Systems and methods for sequencing dissociation spectra of oligonucleotides obtained by negative electron activated dissociation

Abstract

A system for analyzing a mass spectrum of a deprotonated oligonucleotide, comprising: a mass spectrometer configured to collect mass spectrometry data; and an analyzer module configured to receive and analyze mass spectrometry data by: identifying experimental isotope peaks corresponding to precursor ions generated from the deprotonated oligonucleotides; determining characteristics of the precursor ions; identifying experimental isotope peaks corresponding to fragment ions generated from the precursor ions; determining characteristics of fragment ions; selecting candidate fragments for fragment ions; determining isotopic peaks of mass shifts for the candidate fragments based on data including characteristics of the precursor ions and characteristics of the fragment ions; comparing the experimental isotope peak corresponding to the fragment ion with the isotope peak of the mass shift of the candidate fragment; and identifying fragment ions as candidate fragments based on the comparison.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 347,946, filed on June 1, 2022, entitled “Systems and Methods for Sequencing Dissociation Spectra of Oligonucleotides Obtained by Negative Electron Activated Dissociation,” which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to mass spectrometry of oligonucleotides, and more particularly to methods and systems for analyzing dissociation spectra of oligonucleotides obtained by electron separation dissociation and negative electron transfer dissociation to, for example, determine the nucleotide sequence of the oligonucleotide. Background Art

[0004] Mass spectrometry (MS) is an analytical technique for determining the elemental composition of a substance. Specifically, MS measures the mass-to-charge ratio (m / z) of ions generated by a test substance. MS can be used to identify unknown compounds, to determine the isotopic composition of elements in molecules, to determine the structure of a specific compound by observing its fragments (hereinafter also alternatively referred to as dissociation), 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 an ionic form. In general, a mass spectrometer may include an ion source and a mass analyzer. The ion source converts the test sample into gaseous ions and the mass analyzer obtains a mass spectrum based on their m / z ratios (m / z). In some cases, a mass spectrometer may also include one or more isolation devices installed between the ion source and the mass analyzer; or one or more dissociation devices may be additionally included between the isolation device and the mass analyzer.

[0005] A mass spectrometer can employ dissociation to fragment large analytes (e.g., oligonucleotides, DNA, RNA, etc.) into smaller fragment ions (e.g., fragments containing individual nucleic acid bases). These smaller fragment ions can then be separated and quantified based on their m / z ratios.

[0006] For dissociation, different techniques can be used. For example, a common dissociation technique for biomolecule analysis is collision-induced dissociation (CID). In addition, some other techniques utilizing free radical-driven dissociation methods can be used to obtain information that supplements the information obtained from CID. Such other techniques can include electron capture dissociation (ECD), electron transfer dissociation (ETD), electron separation dissociation (EDD), or photodissociation (UVPD) using UV lasers.

[0007] However, the process of electron-based dissociation may generate a cascade of precursor species with reduced charge. For example, after a precursor loses an electron, the resulting precursor ion may be dissociated, or one or more electrons may be lost before dissociation. Therefore, multiple progeny of precursors with different charges may coexist and appear in the spectrum results. Moreover, these ions and fragments generated from different charges may appear at different positions of the m / z scale in the mass spectrum. Therefore, this complexity in the case of electron-driven dissociation may challenge the interpretation of the spectrum. Summary of the invention

[0008] In some embodiments, the technology described herein relates to a method for analyzing a mass spectrum of a deprotonated oligonucleotide, the method comprising: identifying an experimental isotope peak corresponding to a precursor ion generated from the deprotonated oligonucleotide; determining one or more characteristics of the precursor ion; identifying an experimental isotope peak corresponding to a fragment ion generated from the precursor ion; determining one or more characteristics of the fragment ion; selecting candidate fragments for the fragment ion; determining a mass-shifted isotope peak of the candidate fragment based on data including one or more characteristics of the precursor ion and one or more characteristics of the fragment ion; comparing the experimental isotope peak corresponding to the fragment ion and the mass-shifted isotope peak of the candidate fragment; and identifying the fragment ion as a candidate fragment based on the comparison.

[0009] In some embodiments, the technology described herein relates to a method in which fragment ions are generated from precursor ions as a result of negative electron activated dissociation.

[0010] In some embodiments, the techniques described herein relate to a method wherein the one or more characteristics of the precursor ion include a charge of the precursor ion and a mass of the precursor ion.

[0011] In some embodiments, the technology described herein relates to a method further comprising determining a length of a precursor ion based on a mass of the precursor ion.

[0012] In some embodiments, the techniques described herein relate to a method wherein the one or more characteristics of the fragment ions include a charge of the fragment ions and a mass of the fragment ions.

[0013] In some embodiments, the technology described herein relates to a method further comprising determining a dissociation site corresponding to the fragment ion based on the mass of the fragment ion.

[0014] In some embodiments, the technology described herein relates to a method wherein determining a mass-shifted isotopic peak of a candidate fragment comprises estimating a mass shift equal to an integer portion of [z-(|Z|-1)i / N], where z is the charge of the fragment ion, i is the number of nucleotides in the fragment ion, |Z| is the absolute value of the charge of the precursor ion, and N is the number of nucleotides in the precursor ion.

[0015] In some embodiments, the technology described herein relates to a method wherein a deprotonated oligonucleotide is multiply deprotonated.

[0016] In some embodiments, the techniques described herein relate to a method in which fragment ions are generated from precursor ions after removing two or more electrons from the precursor ions.

[0017] In some embodiments, the techniques described herein relate to a method in which fragment ions are generated from precursor ions due to negative electron activated dissociation after removal of two or more electrons from the precursor ions.

[0018] In some embodiments, the technology described herein relates to a system for analyzing a mass spectrum of a deprotonated oligonucleotide, the system comprising: a mass spectrometer configured to collect mass spectrometry data of the deprotonated oligonucleotide; and an analyzer module configured to receive the mass spectrometry data and analyze the mass spectrometry data, wherein analyzing the mass spectrometry data comprises: identifying an experimental isotope peak corresponding to a precursor ion generated from the deprotonated oligonucleotide; determining one or more characteristics of the precursor ion; identifying an experimental isotope peak corresponding to a fragment ion generated from the precursor ion; determining one or more characteristics of the fragment ion; selecting candidate fragments for the fragment ion; determining a mass-shifted isotope peak of the candidate fragment based on data including one or more characteristics of the precursor ion and one or more characteristics of the fragment ion; comparing the experimental isotope peak corresponding to the fragment ion and the mass-shifted isotope peak of the candidate fragment; and identifying the fragment ion as a candidate fragment based on the comparison.

[0019] In some embodiments, the technology described herein relates to a system in which fragment ions are generated from precursor ions as a result of negative electron activated dissociation.

[0020] In some embodiments, the technology described herein relates to a system wherein the one or more characteristics of the precursor ion include a charge of the precursor ion and a mass of the precursor ion.

[0021] In some embodiments, the technology described herein relates to a system wherein analyzing mass spectrometry data further comprises determining a length of a precursor ion based on a mass of the precursor ion.

[0022] In some embodiments, the technology described herein relates to a system wherein the one or more characteristics of the fragment ions include a charge of the fragment ions and a mass of the fragment ions.

[0023] In some embodiments, the technology described herein relates to a system wherein analyzing mass spectrometry data further comprises determining dissociation sites corresponding to the fragment ions based on the masses of the fragment ions.

[0024] In some embodiments, the technology described herein relates to a system wherein determining a mass-shifted isotopic peak of a candidate fragment comprises estimating a mass shift equal to an integer portion of [z-(|Z|-1)i / N], where z is the charge of the fragment ion, i is the number of nucleotides in the fragment ion, |Z| is the absolute value of the charge of the precursor ion, and N is the number of nucleotides in the precursor ion.

[0025] In some embodiments, the technology described herein relates to a system in which a deprotonated oligonucleotide is multiply deprotonated.

[0026] In some embodiments, the techniques described herein relate to a system in which fragment ions are generated from precursor ions after removing two or more electrons from the precursor ions.

[0027] In some embodiments, the technology described herein relates to a system in which fragment ions are generated from precursor ions due to negative electron activated dissociation after removal of two or more electrons from the precursor ions.

[0028] A further understanding of various embodiments of the embodiments may be obtained by referring to the following detailed description in conjunction with the associated drawings, which are briefly described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are not necessarily drawn to scale or detailed. Instead, the emphasis is generally placed on illustrating the principles of the embodiments described herein. The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate several embodiments consistent with the present disclosure. The accompanying drawings, together with the description, are used to explain the principles of the present disclosure.

[0030] In the figure:

[0031] Figure 1 A diagram showing different dissociation sites and naming conventions for fragments of exemplary oligonucleotides as utilized in some embodiments is shown.

[0032] Figure 2Included is a diagram 200 illustrating the process of deprotonation and fragmentation caused by collision-induced dissociation (CID) of a precursor species 210, the corresponding naming convention, and the structures and fragments of the resulting ions, as utilized in some embodiments.

[0033] Figure 3 Panel 300 is shown including five mass spectrum segments 310 - 350 , each mass spectrum segment indicating fragmentation peaks generated from DNA by EDD, according to some embodiments.

[0034] Figure 4A-4C Examples of dissociation according to various embodiments are illustrated.

[0035] Figure 5 Shown is a block diagram of a mass spectrometry system 500 that collects experimental data and analyzes that data, according to some embodiments.

[0036] Figure 6 A flow chart is shown of a method 600 for determining a mass shift of a CRS detected by a mass spectrometry system, according to some embodiments.

[0037] Figure 7 A flow chart of a method 700 for determining mass shifts of fragments produced by nExD is shown in accordance with some embodiments.

[0038] Figure 8 13C peak profile panel 800 is shown corresponding to panel 300 after the analyzer module determined the mass shift for each isotope of the fragments identified for each bin in panel 300, according to some embodiments.

[0039] Fig. 9 An example of an implementation of a module 900 according to some embodiments is schematically depicted. DETAILED DESCRIPTION

[0040] It will be appreciated that, for the sake of clarity, the following discussion will set forth various aspects of the embodiments of the present disclosure, while omitting certain specific details where convenient or appropriate. For example, the discussion of similar or similar features in alternative embodiments may be shortened. For the sake of brevity, well-known ideas or concepts may also not be discussed in detail. Those of ordinary skill in the art will recognize that some embodiments of the present disclosure may not require certain aspects of the details to be specifically described in each embodiment, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be clear that the described embodiments may be altered or varied according to well-known 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.

[0041] Various embodiments provide systems and methods for identifying different fragments or species that may result from dissociation at different locations, with different ionization amounts or locations.

[0042] In various embodiments, the technology described herein relates to methods for analyzing mass spectra of deprotonated oligonucleotides produced by electron separation dissociation (EDD), negative electron transfer dissociation (negative ETD), electron photodissociation dissociation (EPD), and other dissociation methods initiated by electron removal.

[0043] The present disclosure generally defines electron removed species (ERS) as one or more electron removed fragment species or charge reduced precursor species (CRS), and further defines nExD (negative electron activated dissociation) as a general term for dissociations that include EDD, negative ETD, EPD, or other dissociations that can be initiated by electron removal.

[0044] Figure 1 A diagram illustrating different dissociation sites and naming conventions for fragments of an exemplary oligonucleotide precursor species 100 as utilized in some embodiments is shown. In particular, Figure 1 The commonly used nomenclature for ions generated at the dissociation site along the phosphodiester chain of an oligonucleotide species is illustrated.

[0045] More specifically, the precursor 100 includes two ends marked by terminal 3' (right hand side) and terminal 5' (left hand side). Moreover, the oligonucleotide precursor 100 includes four nucleotides, the bases are B1-B4. In addition, Figure 1 The diagram shows three different dissociation sites 112, 114 and 116 at which the precursor 100 can be fragmented by nExD, and the naming convention for the corresponding fragments. For example, if the precursor 100 is fragmented at the dissociation site 112, the resulting left fragment is named a 1 fragment because it includes a base B1 corresponding to the 5' end of the precursor 100. Furthermore, the resulting right fragment is named w 3 fragment, because it includes three bases B2-B4 corresponding to the 3' terminal end of the precursor 100. In a similar manner, if the precursor 100 is fragmented at the dissociation site 114, the left and right fragments are named a 2 and w 2 ; and if the precursor 100 is fragmented at the dissociation site 116, the left and right fragments are named a 3 and w 1 . Figure 1 The naming convention for the fragments generated if dissociation occurs at other sites is also shown. More precisely, the a fragment generated by nExD is denoted as a ·The dot in the superscript indicates the missing electron that generates the free radical.

[0046] Figure 2 Included is a diagram 200 illustrating the process of deprotonation and fragmentation obtained by collision-induced dissociation (CID) of a precursor species 210, the corresponding naming convention, and the structure and fragmentation of the resulting ions, which are utilized in some embodiments. More specifically, the precursor 210 includes 11 bases, labeled B. Moreover, the precursor 210 includes 10 phosphate groups, each of which is illustrated by a solid circle labeled with a minus sign attached to another solid circle labeled P, which represents a proton equivalent to a hydrogen ion. In addition, Figure 1 The two ends of the precursor 100 are similar, and the right and left ends of the precursor 210 are labeled 3' and 5', respectively. The precursor 210 is a neutral molecule and can be represented by M.

[0047] Diagram 200 also includes precursor ions 220. Precursor ions 220 may be generated from precursor 210 by deprotonation. Thus, in some embodiments, such precursor ions may also be referred to as deprotonated oligonucleotide precursor species, or more succinctly as deprotonated oligonucleotides.

[0048] More specifically, precursor ion 220 can be generated from precursor 210 by electrospray ionization in negative mode, which can remove protons from the precursor ion. In diagram 200, five protons located at positions 211-215 are removed from precursor 210 to generate precursor ion 220. As a result, precursor ion 220 has an overall charge of negative 5 (Z=5-). Therefore, in some embodiments, precursor ion 220 is represented by the notation or symbol [M-5H] 5- , where the [M-5H] portion indicates that the precursor ion 220 is generated by subtracting (removing) five H (hydrogen ions, i.e., protons) from M (precursor 210), and the superscript 5- indicates that its total charge is negative 5 (Z=5-). The details shown in diagram 200 and subsequent structures correspond to the ionization or fragmentation of DNA-type oligonucleotides. Similar concepts and methods can be applied to other types of oligonucleotides, such as RNA with direct modifications.

[0049] Diagram 200 also illustrates the fragmentation of DNA via CID that may occur for precursor ion 220 according to some embodiments. More specifically, in the example of diagram 200, precursor ion 220 may be fragmented at dissociation site 222. The fragmentation may result from processes such as molecular vibration or thermal excitation. This fragmentation may generate two fragment ions 230 and 240, as shown in diagram 200. Fragment ion 230 is labeled [a 6 -B] 3- , and the fragment ion 240 is labeled In the [a 6 -B] 3- In the notation, the letter indicates its type and the subscript 6 indicates its dissociation site or length (having 6 bases), such as Figure 1 In the case of CID applied to DNA, such as shown in diagram 200, fragment ion 230 also loses the 6th base at position 231. This base loss is often indicated by "-B", as seen here. Moreover, the charge superscript 3- indicates that fragment ion 230 has a charge of negative 3 (z=3-). By convention, for such fragments, the charge superscript also indicates the number of protons missing from the fragment. In other words, the notation [a 6 -B] 3- The superscript 3- in indicates that fragment ion 230 is missing 3 protons, corresponding to the missing protons at positions 211-213 in this example. Similarly, the superscript 3- in fragment ion 240 is The notation indicates that fragment ion 240 is of type w, comprises 5 bases, and is missing two protons (in this case from positions 214 and 215), so it has a charge of minus 2.

[0050] Various embodiments use mass spectrometry results to find the fragments produced by the dissociation caused by free radicals. In certain embodiments, the dissociation or nExD caused by free radicals applicable to oligonucleotides includes electron separation dissociation (EDD), negative electron transfer dissociation (ETD) and electron photo separation dissociation (EPD). Each of these dissociations can be triggered by removing one or more electrons. In EDD, the electrons in the precursor ions are "separated" by the irradiation of high-energy electron beams, and the kinetic energy of electrons is usually higher than 10eV. In negative ETD, positive reagent ions are often trapped in ion traps with precursor anions at the same time to cause electron transfer from precursor ions to reagent ions. In EPD, UV laser beams are irradiated to precursor ions to cause photoelectric electron separation. In certain embodiments, the term "electronic removal" can be generally used to represent the technology used to remove electronics, including electron transfer electron separation, electron photo separation, etc.

[0051] Figure 3 Panel 300 is shown including five mass spectrum segments 310-350, each of which indicates an isotopic peak profile of fragment ions generated from DNA by EDD (explained below), according to some embodiments. More specifically, segments 310, 320, 330, 340, and 350 show isotopic peak profiles of fragment ions generated from DNA by EDD, respectively. 14 In general, the isotope peak profile (herein alternatively referred to as 13The isotopic peak profile (C peak profile) can correspond to a collection of peaks (in this case five peaks, as seen in each of segments 310-350) that correspond to different amounts of carbon 13 isotopes in the same fragment. The isotopic peak profile can be identified as a group of peaks that are clustered together compared to other peaks, are spread out at equal distances (as explained below), and may form a pattern depending on the abundance of the particular fragment species. 13 Poisson distribution of the average amount of C isotopes.

[0052] In particular, section 310 shows a spectrum including five peaks 311-315 according to an embodiment, which peaks correspond to the a-type fragments contained in 13 The intensity of each segment shows that the spectrum varies according to the mass-to-charge ratio (m / z), the value of which is indicated on the horizontal axis. For the m / z values ​​on the horizontal axis, the mass m is measured in atomic mass units (amu) and the charge z is measured in elementary charge units (e), so the value is in amu / e. More specifically, peaks 311-315 correspond to fragments The five isotopes of carbon correspond to the ionized fragments of a, i.e., type a, which includes 14 bases and lacks 5 protons, so it carries a negative charge of 5 (Z = -5). The difference between these five isotopes is that the carbon 13 ( 13 C) The number of isotopes. More specifically, peaks 311-315 correspond to isotopes including 0-4 carbon 13 isotopes, respectively. The first peak, peak 311, is located at an m / z value of about 834.1, and the last peak, peak 315, is located at an m / z value of about 834.9. Therefore, the distance between consecutive peaks is about 13 C and 12 The mass difference between the Cs (approximately 1.00335 [amu]) divided by the charge of the fragment (5 in this example) corresponds to 0. As explained below, this relationship can be used to determine the charge of a detected species, such as a precursor ion, CRS, or fragment ion, based on the distance between consecutive peaks.

[0053] In some embodiments, one or more electrons are removed from the precursor ion without dissociation by multiple electron separation or electron transfer processes. The removal of one or more electrons reduces the charge of the precursor ion but does not change the degree of deprotonation. For example and in some embodiments, such as embodiments utilizing EDD, such as [M-7H] 7- A precursor ion such as may undergo one or more of the following electron separation reaction chains, during which at each stage a new precursor ion (or a reduced charge species) is generated that has the same mass as the starting precursor ion but with one less electron, and thus a total charge value that differs by -1:

[0054]

[0055] For example, in the first electron separation, [M-7H] 7- Loses an electron and becomes the precursor ion [M-7H] 6- -. In some embodiments, this second generation precursor ion [M-7H] 6- can be detected by mass spectrometry. Alternatively, and in some embodiments, this second generation precursor ion [M-7H] 6- itself can undergo electron separation according to the second reaction below, thereby generating the precursor ion [M-7H] 5- , and so on.

[0056] Alternatively, in some embodiments utilizing negative ETD, such as [M-7H] 7- Precursor ions such as can undergo one or more of the following electron transfer reaction chains using nitrogen ions as reagents. Similar to the above electron separation reaction chain, new precursor ions (or species with reduced charge) are also generated at each stage, with the same mass but one less electron compared to the starting precursor ion, so the value of the total charge differs by -1.

[0057]

[0058] In some embodiments, after a precursor ion loses one or more electrons, it may dissociate and generate one or more fragments that also lack those electrons. Figure 4A-4C Examples of such dissociations according to various embodiments are illustrated. More specifically, Figure 4A Included is a diagram 400 illustrating a precursor ion losing electrons through one of the electron removal techniques and then undergoing nExD due to the loss of electrons. Figure 4B and Figure 4C Graphs 430 and 460 are included, respectively, illustrating a precursor ion that first loses an electron without dissociating and then loses a second electron that triggers nExD.

[0059] More specifically, in Figure 4A In the diagram 400 of FIG. 4 , the precursor ion 410 loses an electron from position 421. Due to the removal of the electron, the precursor ion 410 is transformed into a CRS 420 (a charge-reduced species). CRS 420 also loses 5 protons at the same positions as the precursor ion 410. Therefore, its total charge is negative 4 and is represented by the notation [M-5H]. ·4- This notation shows that CRS420 has an overall charge of negative 4 and has also lost five protons. Balancing the loss of four protons (which adds a charge of negative 5 to the neutral precursor M) and the overall charge of negative 4 is indicated by the notation [M-5H] 5-Compared to the precursor ion 410, the CRS 420 must also lose an electron. Figure 4A In the notation [M-5H], the location where the electron is lost is indicated by an electron hole in the molecular ion (solid circle with a plus sign). ·4- In the figure, the dot before the charge (4-) in the superscript indicates the presence of an unpaired electron.

[0060] nExD initiated after the loss of the first electron (such as dissociation at position 421) produces a 5' terminal fragment 423 and a 3' terminal fragment 425. Figure 4A In the case of the DNA shown, the 5' terminal fragment 423 is a6 ·2- , and the 3' terminal fragment 425 is w 5 2- The a-type fragment ion 423 has an unpaired electron or electron hole 424 .

[0061] In some embodiments, the CRS itself acts as a precursor ion, as the CRS can undergo one or more additional electron removals and generate species referred to herein as second reduced species. Those second reduced species can then undergo dissociation such as nExD and create fragment ions. Figure 4B The diagram 430 in shows such an nExD process according to some embodiments. More specifically, the diagram 430 shows a CRS 440 generated by removing a first electron from position 441, but not dissociated at this time. The position of the electron hole (here at position 441) can be random on the oligonucleotide chain. The CRS 440 can then lose a second electron from position 451, thereby generating a second reduced species 450, which includes a second unpaired electron or electron hole at position 451. The second reduced species 450 can then undergo fragmentation at position 451. The position of the second electron hole can also be random on the oligonucleotide chain.

[0062] Fragmentation by second electron removal creates two fragment ions, fragment ion 453 and fragment ion 456. Similar to fragment ion 425, fragment ion 456 is On the other hand, although fragment ion 453 lacks three protons similarly to fragment ion 423, it differs from fragment ion 423 in that fragment ion 453 also lacks two electrons at positions 454 and 455, while fragment ion 423 lacks one electron at position 424. Therefore, fragment ion 423 has a charge of negative 2, while fragment ion 453 has a charge of negative 1. To illustrate the difference with fragment ion 423 (shown as ) and the fragment ion 453 is marked with [a 6 -H] -Display. The symbol [a 6 -H] - The following can be interpreted as a convention. The combination of the letter a and the subscript 6 indicates an a-type fragment having 6 bases. In addition, the superscript "-" indicates that fragment 423 has an overall charge of negative 1, which is obtained by comparing the standard a · Fragment a 6 ·- Remove one proton to get, that is, the total number of protons is missing 2, and there is also a lack of electrons.

[0063] Next, consider Figure 4C 460, which is similar to Figure 4B Similarly, a diagram illustrates the process by which a precursor ion first undergoes electron depletion and then undergoes nExD according to some embodiments. Diagram 460 shows that the precursor ion loses electrons by removing electrons from position 471 near the 3' terminal. Due to the removal of electrons, the precursor ion transforms into CRS 470 (which also lacks 5 protons at the same position as the precursor ion) with an overall charge of negative 4. As a result, CRS 470 is labeled [M-5H] ·4- The display indicates that CRS 470 has an overall charge of minus 4, which is obtained by losing 5 protons and one electron.

[0064] Diagram 460 also shows that CRS 470 can also lose another electron at position 482 and generate a second reduced species 480, which can then undergo nExD at position 482. The fragmentation creates two fragment ions, fragment ion 483 and fragment ion 485. Similar to fragment ion 423, fragment ion 483 is The fragment ion 485 comprises six bases and has a charge of negative 2. On the other hand, the fragment ion 485, although similar to the fragment ion 425, lacks two protons, but is different from the fragment ion 425 because the fragment ion 485 also lacks an electron at position 486. Therefore, the fragment ion 425 has a charge of negative 2, while the fragment ion 485 instead has a charge of negative 1. To illustrate the difference between the fragment ion 425 (shown as ) and the fragment ion 485 is marked with [w 5 -H] ·- As mentioned above, the notation [w 5 -H] ·- The indicated fragment ion is of type w, including 5 bases, and lacks 2 protons and 1 electron, thus having a charge of -1.

[0065] The phenomenon of electron depletion of precursor ions or electron depletion of fragments in the above-mentioned CRS to generate fragment ions may cause errors in analyzing mass spectrometry results. Such errors may lead to false positives or false negatives. In some embodiments, the nExD product generated from the precursor species by the first electron separation or the first electron transfer may cause a shift in the profile associated with the fragment. In some embodiments, the shift is caused by the difference between the expected mass of the fragment and the actual mass of the fragment. In some embodiments, this shift is called mass shift, which will be further described below.

[0066] Figure 3 Panel 300 of FIG. 1 illustrates examples of possible errors that may occur and be corrected according to some embodiments. For example, section 320 shows a fragment [a 14 -H] 4- 0 to 4 13 The five peaks 321-325 produced by C isotopes. As explained above, [a 14 -H] 4- The mass is equal to However, the fragment ion with a charge of minus 5 Different, fragment ions [a 14 -H] 4- has a charge of minus 4 (due to the removal of one electron). Therefore, since m / z is mass divided by charge, the fragment ion The m / z value of the fragment ion [a 14 -H] 4- By comparing peak 311 (corresponding to the zeroth isotope, i.e., fragment ion The m / z value of peak 321 (without carbon 13 isotope or monoisotope peak) (approximately 834.1) is similar to that of peak 321 (with fragment ion [a 14 -H] 4- This relationship can be verified by looking at the m / z value of the zeroth isotope of

[0067] Therefore, the fragment ion [a 14 -H] 4- The isotopic peak profile is located at the same position as the fragment ion in the same m / z range because they have the same charge (minus 4 units) and approximately the same mass. 14 -H] 4- The zeroth isotope of isotope of , except for the lack of an additional proton (indicated by the "-H" in brackets). 14 -H] 4- The isotopic peak of peak 321 and the fragment ion The position of the isotope peak is shifted to the left, while the fragment ion The position of the isotope peak of is located at the position of peak 322. The magnitude of the shift, which is the distance between peaks 321 and 322, is equal to the mass difference (1 amu) divided by the magnitude of the charge (here 4). It is worth noting that the magnitude of this shift is the same as the magnitude of the shift between consecutive isotopes of the same charge. Therefore, the fragment ion [a 14 -H] 4- The peaks of the 1st to 4th isotopes are also located in the fragment ion In other words, the fragment ion [a 14 -H] 4- The five peaks 321-325 in the isotopic peak profile can be found by fragment ions The five peaks in the isotopic peak profile of the fragment ion [a] are obtained by shifting them to the left by one shift unit (defined as 1 amu divided by the magnitude of the charge, which for this set of peaks is 1 divided by 4 = 0.25). Therefore, since the shift is much smaller than the m / z value of each peak, the fragment ion [a 14 -H] 4- The isotopic peak profile can be mistaken for fragment ions isotopic peak profile and is incorrectly counted as a fragment ion isotope peak profile.

[0068] Sections 330-350 illustrate fragment a 14 More specifically, in segment 330, peaks 331-335 are respectively associated with [a 14 -2H] 3- The zeroth to fourth isotope of the fragment ion corresponds to the fragment ion after being shifted to the left by two shift units (in this case, the shift unit is 1 divided by 3 = 0.33). The positions of the zeroth to fourth isotopes of (indicated by dotted lines, generally located at positions 333-337) overlap. Similarly, segment 340 shows mass spectrum peaks at positions 341-345, which overlap with [a 14 -3H] 2- These peaks appear shifted to the left by three shift units (in this case, 1 amu divided by 2 = 0.5) and may be miscalculated as fragment ions. The peaks corresponding to the isotopes of (indicated by dotted lines, generally located at positions 344-348). Moreover, segment 350 shows the mass spectrum peaks at positions 351-355, which are consistent with [a 14 -4H] -These peaks appear shifted to the left by 4 shift units (in this case, 1 shift unit) and may be miscalculated as fragment ions. The peaks corresponding to the isotopes of (indicated by dotted lines, usually located at positions 355-349).

[0069] Various embodiments address errors such as those described above that may be introduced when analyzing mass spectra. Figure 5 A block diagram of a mass spectrometry system 500 that collects experimental data and analyzes such data is shown in accordance with some embodiments. More specifically, the mass spectrometry system 500 includes a mass spectrometer 510 and an analyzer module 520.

[0070] The mass spectrometer 510 can collect mass spectrometry data and send it to the analyzer module 520. On the other hand, the analyzer module 520 can receive mass spectrometry data from the mass spectrometer 510 and analyze these data to derive a histogram or intensity of different fragments present in the spectrum. More specifically, the analyzer module 520 can identify the experimental isotopic peaks corresponding to the isotopic species of the fragments of the deprotonated oligonucleotide, detect the CRS generated by the deprotonated oligonucleotide from the peak, or detect one or more expected fragments generated via the nExD of the CRS. In addition, the analyzer module 520 can determine the mass shift associated with the nExD and ultimately identify the isotopic species corresponding to the experimental isotopic peaks.

[0071] For example, the analyzer module 520 can analyze mass spectrometry results, such as the mass spectrometry results in the mass spectrometry panel 300, and accordingly identify isotopic peaks, such as the isotopic peaks shown in the sections 320-350, which correspond to the correct fragments (identified in the description of each section above). Thus, the analyzer module can correct the above-mentioned overcounting or undercounting.

[0072] In particular, Figure 6 600 is a flow chart showing a method 600 for determining a mass shift of a CRS detected by a mass spectrometry system according to some embodiments. Figure 7 A flow chart of a method 700 for determining mass shifts of fragment ions produced by nExD according to some embodiments is shown. In some embodiments, ions generated after one or more electron removals and detected before nExD (as in the case of detected CRS) or after nExD (as in the case of fragment ions 453 or 485) can be generally referred to as electron-removed species (ERS).

[0073] As a first step, at step 602 of method 600 or step 702 of method 700, the analyzer module can determine the charge (Z) of the precursor ion corresponding to the ERS. To do this, the analyzer module can first detect a set of peaks corresponding to the isotopic profile of the precursor ion. The analyzer module can identify such a set of peaks based on their pattern, for example, as a set of peaks that are clustered together compared to other peaks, or a set of peaks that together form the Poisson peak height distribution discussed above.

[0074] The analyzer module can then determine Z by using the relationship shown in equation (1):

[0075] Z=M (13C-12C) / Δm (1).

[0076] In equation (1), M (13C-12C) There are two carbon isotopes ( 13 C and 12 C), and Δm is the difference between the m / z values ​​of consecutive peaks in the isotope profile of the precursor ion.

[0077] Next, the analyzer module can detect the isotope profile. Figure 6 At step 604 of method 600 in FIG. 1 , the analyzer module detects a set of peaks corresponding to the isotopic profile of the CRS. Similarly, in Figure 7 At step 704 of method 700 in , the analyzer module detects one or more groups of peaks corresponding to the isotopic profile of the one or more fragment ions, such as a group of five peaks seen in any of the sections 320 - 350 of panel 300 .

[0078] Next, at step 606 of method 600 or step 706 of method 700, the analyzer module can determine the charge for the corresponding ERS(z). In some embodiments, the analyzer module can determine z in a similar manner as Z, i.e., by using the relationship in equation (1) after interpolating the difference between the m / z values ​​of consecutive peaks in the isotope profile of the previously detected ERS for the value of Δm.

[0079] Next, at step 608 of method 600, the analyzer module 520 determines the mass shift of the CRS. In some embodiments, the analyzer module 520 determines this mass shift by using the relationship shown in equation (2):

[0080] Mass shift for CRS = Zz (2),

[0081] where the mass shift is measured in shift units for CRS, as defined above.

[0082] In some embodiments, equation (2) can be interpreted as follows. The difference between the charge of the precursor ion and the CRS can correspond to the number of electrons removed from the precursor ion to generate the CRS. Moreover, as explained above in the detailed description of sections 320-350 of panel 300, the number of electrons removed can be converted into the number of mass shifts in the corresponding isotope peak profile.

[0083] on the other hand, Figure 7 The remainder of method 700 in FIG. 7 shows steps that an analyzer module may perform to determine one or more mass shifts of one or more fragment ions according to some embodiments. More specifically, at step 708, the analyzer module determines the number (or length) of nucleotides (N) in the analyte oligonucleotide. The analyzer module may determine N by first determining the mass of the precursor ion M using equation (3):

[0084] M=MOZ*Z+M H *Z (3).

[0085] In equation (3), MOZ is the m / z value of the zeroth isotopic peak in the isotopic profile of the precursor ion, Z is the charge of the precursor ion previously determined, and M H is the mass of a hydrogen atom (or proton) measured in atomic mass units (amu).

[0086] The analyzer module can then use the determined M and the known average mass (M) of the nucleotides (e.g., A, T, C, G in the case of DNA and A, U, C, G in the case of RNA) to calculate the average mass of the nucleotides. nt ) to estimate N based on equation (4):

[0087] N≈M / M nt (4).

[0088] Next, at step 710, the analyzer module can determine the location of the dissociation site (i) that results in the creation of a fragment ion from the precursor ion. The analyzer module can determine i by first determining the mass (m) of the fragment ion using equation (5):

[0089] m=moz*z+MH*z (5).

[0090] In equation (5), moz is the m / z value of the zeroth isotopic peak in the isotopic profile of the fragment ion, and z is its previously determined charge. The analyzer module can then estimate i using equation (6):

[0091] i≈m / M nt (6).

[0092] In some embodiments, as described in detail below, the analyzer module needs to determine the value of the ratio of i to N (i / N). In some embodiments, where the candidate sequences of the oligonucleotides and fragment ions are known in advance, the analyzer module can determine the ratio (i / N) based on a first method that uses the exact number of nucleotides in the candidate sequence as N and the exact number of nucleotides in the fragments as the value of i. Otherwise, the analyzer module can determine the ratio based on a second method that combines equations (4) and (6) to derive equation (7) as an approximation of this ratio:

[0093] i / N≈m / M (7).

[0094] The analyzer module can then use the value of the ratio (i / N) determined by one of the first or second methods described above to derive the mass shift of the fragment ion as follows. When no mass shift is induced, which is the case of the first nExD caused by only one electron removal, the analyzer module can estimate the charge of the conventional (or non-shifted) fragment using the approximation shown in equation (8):

[0095] The charge of a regular fragment is ≈(|Z|-1)*i / N(8).

[0096] Based on equation (8), at step 712, the analyzer module can determine the mass shift of the isotopic profile of the fragment ions. In some embodiments, the analyzer module determines this mass shift by using the relationship shown in equation (9):

[0097] The mass shift for a fragment ion ≈ the integer part [z-(|Z|-1)i / N] (9) where the mass shift is measured in the shift units of the fragment ion, as defined above.

[0098] In some embodiments, determining the mass shift for a CRS or fragment enables the analyzer module to correct for the possible shift in the spectral results and accordingly provide a corrected mass shift for the detected CRS or fragment. 13 C profile. Thus, the analyzer module will produce more accurate results for the structure or chemical composition of the oligonucleotide.

[0099] More specifically, in various embodiments, the analyzer module can first enumerate different possible candidate fragments and different possible candidate fragment ions. In addition, using the above method or system, the analyzer module can apply mass shifts to the profiles of the candidate fragments to determine the position of the profiles of the candidate fragment ions. Then, the analyzer module can search for spectral results that match one or more of the predicted profiles of the fragment ions, and if found, these profiles are identified as corresponding to the candidate fragment ions, thereby more accurately determining the structure of the precursor.

[0100] Figure 8300 after the analyzer module determines the mass shift for each isotope of the fragments identified in each segment and applies these mass shifts to the corresponding peaks to determine the correct position of the peaks. 13 C peak profile panel 800, which corresponds to the fragment ions mentioned with respect to panel 300. More specifically, segments 820-850 respectively show the predicted positions of the peaks of the profiles of the fragment ions previously associated with segments 320-350. In each segment, the dotted lines marked with numbers indicate the positions of the corrected peaks for the corresponding isotopes. As seen in each segment, especially segments 820-850, the corrected positions are consistent with the experimental data observed in segments 320-350, respectively. More specifically, the corrected positions of peaks 821-825 are consistent with the experimental positions of peaks 321-325, respectively; the corrected positions of peaks 831-835 are consistent with the experimental positions of peaks 331-335, respectively; the corrected positions of peaks 841-845 are consistent with the experimental positions of peaks 341-345, respectively; and the corrected positions of peaks 851-855 are consistent with the experimental positions of peaks 351-355, respectively. Some of these bins include additional data that is believed to possibly correspond to contamination (eg, as seen in bin 810 around 833.1 moz, bin 830 just below 1390.0 or close to 1392.2, bin 840 just below 2085.0, etc.).

[0101] Thus, the disclosed systems and methods are able to identify and correct errors that may result from the removal of one or more electrons from precursor ions or other species detected by a mass spectrometer.

[0102] In various embodiments, one or more of the disclosed modules may be implemented via one or more computer programs for performing the functions of the corresponding modules or via a computer processor executing those programs. In some embodiments, one or more of the disclosed modules may be implemented via one or more hardware units that execute firmware for performing the functions of the corresponding modules. In various embodiments, one or more of the disclosed modules may include a storage medium for storing data used by the module or a software or firmware program executed by the module. In various embodiments, one or more of the disclosed modules or the disclosed storage medium may be inside or outside the disclosed system. In some embodiments, one or more of the disclosed modules or storage media may be implemented via a computing "cloud", and the disclosed system is connected to the computing "cloud" via a network connection and uses external modules or storage media accordingly. In some embodiments, the disclosed storage medium for storing information may include a non-transient computer-readable medium, such as a CD-ROM, a computer memory, for example, a hard disk or a flash memory. In addition, in various embodiments, one or more of the storage media may be a non-transient computer-readable medium that stores data or computer programs executed by various modules, or implements various techniques or flow charts disclosed herein.

[0103] Fig. 9 Schematically depicted is an example of an implementation of a module 900 according to some embodiments. The module 900 comprises a processor 910 (e.g., a microprocessor), at least one permanent memory module (e.g., ROM 920), at least one transient memory module (e.g., RAM) 930, a bus 940, and a communication module 950.

[0104] Processor 910, ROM 920, and RAM 930 may be used to store and execute instructions for performing the functions of module 900. In addition, bus 940 may allow communication between the processor and various other components of the controller. Communication module 950 may be configured to allow sending and receiving signals.

[0105] Although some aspects have been described in the context of systems and / or devices, it is clear that these aspects can also represent the description of corresponding methods, where blocks or devices correspond to method steps or features of method steps. Similarly, the aspects described in the context of method steps also represent the description of corresponding blocks or items or features of corresponding devices. Some or all of the method steps can be performed by (or using) hardware devices such as processors, microprocessors, programmable computers or electronic circuits. In some embodiments, some or more of the most important method steps can be performed by such devices.

[0106] Those skilled in the art will appreciate that various changes can be made to the above embodiments without departing from the scope of the present invention.

[0107] The above detailed description refers to the accompanying drawings. The same or similar reference numerals may have been used in the drawings or the specification to refer to the same or similar parts. Moreover, unless otherwise specified, similarly named elements may perform similar functions and may be similarly designed. The details are set forth to provide an understanding of the exemplary embodiments. Embodiments may be practiced without some of these details, such as alternative embodiments. In other cases, well-known techniques, processes, and components are not described in detail to avoid confusing the described embodiments.

[0108] The description of the aforementioned embodiment is given for the purpose of illustration only. It is not exhaustive and does not limit the embodiment to the disclosed precise form. Although several exemplary embodiments and features are described, modifications, adjustments and other implementations may also be possible without departing from the spirit and scope of the embodiment. Therefore, unless otherwise clearly stated, the description relates to one or more embodiments and should not be interpreted as limiting the embodiment to an integral whole. Whether the present disclosure states that a feature is related to "one", "the", "one", "one or more", "some" or "each" embodiment, this is correct. As used herein, the singular forms "one", "an" and "the" may include plural forms, unless the context clearly indicates otherwise. In addition, the term "coupling" does not exclude the presence of intermediate elements between coupling items. Moreover, stating that a certain feature may exist indicates that the feature may exist in one or more embodiments.

[0109] In this disclosure, the terms "comprises," "including," "contains," and "having," when used after a set or system, mean open inclusion and do not exclude the addition of other, unrecited members to the set or system. Furthermore, unless otherwise stated or inferred from the context, the conjunction "or," if used, is not exclusive but rather inclusive meaning and / or. Moreover, if these terms are used, a subset of a set may include one or more than one member of the set, including all members.

[0110] Additionally, if used in this disclosure, and unless otherwise specified or inferred, if a first variable does not decrease but generally increases when a second variable increases, then the first variable is an increasing function of the second variable. On the other hand, if the first variable does not increase but generally decreases when the second variable increases, then the first variable is a decreasing function of the second variable. In some embodiments, if the first variable is directly proportional or inversely proportional to the second variable, then the first variable can be an increasing function or a decreasing function of the second variable.

[0111] The disclosed systems, methods and devices are not limited to any specific aspect or feature or combination thereof, nor do the disclosed systems, methods and devices require the existence of any one or more specific advantages or solutions to problems. Any operating theory is for ease of explanation, but the disclosed systems, methods and devices are not limited to such operating theories.

[0112] In light of the above teachings, modifications and variations are possible, or may be obtained by practicing the embodiments. For example, the steps described need not be performed in the same order discussed or with the same degree of separation. Similarly, individual steps may be omitted, repeated, combined, or performed in parallel as needed to achieve the same or similar purposes. Similarly, the described system does not necessarily include all the parts described in the embodiments, and may also include other parts not described in the embodiments. Thus, the embodiments are not limited to the above details, but are defined by the appended claims according to the full scope of their equivalents. In addition, the present disclosure is directed to all novel and non-obvious features and aspects of the various disclosed embodiments, individually and in various combinations and sub-combinations with each other.

[0113] Although the present disclosure has been particularly described in conjunction with specific embodiments, many alternatives, modifications and variations will be apparent from the foregoing description. Therefore, it is intended that the appended claims will cover any such alternatives, modifications and variations that fall within the true spirit and scope of the present disclosure.

Claims

1. A method for analyzing a mass spectrum of a deprotonated oligonucleotide, the method comprising: identifying experimental isotope peaks corresponding to precursor ions generated from deprotonated oligonucleotides; determining one or more characteristics of the precursor ion; identifying experimental isotope peaks corresponding to fragment ions generated from precursor ions; determining one or more characteristics of the fragment ions; selecting candidate fragments for fragment ions; determining a mass-shifted isotope peak of the candidate fragment based on data including one or more characteristics of the precursor ion and one or more characteristics of the fragment ion; comparing an experimental isotope peak corresponding to the fragment ion and a mass-shifted isotope peak of the candidate fragment; and The fragment ions are identified as candidate fragments based on the comparison.

2. The method according to claim 1, wherein: Fragment ions are generated from precursor ions due to negative electron activated dissociation.

3. The method according to any one of claims 1 to 2, wherein: The one or more characteristics of the precursor ion include a charge of the precursor ion and a mass of the precursor ion. The method of claim 3 , further comprising determining a length of the precursor ion based on a mass of the precursor ion.

5. The method according to any one of claims 1 to 4, wherein: The one or more characteristics of the fragment ions include the charge of the fragment ions and the mass of the fragment ions. The method according to claim 5 , further comprising determining the dissociation sites corresponding to the fragment ions based on the masses of the fragment ions.

7. The method according to any one of claims 1 to 6, wherein: Determining the mass-shifted isotopic peak of a candidate fragment includes estimating the mass shift equal to the integer part of [z-(|Z|-1)i / N], where z is the charge of the fragment ion, i is the number of nucleotides in the fragment ion, |Z| is the absolute value of the charge of the precursor ion, and N is the number of nucleotides in the precursor ion.

8. The method according to any one of claims 1 to 7, wherein: The deprotonated oligonucleotides are multiply deprotonated.

9. The method according to any one of claims 1 to 8, wherein: Fragment ions are generated from precursor ions after two or more electrons are removed from the precursor ions.

10. The method according to claim 9, wherein: Fragment ions are generated from precursor ions due to negative electron activated dissociation after two or more electrons are removed from the precursor ions.

11. A system for analyzing a mass spectrum of a deprotonated oligonucleotide, the system comprising: a mass spectrometer configured to collect mass spectrometry data of the deprotonated oligonucleotide; as well as an analyzer module configured to receive the mass spectrometry data and analyze the mass spectrometry data, wherein analyzing the mass spectrometry data comprises: identifying experimental isotope peaks corresponding to precursor ions generated from deprotonated oligonucleotides; determining one or more characteristics of the precursor ion; identifying experimental isotope peaks corresponding to fragment ions generated from precursor ions; determining one or more characteristics of the fragment ions; selecting candidate fragments for fragment ions; determining a mass-shifted isotope peak of the candidate fragment based on data including one or more characteristics of the precursor ion and one or more characteristics of the fragment ion; comparing an experimental isotope peak corresponding to the fragment ion and a mass-shifted isotope peak of the candidate fragment; and The fragment ions are identified as candidate fragments based on the comparison.

12. The system according to claim 11, wherein: Fragment ions are generated from precursor ions due to negative electron activated dissociation.

13. The system according to any one of claims 11-12, wherein: The one or more characteristics of the precursor ion include a charge of the precursor ion and a mass of the precursor ion.

14. The system according to claim 13, wherein: Analyzing the mass spectrometry data also includes determining the length of the precursor ion based on the mass of the precursor ion.

15. The system according to any one of claims 11 to 14, wherein: The one or more characteristics of the fragment ions include the charge of the fragment ions and the mass of the fragment ions.

16. The system of claim 15, wherein: Analyzing the mass spectrometry data also includes determining dissociation sites corresponding to the fragment ions based on the masses of the fragment ions.

17. The system according to any one of claims 11 to 16, wherein: Determining the mass-shifted isotopic peak of a candidate fragment includes estimating the mass shift equal to the integer part of [z-(|Z|-1)i / N], where z is the charge of the fragment ion, i is the number of nucleotides in the fragment ion, |Z| is the absolute value of the charge of the precursor ion, and N is the number of nucleotides in the precursor ion.

18. The system according to any one of claims 11 to 17, wherein: The deprotonated oligonucleotides are multiply deprotonated.

19. The system according to any one of claims 11 to 18, wherein: Fragment ions are generated from precursor ions after two or more electrons are removed from the precursor ions.

20. The system of claim 19, wherein: Fragment ions are generated from precursor ions due to negative electron activated dissociation after two or more electrons are removed from the precursor ions.

Images