDD).

[0032] In some embodiments, the negRID can include plasma electron detachment dissociation (EDD).

[0033] In some embodiments, the negRID can include simultaneous electron detachment dissociation (EDD).

[0034] In some embodiments, the negRID can include simultaneous plasma electron detachment dissociation (EDD).

[0035] In some embodiments, the negRID can include negative electron-transfer dissociation (ETD).

[0036] In some embodiments, the negRID can include negative beam-type electrontransfer dissociation (ETD).

[0037] In some embodiments, the negRID can include simultaneous negative electrontransfer dissociation (ETD).

[0038] In some embodiments, the negRID can include simultaneous negative beam -type electron-transfer dissociation (ETD).

[0039] In some embodiments, the negRID can include electron photodetachment dissociation (EPD).

[0040] In another general aspect, a computer program product can include a non- transitory tangible computer-readable storage medium whose contents cause a processor to perform a method for dissociating an oligonucleotide. The methodcan include: providing a system comprising one or more distinct software modules, wherein the distinct software modules comprise a control module; (a) loading a plurality of precursor ions of one or more oligonucleotides into an ion trap using the control module; (b) applying negative radical-induced dissociation (negRID) to the plurality of precursor ions in the ion trap during a first time period, thereby producing charge-reduced ions from the plurality of precursor ions using the control module; (c) applying resonant collision-induced dissociation (CID) in the ion trap during a second time period to dissociate the charge-reduced ions using the control module; (d) pausing without any dissociation in the ion trap during a third time period to cool ions produced from the previous resonant CID or applying again negRID in the ion trap during the third time period to again produce charge-reduced ions from the plurality of precursor ions while at a same time allowing ions produced from the previous resonant CID to be cooled using the control module; (e) repeating steps (c) and (d) one or more times using the control module; and (f) transferring product ions produced from steps (b)-(e) to a mass analyzer for mass analysis to obtain a mass spectrum using the control module.

[0041] In another general aspect, a system for dissociating an oligonucleotide can include an ion trap that: (a) loads a plurality of precursor ions of one or more oligonucleotides; (b) applies negative radical-induced dissociation (negRID) to the plurality of precursor ions during a first time period, thereby producing charge- reduced ions from the plurality of precursor ions; (c) applies resonant collision- induced dissociation (CID) during a second time period to dissociate the charge- reduced ions; (d) pauses without any dissociation during a third time period to cool ions produced from the previous resonant CID or applies again negRIDduring the third time period to again produce charge -reduced ions from the plurality of precursor ions while at a same time allowing ions produced from the previous resonant CID to be cooled; (e) repeats steps (c) and (d) one or more times; and (f) transfers product ions produced from steps (b)-(e) to a mass analyzer for mass analysis to obtain a mass spectrum.

[0042] These and other features of the applicant’s teachings are set forth herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0044] Figure 1 is a block diagram that illustrates a computer system, upon which embodiments of the present teachings may be implemented.

[0045] Figure 2 is an exemplary plot of an EDD spectrum showing a PO-DNA precursor ion and its four CRS, upon which embodiments of the present teachings may be implemented.

[0046] Figure 3 is an exemplary diagram showing the chemical formulas that describe how the four CRS of Figure 2 are produced during radical -induced dissociation, upon which embodiments of the present teachings may be implemented.

[0047] Figure 4 is an exemplary plot showing the intensity versus post-activation energies of a PS-DNA precursor ion and its four CRS, upon which embodiments of the present teachings may be implemented.

[0048] Figure 5 is an exemplary diagram showing the chemical structure of a portion of PS-DNA, upon which embodiments of the present teachings may be implemented.

[0049] Figure 6 is an exemplary plot showing the intensity versus post-activation energies of a PO-DNA precursor ion and its four CRS, upon which embodiments of the present teachings may be implemented.

[0050] Figure 7 is an exemplary diagram showing the chemical structure of a portion of PO-DNA, upon which embodiments of the present teachings may be implemented.

[0051] Figure 8 is an exemplary timing diagram showing a long period of negRID activation of precursor ions followed by repeated shorter periods of resonant CID post-activation of CRS ions and cooling, in accordance with various embodiments.

[0052] Figure 9 is an exemplary timing diagram showing a short period of negRID activation of precursor ions followed by repeated short periods of resonant CID post-activation of CRS ions and negRID activation of precursor ions, in accordance with various embodiments.

[0053] Figure 10 is an exemplary diagram of a branched ion trap showing precursor ion loading into the electron branch, upon which embodiments of the present teachings may be implemented.

[0054] Figure 11 is an exemplary diagram of a branched ion trap showing negRID activation in the electron branch, upon which embodiments of the present teachings may be implemented.

[0055] Figure 12 is an exemplary diagram of a branched ion trap showing precursor ion loading and negRID activation of precursor ions occurring simultaneously, in accordance with various embodiments.

[0056] Figure 13 is an exemplary diagram of a branched ion trap in which a magnetic field is established parallel to the electron branch, in accordance with various embodiments.

[0057] Figure 14 is an exemplary plot showing that two resonant frequencies are found in an ion motion simulation when an AC frequency is scarmed in the branch of the branched ion trap of Figure 13 parallel to the magnetic field.

[0058] Figure 15 is an exemplary plot showing that one resonant frequency is found in an ion motion simulation when an AC frequency is scarmed in the branch of the branched ion trap of Figure 13 perpendicular to the magnetic field.

[0059] Figure 16 is an exemplary top view of a branched ion trap showing auxiliary T-bar electrodes used to apply resonant CID, upon which embodiments of the present teachings may be implemented.

[0060] Figure 17 is an exemplary side view of a branched ion trap showing auxiliary T- bar electrodes used to apply resonant CID, upon which embodiments of the present teachings may be implemented.

[0061] Figure 18 is an exemplary diagram graphically showing the sequence of a short period of resonant CID post-activation followed by a short period of pause or of negRID activation performed repeatedly in the same branched ion trap, in accordance with various embodiments.

[0062] Figure 19 is an exemplary schematic diagram of a system for dissociating an oligonucleotide, in accordance with various embodiments.

[0063] Figure 20 is an exemplary flowchart showing a method for dissociating an oligonucleotide, in accordance with various embodiments.

[0064] Figure 21 is an exemplary schematic diagram of a system that includes one or more distinct software modules and that performs a method for dissociating an oligonucleotide, in accordance with various embodiments.

[0065] Before one or more embodiments of the present teachings are described in detail, one skilled in the art will appreciate that the present teachings are not limited in their application to the details of construction, the arrangements of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.DESCRIPTION OF VARIOUS EMBODIMENTS COMPUTER-IMPLEMENTED SYSTEM

[0066] Figure 1 is a block diagram that illustrates a computer system 100, upon which embodiments of the present teachings may be implemented. Computer system 100 includes a bus 102 or other communication mechanism for communicating information, and a processor 104 coupled with bus 102 for processing information. Computer system 100 also includes a memory 106, which can be a random-access memory (RAM) or other dynamic storage device, coupled to bus 102 for storing instructions to be executed by processor 104. Memory 106 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 104. Computer system 100 further includes a read only memory (ROM) 108 or other static storage device coupled to bus 102 for storing static information and instructionsfor processor 104. A storage device 110, such as a magnetic disk or optical disk, is provided and coupled to bus 102 for storing information and instructions.

[0067] Computer system 100 may be coupled via bus 102 to a display 112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 114, including alphanumeric and other keys, is coupled to bus 102 for communicating information and command selections to processor 104. Another type of user input device is cursor control 116, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 104 and for controlling cursor movement on display 112.

[0068] A computer system 100 can perform the present teachings. Consistent with certain implementations of the present teachings, results are provided by computer system 100 in response to processor 104 executing one or more sequences of one or more instructions contained in memory 106. Such instructions may be read into memory 106 from another computer-readable medium, such as storage device 110. Execution of the sequences of instructions contained in memory 106 causes processor 104 to perform the process described herein.

[0069] Alternatively, hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. For example, the present teachings may also be implemented with programmable artificial intelligence (Al) chips with only the encoder neural network programmed - to allow for performance and decreased cost. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0070] The term “computer-readable medium” or “computer program product” as used herein refers to any media that participates in providing instructions to processor 104 for execution. The terms “computer-readable medium” and “computer program product” are used interchangeably throughout this written description. Such a medium may take many forms, including but not limited to, non-volatile media and volatile media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device 110. Volatile media includes dynamic memory, such as memory 106.

[0071] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD- ROM, digital video disc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0072] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 104 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 100 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 102 can receive the data carried in the infra-red signal and place the data on bus 102. Bus 102 carries the data to memory 106, from which processor 104 retrieves and executes the instructions. Theinstructions received by memory 106 may optionally be stored on storage device 110 either before or after execution by processor 104.

[0073] In accordance with various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0074] The following descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.PULSED RESONANT POST-ACTIVATION

[0075] As described above, radical-induced dissociation methods, such as EDD, ETD, and EPD have been used to dissociate modified oligonucleotides, or PS-DNAs and PS-RNAs. However, when these methods are applied to regular DNAs andRNAs, or PO-DNAs and PO-RNAs, the dissociation efficiency is significantly reduced.

[0076] As a result, additional systems and methods are needed for dissociating PO-DNAs and PO-DNAs using radical-induced dissociation methods, such as EDD, ETD, and EPD.

[0077] More specifically, in oligonucleotide dissociation, charge-reduced species or CRS ions are dominant. However, the post-activation energy required to dissociate CRS in PS-DNAs and PS-RNAs is different from the post-activation energy required to dissociate CRS in PO-DNAs and PO-RNAs.

[0078] Figure 2 is an exemplary plot 200 of an EDD spectrum showing a PO-DNA precursor ion and its four CRS, upon which embodiments of the present teachings may be implemented. In plot 200, peak 210 is the precursor ion. Peak 221 is the first CRS, peak 222 is the second CRS, peak 223 is the third CRS, and peak 224 is the fourth CRS.

[0079] Figure 3 is an exemplary diagram 300 showing the chemical formulas that describe how the four CRS of Figure 2 are produced during radical-induced dissociation, upon which embodiments of the present teachings may be implemented. Figure 3 shows that first CRS 321 is produced from precursor ion 310. Second CRS 322 is produced from first CRS 321. Third CRS 323 is produced from second CRS 322. Fourth CRS 324 is produced from third CRS 323.

[0080] Figure 4 is an exemplary plot 400 showing the intensity versus post-activation energies of a PS-DNA precursor ion and its four CRS, upon which embodiments of the present teachings may be implemented. In plot 400, curve 410 depicts the threshold of the collisional dissociation for the precursor ions as 10 eV. Theprecursor was completely dissociated at 18 eV. Curve 421 depicts the threshold of the collisional dissociation for CRS1 as ~ 0 eV. The precursor was near- completely dissociated at 10 eV. Curve 422 depicts the threshold of the collisional dissociation for CRS2 as ~ 10 eV. The precursor was near-completely dissociated at ~23 eV. Curve 423 depicts the threshold of the collisional dissociation for CRS3 as ~ 4 eV. The precursor was near-completely dissociated at ~15 eV. Curve 424 depicts the threshold of the collisional dissociation for CRS4 as ~ 25 eV. Plot 400 shows that for PS-DNA the post-activation energies of the four CRS are quite different from the post-activation energy of the precursor ion. A comparison of curves 421 and 410, for example, shows that the first CRS can be dissociated using a much lower activation energy than the precursor ion.

[0081] Figure 5 is an exemplary diagram 500 showing the chemical structure of a portion of PS-DNA, upon which embodiments of the present teachings may be implemented. Ovals 510 and 520 highlight portions of the PS-DNA that differ from PO-DNA.

[0082] Figure 6 is an exemplary plot 600 showing the intensity versus post-activation energies of a PO-DNA precursor ion and its four CRS, upon which embodiments of the present teachings may be implemented. In plot 600, curve 610 depicts the post-activation of the precursor ion. Curve 621 depicts the post-activation of the first CRS. Curve 622 depicts the post-activation energy of the second CRS. Curve 623 depicts the post-activation of the third CRS. Curve 624 depicts the post-activation of the fourth CRS. Figure 6 shows that for PO-DNA the postactivation energies of the four CRS are quite similar to the post-activation energy of the precursor ion. This similarly explains the poor dissociation efficiency ofradical-induced dissociation in dissociating PO-DNA. When post activation or CID is applied to the remaining precursor ions and all CRS, fragments by difference dissociation mechanism (i.e., non-radical induced dissociation and radical induced dissociation) are overlapped. Such non-mass-selective post activation provides different types of fragments and makes the interpretation of the spectrum difficult.

[0083] Figure 7 is an exemplary diagram 700 showing the chemical structure of a portion of PO-DNA, upon which embodiments of the present teachings may be implemented. Ovals 710 and 720 highlight portions of PO-DNA that differ from PS-DNA.

[0084] In various embodiments, pulsed resonant collision-induced dissociation (CID) is repeatedly applied to the 1stCRS ions (CRS1) ions after negative radical-induced dissociation (negRID) to dissociate the CRS ions. In other words, post-activation is applied to the non-dissociated 1stcharge-reduced species produced by negative radical-induced dissociation, such as EDD, neg ETC, or EPD. Post-activation dissociates the non-sequence-informative charge-reduced species and produces sequence -informative fragment ions. This method provides higher sensitivity and higher sequence coverage of oligonucleotides with less sample consumption.

[0085] In various embodiments, negRID is applied once followed by repeated pulses of resonant CID. Each pulse of resonant CID is followed by a pause or cooling time period.

[0086] Figure 8 is an exemplary timing diagram 800 showing a long period of negRID activation of precursor ions followed by repeated shorter periods of resonant CID post-activation of CRS ions and cooling, in accordance with various embodiments. In diagram 800, for example, negRID activation of precursor ionsis applied during time period Ti followed by repeated time periods T2 and T3, during which resonant CID and a pause are applied, respectively, to activate and cool CRS ions. The pause in resonant CID during time period T3 allows for ion cooling. The negRID period, Tl, is determined to maximize the CRS1 intensity. This duration depends on the electron beam intensity actually used. In a nonlimiting example, time periods T2 and T3 are each 2 ms. This improves the dissociation efficiency provided by resonant CID.

[0087] In various alternative embodiments, alternating periods of negRID and resonant CID are applied. In this method, the pause or cooling time for the CRS ions occurs during the subsequent negRID period.

[0088] Figure 9 is an exemplary timing diagram 900 showing a short period of negRID activation of precursor ions followed by repeated short periods of resonant CID post-activation of CRS ions and negRID activation of precursor ions, in accordance with various embodiments. In diagram 800, for example, negRID activation of precursor ions is applied during time period T 1 followed by repeated time periods T2 and T3, during which resonant CID and a repeat negRID activation are applied, respectively. During the time period T2, CRS ions are activated. During time period T3, CRS ions are cooled while precursor ions are activated using negRID. As a result, cooling is not required until time period T4, for example, which is after the last post-activation of CRS ions. In a nonlimiting example, time period Ti is 2 ms, time periods T2 and T3 are each 2 ms, and time period T4 is 2 ms.

[0089] In various embodiments, negRID activation and resonant CID post-activation are performed in the same ion trap. This ion trap can be, but is not limited to, a three- dimensional RFQ ion trap, a linear RFQ ion trap, or a branched ion trap.

[0090] In various embodiments, negRID activation and resonant CID post-activation are performed in a different ion trap from the electron-induced dissociation device. This ion trap can be, but is not limited to, a three-dimensional RFQ ion trap, a linear RFQ ion trap, or a branched ion trap. This ion trap can be placed in the electron beam path or in the upstream or the downstream in the ion path.

[0091] Figure 10 is an exemplary diagram 1000 of a branched ion trap showing precursor ion loading into the electron branch, upon which embodiments of the present teachings may be implemented. In diagram 1000, precursor ions 1001 are loaded into the ion trap through ion branch 1010 and into electron branch 1020.

[0092] Figure 11 is an exemplary diagram 1100 of a branched ion trap showing negRID activation in the electron branch, upon which embodiments of the present teachings may be implemented. In diagram 1000, negRID is applied to precursor ions 1001 in electron branch 1020. The negRID is applied using EDD for example.

[0093] In various embodiments, precursor loading and negRID activation are performed simultaneously.

[0094] Figure 12 is an exemplary diagram 1200 of a branched ion trap showing precursor ion loading and negRID activation of precursor ions occurring simultaneously, in accordance with various embodiments. In diagram 1200, precursor ions 1001 are loaded into the ion trap through ion branch 1010 and into electron branch 1020. Simultaneously, negRID is applied to precursor ions 1001 in electron branch 1020. The negRID is applied using EDD for example.

[0095] In various embodiments, negRID activation and resonant CID post-activation are performed in a branched ion trap with a magnetic field that is established parallel to at least one branch of the branched ion trap.

[0096] Figure 13 is an exemplary diagram 1300 of a branched ion trap in which a magnetic field is established parallel to the electron branch, in accordance with various embodiments. In diagram 1300, magnetic field 1310 is established parallel to electron branch 1020 and perpendicular to ion branch 1010. In order to determine which branch of the branched ion trap of Figure 13 should be used for resonant CID post-activation, an AC frequency is scanned in both branches.

[0097] Figure 14 is an exemplary plot 1400 showing that two resonant frequencies are found in an ion motion simulation when an AC frequency is scanned in the branch of the branched ion trap of Figure 13 parallel to the magnetic field. Trace 1410 of plot 1400 shows two resonant frequencies when magnetic field 1310 is parallel to electron branch 1020 of the branched ion trap of Figure 13.

[0098] Figure 15 is an exemplary plot 1500 showing that one resonant frequency is found in an ion motion simulation when an AC frequency is scarmed in the branch of the branched ion trap of Figure 13 perpendicular to the magnetic field. Trace 1510 of plot 1500 shows one resonant frequency when magnetic field 1310 is perpendicular to ion branch 1010 of the branched ion trap of Figure 13. In addition, the amplitude of the one peak in plot 1500 is much larger than the amplitudes of the peaks in plot 1400 of Figure 14.

[0099] As a result, and in various embodiments, resonant CID post-activation is performed in the branch of a branched ion trap that is perpendicular to the applied magnetic field. More specifically, in various embodiments, resonant CID postactivation is performed in the ion branch of a branched ion trap that is perpendicular to the applied magnetic field.

[0100] In various embodiments, resonant CID post-activation using auxiliary T-bar electrodes.

[0101] Figure 16 is an exemplary top view 1600 of a branched ion trap showing auxiliary T-bar electrodes used to apply resonant CID, upon which embodiments of the present teachings may be implemented. Top view 1600 shows the location of auxiliary ion branch T-bars 1610 in ion branch 1010 and auxiliary electron branch T-bars 1620 in electron branch 1020. Auxiliary electrodes are electrodes that are not the electrodes used to provide the radial confinement. In Figure 16, for example, electrodes 1630 and 1640 are the electrodes used to provide the radial confinement .

[0102] Figure 17 is an exemplary side view 1700 of a branched ion trap showing auxiliary T-bar electrodes used to apply resonant CID, upon which embodiments of the present teachings may be implemented. Side view 1700 also shows the location of ion branch T-bars 1610 and electron branch T-bars 1620. In addition, side view 1700 shows how the resonant excitation is applied.

[0103] In various embodiments, resonant CID post-activation is performed in a pulsed manner. The resonant CID post-activation is performed in a first time period followed by either a pause or a subsequent pulse of negRID activation. In various preferred embodiments, the sequence of a short period of resonant CID postactivation followed by a short period of pause or of negRID activation is repeated multiple times, as described above.

[0104] Figure 18 is an exemplary diagram 1800 graphically showing the sequence of a short period of resonant CID post-activation followed by a short period of pause or of negRID activation performed repeatedly in the same branched ion trap, in accordance with various embodiments.System for dissociating an oligonucleotide

[0105] Figure 19 is an exemplary schematic diagram 1900 of a system for dissociating an oligonucleotide, in accordance with various embodiments. The system includes ion trap 1935 of mass spectrometer 1930.

[0106] In various embodiments, ion trap 1935 and mass spectrometer 1930 are controlled by processor 1940. Processor 1940 represents just one of one or more processors that can be used in various embodiments. In other words, various embodiments are not limited to performing the steps described herein with a single processor. Processor 1940 can be, but is not limited to, a controller, a computer, a microprocessor, the computer system of Figure 1, or any device capable of sending and receiving control signals and data and capable of analyzing data.

[0107] Ion trap 1935 performs a number of sequential steps. In step (A), ion trap 1935 loads a plurality of precursor ions of one or more oligonucleotides. In step (B), ion trap 1935 applies negRID to the plurality of precursor ions during a first time period, producing charge-reduced ions from the plurality of precursor ions. In step (C), ion trap 1935 applies resonant CID during a second time period to dissociate the charge-reduced ions. In step (D), ion trap 1935 pauses without any dissociation during a third time period to cool ions produced from the previous resonant CID or applies again negRID during the third time period to again produce charge-reduced ions from the plurality of precursor ions while at the same time allowing ions produced from the previous resonant CID to be cooled. In step(E), ion trap 1935 repeats steps (C) and (D) one or more times. Finally, in step(F), product ions produced from steps (B)-(E) are transferred to mass analyzer 1937 to obtain product mass spectrum 1941.

[0108] In various embodiments, the one or more oligonucleotides include deoxyribonucleic acid (DNA) with one to all phosphodiester (PO).

[0109] In various embodiments, the one or more oligonucleotides include ribonucleic acid (RNA) with one to all phosphodiester (PO).

[0110] In various embodiments, the negRID includes one of EDD, plasma EDD, simultaneous EDD, or simultaneous plasma EDD.

[0111] In various embodiments, the negRID includes one of negative ETD or negative beam-type ETD.

[0112] In various embodiments, the negRID includes EPD.

[0113] In various embodiments, ion trap 1935 includes a branched ion trap and the resonant CID is applied in an ion branch of the branched ion trap that is perpendicular to an applied magnetic field.

[0114] In various embodiments, ion source device 1932 of mass spectrometer 1930 ionizes oligonucleotide 1910, producing an ion beam. Ion source device 1932 is controlled by processor 1940, for example. Ion source device 1932 is shown as a component of mass spectrometer 1930. In various alternative embodiments, ion source device 1932 is a separate device. Ion source device 1932 can be, but is not limited to, an electrospray ion source (ESI) device or a chemical ionization (CI) source device such as an atmospheric pressure chemical ionization source (APCI) device or an atmospheric pressure photoionization (APPI) source device.

[0115] Mass spectrometer 1930 selects and fragments precursor ions of oligonucleotide 1910 and mass analyzes product ions of the precursor ion from the ion beam. Mass spectrometer 1930 further includes mass analyzer 1937. Mass spectrometer 1930 produces mass spectrum 1941 using mass analyzer. Mass spectrum 1941 is, for example, used to determine the sequence of oligonucleotide 1910.

[0116] In Figure 19, mass analyzer 1937 is shown as a time-of-flight (TOF) device. One of ordinary skill in the art can appreciate that mass analyzer 1937 can be any type of mass analyzer including, but not limited to, a quadrupole, an ion trap, an orbitrap, or a Fourier transform ion cyclotron resonance (FT-ICR) device.

[0117] In various embodiments, the system of Figure 19 further includes a separation device 1920 that separates oligonucleotide 1910 from a sample. As shown in Figure 1920, additional device 1920 is an LC device. In various alternative embodiments, additional device 1920 can be, but is not limited to, a capillary electrophoresis (CE) device, or an ion mobility spectrometry (IMS) device.Method for dissociating an oligonucleotide

[0118] Figure 20 is an exemplary flowchart showing a method 2000 for dissociating an oligonucleotide, in accordance with various embodiments.

[0119] In step 2010 of method 2000, a plurality of precursor ions of one or more oligonucleotides is loaded into an ion trap.

[0120] In step 2020, negRID is applied to the plurality of precursor ions in the ion trap during a first time period, producing charge-reduced ions from the plurality of precursor ions.

[0121] In step 2030, resonant CID is applied in the ion trap during a second time period to dissociate the charge-reduced ions.

[0122] In step 2040, a pause without any dissociation in the ion trap is performed during a third time period to cool ions produced from the previous resonant CID, or again negRID is applied in the ion trap during the third time period to again produce charge-reduced ions from the plurality of precursor ions while at the same time allowing ions produced from the previous resonant CID to be cooled.

[0123] In step 2050, steps 2030 and 2040 are repeated one or more times.

[0124] In step 2060, product ions produced from steps 2020-2050 are transferred to a mass analyzer for mass analysis to obtain a mass spectrum.Computer program product for dissociating an oligonucleotide

[0125] In various embodiments, a computer program product includes a non-transitory tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method for dissociating an oligonucleotide. This method is performed by a system that includes one or more distinct software modules.

[0126] Figure 21 is an exemplary schematic diagram of a system 2100 that includes one or more distinct software modules and that performs a method for dissociating an oligonucleotide, in accordance with various embodiments. System 2100 includes control module 2110.

[0127] In step (A), control module 2110 loads a plurality of precursor ions of one or more oligonucleotides.

[0128] In step (B), control module 2110 applies negRID to the plurality of precursor ions during a first time period, producing charge-reduced ions from the plurality of precursor ions.

[0129] In step (C), control module 2110 applies resonant CID during a second time period to dissociate the charge-reduced ions.

[0130] In step (D), control module 2110 pauses without any dissociation during a third time period to cool ions produced from the previous resonant CID or applies again negRID during the third time period to again produce charge-reduced ions fromthe plurality of precursor ions while at the same time allowing ions produced from the previous resonant CID to be cooled.

[0131] In step (E), control module 2110 repeats steps (C) and (D) one or more times.

[0132] Finally, in step (F), control module 2110 transfers product ions produced from steps (B)-(E) to a mass analyzer for mass analysis to obtain a mass spectrum.

[0133] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0134] Further, in describing various embodiments, the specification may have presented a method and / or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and / or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

Claims

WHAT IS CLAIMED IS:

1. A method for dissociating an oligonucleotide, comprising:(a) loading a plurality of precursor ions of one or more oligonucleotides into an ion trap;(b) applying negative radical-induced dissociation (negRID) to the plurality of precursor ions in the ion trap during a first time period, thereby producing charge -reduced ions from the plurality of precursor ions;(c) applying resonant collision-induced dissociation (CID) in the ion trap during a second time period to dissociate the charge-reduced ions;(d) pausing without any dissociation in the ion trap during a third time period to cool ions produced from the previous resonant CID or applying again negRID in the ion trap during the third time period to again produce charge-reduced ions from the plurality of precursor ions while at a same time allowing ions produced from the previous resonant CID to be cooled;(e) repeating steps (c) and (d) one or more times; and(f) transferring product ions produced from steps (b)-(e) to a mass analyzer for mass analysis to obtain a mass spectrum.

2. The method of claim 1, wherein the one or more oligonucleotides comprise deoxyribonucleic acid (DNA) with one to all phosphodiester (PO).

3. The method of claim 1, wherein the one or more oligonucleotides comprise ribonucleic acid (RNA) with one to all phosphodiester (PO).

4. The method of any one of claims 1 to 3, wherein the negRID comprises electron detachment dissociation (EDD).

5. The method of any one of claims 1 to 3, wherein the negRID comprises plasma electron detachment dissociation (EDD).

6. The method of any one of claims 1 to 3, wherein the negRID comprises simultaneous electron detachment dissociation (EDD).

7. The method of any one of claims 1 to 3, wherein the negRID comprises simultaneous plasma electron detachment dissociation (EDD).

8. The method of any one of claims 1 to 3, wherein the negRID comprises negative electron-transfer dissociation (ETD).

9. The method of any one of claims 1 to 3, wherein the negRID comprises negative beam-type electron-transfer dissociation (ETD).

10. The method of any one of claims 1 to 3, wherein the negRID comprises simultaneous negative electron-transfer dissociation (ETD).

11. The method of any one of claims 1 to 3, wherein the negRID comprises simultaneous negative beam-type electron-transfer dissociation (ETD).

12. The method of any one of claims 1 to 3, wherein the negRID comprises electron photodetachment dissociation (EPD).

13. The method of any one of claims 1 to 3, wherein the ion trap is a branched ion trap, and the resonant CID is applied in an ion branch of the branched ion trap that is perpendicular to an applied magnetic field.

14. A computer program product, comprising a non-transitory tangible computer-readable storage medium whose contents cause a processor to perform a method for dissociating an oligonucleotide, the method comprising: providing a system comprising one or more distinct software modules, wherein the distinct software modules comprise a control module;(a) loading a plurality of precursor ions of one or more oligonucleotides into an ion trap using the control module;(b) applying negative radical-induced dissociation (negRID) to the plurality of precursor ions in the ion trap during a first time period, thereby producing charge -reduced ions from the plurality of precursor ions using the control module;(c) applying resonant collision-induced dissociation (CID) in the ion trap during a second time period to dissociate the charge-reduced ions using the control module;(d) pausing without any dissociation in the ion trap during a third time period to cool ions produced from the previous resonant CID or applying again negRID in the ion trap during the third time period to again produce charge-reduced ions from the plurality of precursor ions while at a same time allowing ions produced from the previous resonant CID to be cooled using the control module;(e) repeating steps (c) and (d) one or more times using the control module; and(f) transferring product ions produced from steps (b)-(e) to a mass analyzer for mass analysis to obtain a mass spectrum using the control module.

15. A system for dissociating an oligonucleotide, comprising: an ion trap that(a) loads a plurality of precursor ions of one or more oligonucleotides;(b) applies negative radical-induced dissociation (negRID) to the plurality of precursor ions during a first time period, thereby producing charge-reduced ions from the plurality of precursor ions;(c) applies resonant collision-induced dissociation (CID) during a second time period to dissociate the charge-reduced ions;(d) pauses without any dissociation during a third time period to cool ions produced from the previous resonant CID or applies again negRID during the third time period to again produce charge-reduced ions from the plurality of precursor ions while at a same time allowing ions produced from the previous resonant CID to be cooled;(e) repeats steps (c) and (d) one or more times; and(f) transfers product ions produced from steps (b)-(e) to a mass analyzer for mass analysis to obtain a mass spectrum.

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