Electron detachment method and device for oligonucleotide analysis in mass spectrometry

Abstract

Mass spectrometry systems and methods are disclosed. In some embodiments, the mass spectrometry system comprises an ion trap configured to receive a plurality of precursor ions and to load the plurality of precursor ions in a trap region; an electron gun configure to perform an irradiation operation by applying an electron beam to the trap region to produce fragmented ions; an RF gate configured to perform an extraction operation by extracting, from the trap region, a high m / z subset of the fragmented ions with m / z ratios greater than a specific value; and a mass spectrometer configured to perform mass spectrometry analysis on a subset of the fragmented ions remaining in the trap region, wherein an irradiation-extraction operation, including performing the irradiation operation and the extraction operation, is repeated at least twice before performing the mass spectrometry analysis on the subset of the fragmented ions remaining in the trap region.

Description

ELECTRON DETACHMENT METHOD AND DEVICE FOR OLIGONUCLEOTIDEANALYSIS IN MASS SPECTROMETRYRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 534,241 filed on August 23, 2023, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to sequencing of polynucleotides, and in particular, sequencing of RNA and RNA oligonucleotides.BACKGROUND

[0003] Mass spectrometry (MS) is an analytical technique utilized for quantitative and qualitative analysis of some substances such as pharmaceutical products. Specifically, MS may measure a mass-to-charge ratio (m / z) of ions generated from a test substance. MS may be used to identify unknown compounds, to determine isotopic composition of elements in a molecule, to determine the structure of a particular compound by observing its fragmentation (hereinafter also alternatively called dissociation), and / or to quantify the amount of a particular compound in a sample.

[0004] Mass spectrometers detect ions and, as such, a test sample must be converted to an ionic form during mass analysis. Generally, a mass spectrometer may include an ion source and a mass analyzer. The ion source converts a test sample into gaseous ions and the mass analyzer obtains mass spectra based on their m / z. In some cases, the mass spectrometer may further include one or more isolation devices installed between the ion source and the mass analyzer; or additionally one or more dissociation device between the isolation device and the mass analyzer.

[0005] A mass spectrometer may employ dissociation to cause the fragmentation of large analytes (e.g., oligonucleotides, DNA, RNA, etc.) into smaller fragment ions (e.g., fragments containing individual nucleobases). These smaller fragment ions may then be mass analyzed and quantified based on their m / z ratios. The results may be utilized to determine the composition or the structure of the analyzed substance.

[0006] For dissociation, different techniques may be used. For example, a common dissociation technique for biomolecule analysis is the collision induced dissociation (CID). Further, some other techniques that utilize radical driven dissociation methods may be used to obtain information that complement information derived from CID. Such other techniques may include electron capture dissociation (ECD), electron transfer dissociation (ETD), electron detachment dissociation (EDD), and / or photo dissociation using UV laser (UVPD).

[0007] The process of the electron based dissociations may, however, generate a cascade of charge reduced precursor species. For example, after a precursor loses one electron, the resultant precursor ion may be dissociated or may, before being dissociated, lose one or more electrons. Therefore, multiple offspring of the precursors with different charges but the same molecular weight may coexist and appear in the spectrometry results. Moreover, the produced fragments from the different charge-reduced species may have different m / z values, although the dissociation sites and the fragment charge states are the same since the masses are different. The mass differences may be a few multiples of the hydrogen mass, i.e., the m / z values may differ by a few units (-1, -2, -3, etc.). Further, in sequencing of RNAs, in particular, the m / z values for cytosine (C) and uracil (U) nucleotides differ by 1 unit. At the same time, EDD may produce hydrogen shifted fragments for which the m / z values also differ by a few units. This similarity of two different cases may make differentiating between them difficult, thus introducing challenges in analyzing some substances, such as RNA oligonucleotides, in conventional systems.

[0008] Accordingly, it is desirable to devise new systems and methods for confident sequencing of polynucleotides, such as RNA oligonucleotides, including confident de novo sequencing of such polynucleotides.SUMMARY

[0009] Some embodiments relate to a mass spectrometry method including: loading a plurality of precursor ions in a trap region; performing an iteration of irradiation-extraction operations, wherein each irradiation-extraction operation in the iteration includes: applying an electron beam to the trap region to produce fragmented ions, and extracting, from the trap region, a high m / z subset of the fragmented ions with m / z ratios greater than a specific value; and performing mass spectrometry analysis on a subset of the fragmented ions remaining in the trap region, wherein the iteration includes at least two irradiation-extraction operations.

[0010] Some embodiments relate to a method, wherein the specific value is an m / z value of the precursor ion.

[0011] Some embodiments relate to a method, wherein: the loading lasts for a loading duration; and the loading duration depends on a magnitude of charge accumulated in the trap region.

[0012] Some embodiments relate to a method, wherein the magnitude of charge accumulated in the trap region is determined based on a mass spectrometry spectrum of accumulated charge during a preliminary operation.

[0013] Some embodiments relate to a method, wherein the loading duration is inversely related to an intensity of a precursor ion current.

[0014] Some embodiments relate to a method, wherein the loading duration is inversely proportional to the intensity of the precursor ion current.

[0015] Some embodiments relate to a method, wherein the loading duration is determined based on a mass spectrometry spectrum of the precursor ion current during a preliminary operation.

[0016] Some embodiments relate to a method, further including a pausing after applying the electron beam and before extracting the ions.

[0017] Some embodiments relate to a method, wherein a duration of the pausing is less than 5 milliseconds.

[0018] Some embodiments relate to a method, wherein after the extracting, a majority of fragmented ions with m / z values lower than the specific value remain in the trap region.

[0019] Some embodiments relate to a method, further performing a nucleotide sequence determination of the precursor ions.

[0020] Some embodiments relate to a method, wherein the precursor ions are polynucleotides.

[0021] Some embodiments relate to a method, wherein the polynucleotide is an oligonucleotide.

[0022] Some embodiments relate to a method, wherein a z value of the oligonucleotide is in a range of 5-15.

[0023] Some embodiments relate to a method, wherein the polynucleotide is an RNA.

[0024] Some embodiments relate to a method, wherein: the electron beam is applied for an irradiation duration; and the irradiation duration depends on an intensity of second charge reduced ions in the trap region.

[0025] Some embodiments relate to a method, wherein the irradiation duration is determined so that after applying the electron beam, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

[0026] Some embodiments relate to a method, wherein the irradiation duration is in a range of 1-300 ms.

[0027] Some embodiments relate to a method, wherein: the electron beam is also applied at an irradiation intensity; and the irradiation intensity also depends on the intensity of the second charge reduced ions in the trap region.

[0028] Some embodiments relate to a method, wherein the irradiation duration and the irradiation intensity are determined so that after applying the electron beam, the intensity ofthe second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

[0029] Some embodiments relate to a method, wherein: the electron beam is applied at an irradiation intensity; and the irradiation intensity depends on an intensity of second charge reduced ions in the trap region.

[0030] Some embodiments relate to a method, wherein the irradiation intensity is determined so that after applying the electron beam, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

[0031] Some embodiments relate to a method, wherein the irradiation electron beam intensity (or current) is in a range of 0.01 pA to 10 |1A.

[0032] Some embodiments relate to a mass spectrometry system including: an ion trap configured to receive a plurality of precursor ions and to load the plurality of precursor ions in a trap region; an electron gun configured to perform an irradiation operation by applying an electron beam to the trap region to produce fragmented ions; an RF gate configured to perform an extraction operation by extracting, from the trap region, a high m / z subset of the fragmented ions with m / z ratios greater than a specific value; and a mass spectrometer configured to perform mass spectrometry analysis on a subset of the fragmented ions remaining in the trap region, wherein: an irradiation-extraction operation, including performing the irradiation operation and the extraction operation, is repeated at least twice before performing the mass spectrometry analysis on the subset of the fragmented ions remaining in the trap region.

[0033] Some embodiments relate to a system, wherein the specific value is an m / z value of the precursor ion.

[0034] Some embodiments relate to a system, wherein: the loading lasts for a loading duration; and the loading duration depends on a magnitude of charge accumulated in the trap region.

[0035] Some embodiments relate to a system, wherein the mass spectrometer is configured to determine the magnitude of charge accumulated in the trap region based on a mass spectrometry spectrum of accumulated charge during a preliminary operation.

[0036] Some embodiments relate to a system, wherein the loading duration is inversely related to an intensity of a precursor ion current.

[0037] Some embodiments relate to a system, wherein the loading duration is inversely proportional to the intensity of the precursor ion current.

[0038] Some embodiments relate to a system, wherein the loading duration is determined based on a mass spectrometry spectrum of the precursor ion current during a preliminary operation.

[0039] Some embodiments relate to a system, wherein the RF gate is further configured to pause after the irradiation operation before performing the extraction operation.

[0040] Some embodiments relate to a system, wherein a duration of the pause is less than 5 milliseconds.

[0041] Some embodiments relate to a system, wherein after the extraction operation a majority of fragmented ions with m / z values lower than the specific value remain in the trap region.

[0042] Some embodiments relate to a system, wherein: the electron gun is configured to perform the irradiation operation for an irradiation duration; and the irradiation duration depends on an intensity of second charge reduced ions in the trap region.

[0043] Some embodiments relate to a system, wherein the irradiation duration is determined so that after the irradiation operation, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

[0044] Some embodiments relate to a system, wherein the irradiation duration is in a range of 1-300 ms.

[0045] Some embodiments relate to a system, wherein: the electron gun is further configured to perform the irradiation operation at an irradiation intensity; and the irradiation intensity also depends on the intensity of the second charge reduced ions in the trap region.

[0046] Some embodiments relate to a system, wherein the irradiation duration and the irradiation intensity are determined so that after applying the electron beam, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

[0047] Some embodiments relate to a system, wherein: the electron gun is configured to perform the irradiation operation at an irradiation intensity; and the irradiation intensity depends on an intensity of second charge reduced ions in the trap region.

[0048] Some embodiments relate to a system, wherein the irradiation intensity is determined so that after the irradiation operation, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

[0049] Some embodiments relate to a system, wherein the irradiation intensity (or electron beam current) is in a range of 0.01-10 pA.

[0050] Further understanding of various aspects of the embodiments may be obtained by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The drawings are not necessarily to scale or exhaustive. Instead, emphasis is generally placed upon illustrating the principles of the embodiments described herein. The accompanying drawings, which are incorporated in this specification and constitute a part of it, illustrate several embodiments consistent with the disclosure. Together with the description, the drawings serve to explain the principles of the disclosure.

[0052] In the drawings:

[0053] FIG. 1 is a block diagram of a dissociation system 100 according to some embodiments.

[0054] FIG. 2 shows a mass spectrum 200 illustrating generation of some undesired ions as addressed in some embodiments.

[0055] FIG. 3 shows a mass spectrum 300 after one application of the revised electron beam according to some embodiments.

[0056] FIG. 4 shows a flow chart for a recursive method 400 as performed by some embodiments.

[0057] FIG. 5 shows an extraction intensity diagram 500 according to some embodiments.

[0058] FIG. 6 is a mass spectral diagram 600 illustrating the effect of repeating the irradiation-extraction operation up to the desired state according to some embodiments.

[0059] FIGS. 7A and 7B illustrate the effect of the disclosed techniques utilized by different embodiments compared to the conventional techniques.

[0060] FIG. 8A illustrates the problem of variability of the optimum extraction voltage addressed by some embodiments.

[0061] FIG. 8B illustrates the results of utilizing the charge control techniques to address the problem of variability of the optimum extraction voltage according to some embodiments.DETAILED DESCRIPTION

[0062] It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of thespecifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

[0063] The following detailed description refers to the accompanying drawings. The same or similar reference numbers may have been used in the drawings or in the description to refer to the same or similar parts. Also, similarly named elements may perform similar functions and may be similarly designed, unless specified otherwise. Details are set forth to provide an understanding of the exemplary embodiments. Embodiments, e.g., alternative embodiments, may be practiced without some of these details. In other instances, well known techniques, procedures, and components have not been described in detail to avoid obscuring the described embodiments.

[0064] Various embodiments address the above mentioned disadvantages of conventional systems by providing mass spectrometry systems that include mechanisms for controlled fragmentation of precursor ions before introducing the fragments into a mass analyzer.

[0065] FIG. 1 is a block diagram of a dissociation system 100 according to some embodiments. Dissociation system 100 includes an ion trap 130, an electron gun 120, and an ion-guide 150. In some embodiments, dissociation system 100 may be an electron activated dissociation (EAD) device.

[0066] Ion trap 130 includes a trap region 132. Ion trap 130 is configured to receive ions, such as precursor ions, and trap the received ions in the trap region 132. In various embodiments, the precursor ions may correspond to one or more polynucleotides, including oligonucleotides or RNA molecules.

[0067] In addition to the trap region, the ion trap may include mechanisms for selectively releasing ions from the ion trap. In the embodiment shown in FIG. 1, for example, ion trap 130 includes an RF gate 140 that comprises an extraction voltage generator 142 and an RF generator 144. In various embodiments, a DC voltage applied to extraction voltage generator142 generates an electric field that drives some of the ions toward exiting the trap region and entering ion-guide 150. Moreover, an AC voltage applied by RF generator 144 may create a potential barrier, the height of which may be controlled by different characteristics of the AC voltage, such as its amplitude, and thus selectively allowing a subset of the driven ions to exit the ion trap.

[0068] Electron gun 120 is configured to apply an electron beam 122 to trap region 132 causing some of the precursor ions to undergo electron detachment dissociation (EDD). Electron beam 122 may also generate some undesired charge reduced specie (CRS) ions, further described below.

[0069] Ion-guide 150 is configured to receive a subset of the ions from trap region 132 and send them to a mass spectrometer. In some embodiments, those extracted ions may be further analyzed by the mass spectrometer. In some embodiments, ion-guide 150 may be an IQ2B lens.

[0070] In some applications, electron gun 120 may apply electron beam 122 to trap region 132 at a high intensity or for a long duration of time, causing generation of undesired charge reduced species of higher orders, as further explained next.

[0071] FIG. 2 shows a mass spectrum 200 illustrating generation of some undesired ions as addressed in some embodiments. More specifically, mass spectrum 200 may correspond to a subset of the ions in an ion trap after the precursor ions have been exposed to an electron beam with high intensity or for a long duration. In various mass spectra, such as mass spectrum 200, the horizontal axis shows the mass to charge ratio (m / z) of ions (throughout this disclosure in unitless values, in which m is measured in units of Daltons, Da, and Z is measured in units of the absolute value of the electron charge, e). The vertical axis, on the other hand, shows the intensity of detected ions at a given m / z in units of count per second (cps).

[0072] Mass spectrum 200 includes multiple spectral peaks including peaks 210 and 221- 224. Peak 210, at m / z value around 750, corresponds to the intensity of precursor ions. Peaks 221-224, on the other hand, correspond to charge-reduced ions (hereinafter alternatively called charge-reduced species or CRS). More specifically, peaks 221, 222, 223, and 224(respectively located at m / z values around 850, 1000, 1200, and 1500) respectively correspond to the first, second, third, and fourth CRS.

[0073] Regarding FIG. 2, noteworthy is that mass spectrum 200 may illustrate a subsection of the complete mass spectrum of the ions in the ion trap. More specifically, the complete mass spectrum may include additional spectral peaks that are not shown in mass spectrum 200. Some of those peaks may be located outside the range shown in mass spectrum 200 at, for example, m / z values lower than 300. One or more of the peaks at m / z values below the m / z value of the precursor ion (around 750 in mass spectrum 200) may correspond to fragment ions of interest alternatively called desirable ions. Determining the intensities of the desirable ions (as well as the intensity of the precursor ion) may be utilized to determine the characteristics of the analyzed substance.

[0074] As is seen from FIG. 2, unlike the peaks corresponding to the desired ions, CRS peaks 221-224 have m / z values that are greater than the m / z value of precursor ion peak 210. As such, these charge-reduced ions may be fragmented to smaller ions by the next electron detachment process and cause analysis problems by interfering with the mass spectrometry analysis of the fragments of the precursor ions.

[0075] Some embodiments address the above problems by revising one or more characteristics of the electron beam and, further, by removing higher order CRS ions from trap region 132, as detailed below. For example, some embodiments change the duration or intensity of the applied electron beam. More specifically, referring back to FIG. 1, once precursor ions enter trap region 132, electron gun 120 may apply electron beam 122 to the trap region 132 for a short period of time or at low intensity.

[0076] In various embodiments, the appropriate intensity or exposure duration of the electron beam may be determined based on various factors such as the density of the ions in the trap region or the charge state of the precursor ions. For example, in some embodiments, the appropriate exposure duration may be in the range of 1-50 ms. Similarly, in some embodiments, the appropriate intensity of the electron beam may be in the range of 0.01-10 A.

[0077] After each such application of the electron beam, there may exist fewer mass spectral peaks related to undesired ions, such as undesired CRS ions, and each such peak may have a lower intensity compared with what was shown in FIG. 2, as explained below.

[0078] FIG. 3 shows a mass spectrum 300 after one such application of the revised electron beam according to some embodiments. More specifically, mass spectrum 300 shows three intensity peaks, i.e., a precursor ion peak 310, a first CRS peak 321, and a second CRS peak 322, respectively located at m / z values about 750, 850, and 1000. Mass spectrum 300 shows that, as compared to mass spectrum 200 in FIG. 2, of the multiple possible orders of CRS, the revised electron beam only generates a considerable amount of the first CRS (identified by first CRS peak 321), while the intensity of higher order CRS are either negligible (very weak second CRS peak 322) or nonexistent. The absence of higher order CRS may thus prevent undesired dissociation from higher order CRSs, which may produce undesired hydrogen mass shifted fragments.

[0079] In order to prevent undesirable fragmentation of the higher order CRS ions, and in addition to revising the electron beam to avoid or reduce generation of higher order CRS ions, some embodiments further remove the generated undesired CRS ions from the trap region before another application of the electron beam. More specifically, after each irradiation by the revised electron beam, the system may extract the product ions that have m / z values higher than the m / z value of the precursor ion. These extracted product ions may thus include the first CRS.

[0080] In particular, referring back to FIG. 1, after an application of the revised electron beam, RF gate 140 may extract from the trap region a high m / z subset of the ions in the trap region, that is, a subset or all of the ions with m / z values that are greater than a specific value. Such an extraction may, therefore, exclude undesired fragmented ions from the rest of the process. In particular, the specific value may be the m / z value of the precursor ion. As described in relation to mass spectrum 300, these ions typically correspond to the undesired CRS. In the embodiment shown in FIG. 1, for example, the extraction may be achieved by selecting appropriate values for parameters such as the magnitude of the DC voltage applied by extraction voltage generator 142 and the amplitude of the AC voltage applied by RF generator 144. In particular, these values may be selected such that the higher m / z value ionsencounter a lower potential barrier and can escape the ion trap, while the lower m / z value ions encounter a higher potential barrier and remain trapped in the ion trap. In some embodiments, the combination of those parameters determines the threshold m / z value, such that ions with m / z values under that threshold value remain trapped and ions with m / z values above that threshold can escape, i.e., are extracted, from the ion trap.

[0081] Some embodiments may repeat these operations of irradiation followed by extraction multiple times. Utilizing this repeated cycle, the system may therefore consume precursor ion to generate the desired fragments while preventing electron detachment and electron detachment dissociation of the CRS, which may generate the undesired hydrogen shifted fragments. Various embodiments may therefore apply the above-described repeated cycles through a recurring irradiation-extracting operation. FIG. 4 shows a flow chart for a recursive method 400 as performed by some embodiments. Method 400 may be performed by a combination of a dissociation system such as system 100 and a mass analyzer such as a mass spectrometer.

[0082] At a loading step 402 of the method 400, the system receives and traps a plurality of precursor ions in an ion trap. The plurality of precursor ions may be derived from a sample under analysis. In various embodiments, the loading step may last for a loading duration during which the system accumulates the precursor ions in the trap region. In some embodiments, the loading duration is set to be equal or less than a maximum loading duration. In various embodiments, the loading duration or the maximum loading duration may be determined based on different factors such as a desired amount of the accumulated charge or a maximum amount of the accumulated charge in the trap region. The accumulated charge may result from the accumulation of the precursor ions in the trap region. These techniques are further described below.

[0083] Next, at an irradiation step 404, the system starts one iteration of an irradiationextraction operation, which includes irradiation step 404, an extraction step 406, and a decision step 408, as detailed below.

[0084] More specifically, at irradiation step 404, the system applies an electron beam to ions that are trapped in the trap region. In various embodiments, the intensity and / or theduration of the electron beam may be adjusted in the manner described above to avoid or to minimize generation of undesired ions such as high order CRS ions.

[0085] Next, at extraction step 406, the system extracts a subset of the ions in the trap region, tentatively called a high m / z subset of the ions. The high m / z subset of the ions are ions with m / z values that are larger than a specific m / z value. In some embodiments, the specific m / z value is selected to be the m / z value of the precursor ion, as also explained above. In some embodiments, some or all of the ions that are extracted during one or more of the iterations may be mass analyzed to, for example, derive some information about the precursor ions. In some embodiments, after the extraction, a majority of fragmented ions with m / z values lower than the specific value remain in the trap region.

[0086] In some embodiments, in one or more iterations (or even all iterations), the system may include a pause time between the irradiation step and the extraction step. That is, after completion of the irradiation step, the system may wait for the duration of the pause time before performing the extraction step. The pause may, for example, allow the ions in the trap region to reach a steady state more suitable for the extraction step. In some embodiments, each of these three operations (irradiation, pause, extraction) may last for one or more milliseconds. For example, the durations may be 1 ms for the irradiation, 3 ms for the pause, and 2 ms for the extraction. Therefore, in some embodiments, each iteration, which includes these three operations, may last for a few milliseconds, for example, 5 to 10 ms.

[0087] In some embodiments, in order to extract the undesired ions while leaving the desired ions in the trap region, the system may utilize relations between the extraction intensity of different ions with respect to the extraction voltage.

[0088] FIG. 5 shows an extraction intensity diagram 500 that may be utilized by a system for determining the extraction voltage to achieve the selective extraction of undesired ions according to some embodiments. In an extraction intensity diagram such as diagram 500, the X axis corresponds to the values of the extraction voltage in volts (v) and the Y axis corresponds to the intensity of the extracted ions at each extraction voltage in cps. Moreover, different curves in the extraction intensity diagram correspond to the intensity-extraction voltage relations for different extracted ions.

[0089] More specifically, extraction intensity diagram 500 includes 4 curves 510, 520, 530, and 540 respectively corresponding to extraction voltage intensities for the precursor ion, the CRS, the high m / z ion products, and the mid m / z ion products. All three curves 520, 530, and 540 correspond to undesired products that have m / z values higher than the m / z value of the precursor ion. As seen in extraction intensity diagram 500, extraction of different ions may reach a high intensity at different values of the extraction voltage. For example, as seen in extraction intensity diagram 500, the extraction of the CRS (CRS curve 520) reaches its maximum intensity around the extraction voltage of 0.5 v, while the extraction of the precursor ion (precursor ion curve 510) reaches its maximum intensity around the extraction voltage of 1.5 v. Moreover, the extraction of the mid m / z ion products (curve 540) and the high m / z products (curve 530) is also at a plateau level at the extraction voltage of 0.5 v.

[0090] The system may utilize the above-described differences among the extraction voltage intensities of different ions to determine an optimum extraction voltage enabling selective extraction of a large proportion of some ions while leaving a large proportion of other ions in the trap region. The system may achieve the optimum extraction voltage through the appropriate combination of the parameters of the DC voltage and the AC voltage at RF gate 140. In particular, in some embodiments, the AC voltage provides a potential barrier that has an inverse linear m / z dependence; that is, for an ion with a low m / z value the potential barrier is proportionally higher than the potential barrier for an ion with a high m / z value. The DC voltage, on the other hand, may provide an attractive force and thus lower the potential barrier provided by the AC voltage, with no m / z dependence. The combination of these two effects establishes a high m / z pass filter.

[0091] For example, based on the above-discussed differences in the intensity-extraction voltage relations among different ions seen in diagram 500, extraction voltage generator 142 may set an optimum DC extraction voltage of around 0.5 v to extract the CRS (at an intensity around 80,000 cps as seen for curve 520 at 0.5 v) without extracting a significant amount of the precursor ion (reflected in precursor ion curve 510 having the relatively low intensity of 10,000 cps at 0.5 v). Moreover, based on curves 530 and 540, an extraction voltage of 0.5 v also extracts a significant amount of the undesired mid m / z and high m / z ion products.

[0092] Returning to recursive method 400 in FIG. 4, at decision step 408, the system may determine whether another irradiation-extraction iteration is desired or possible, or otherwise the iterations should be ended. In some embodiments, this determination may depend on one or more factors such as factors detailed below. For example, the system may pre-determine a maximum number of desired iterations and then, while performing recursive method 400, at decision step 408, the system may decide to repeat the iterations until the number of iterations reaches that maximum number. In some embodiments, the maximum number of iterations may be based on the consumption of precursor ions. When all or a certain percentage of the precursor ions are consumed, there may be no further production of useful ion fragments needed for MS analysis.

[0093] In some embodiments, in each subsequent iteration, the electron beam may have less intensity or duration from the previous iteration In some embodiments, the system may reduce the intensity of the electron beam in this manner because a smaller concentration of precursor ions will be available in each successive repetition of irradiation followed by and extraction of CRS. As mentioned above, eventually the population of precursor ions may be partially or completely consumed, and the iterations end.

[0094] If, at decision step 408, the system determines that further irradiation and extraction is to be performed, method 400 loops back to irradiation step 404 for performing another iteration of the irradiation-extraction operation. Repetition of this operation may therefore increase the density of the desired ions in the trap region, as illustrated below in the detailed description of FIG. 6.

[0095] Otherwise, if the system determines that no further irradiation and extraction is needed or possible, the system may therefore end the iterations and proceed to an analysis step 410.

[0096] At analysis step 410, the system may extract remaining ions, that is, some or all of the ions that have remained in the ion trap, and analyze them via, for example, the mass spectrometer. The analysis may determine mass spectrometry characteristics of the remaining ions and accordingly analyze the precursor ion. At this point, because of the preceding iterative irradiation and extraction steps, there will be no or very few higher order CRS ionsleft in the system. The analysis may, for example, determine the chemical composition, the nucleotide sequence, or the structure of the precursor ion.

[0097] FIG. 6 is a mass spectral diagram 600 illustrating the effect of repeating the irradiation-extraction operation up to the desired state explained above according to some embodiments. More specifically, to generate diagram 600, method 400 was implemented four different times The four implementations differed by the number of times that the irradiationextraction operation was performed before ending the iterations and performing analysis step 410. In particular, the irradiation-extraction operation was performed one time for the first implementation, two times for the second implementation, five times for the third implementation, and ten times for the fourth implementation.

[0098] As a result, diagram 600 includes an overlap of four spectra 610, 620, 630, and 640 corresponding to the spectra derived for the ions in the trap region respectively after the first to fourth implementation. All four spectra include five peaks located at m / z values that are approximately 1078.2, 1078.7, 1079.2, 1079.7, and 1080.2. The first peak, at 1078.2, has approximately the same height (intensity) in all four spectra and corresponds to the detection of a non-radical a-H type ion. As such, these peaks do not represent a -H product by second electron detachment.

[0099] The 2nd to 5th peaks, on the other hand, indicate that increasing the number of iterations may increase the intensities of the desired ions until the number of iterations reaches an end as described above, after which further iterations may not increase desired ion fragment intensities significantly. For example, the intensity of the second peak for the two iteration curve 620 is almost twice the intensity of the second peak for the one iteration curve 610. On the other hand, the intensity of the second peak for the five iteration curve 630 is less than five times (instead around 3.25 times) the intensity of the second peak for the one iteration curve 610; and the intensity of the second peak for the ten iteration curve 640 is much less than ten times (instead around 3.5 times) the intensity of the second peak for the one iteration curve 610. The third to fifth peaks indicate similar behaviors for the four curves. These results indicate that for the system corresponding to diagram 600, a saturation is reached for the number of iterations of around 5 due to the consumption of the precursor ions as described above.

[0100] In various embodiments, the above techniques reduce the intensities of undesired ion products, such as the CSR ions, in the ion trap. FIGS. 7A and 7B illustrate the effect of the disclosed techniques utilized by different embodiments compared to the conventional techniques. More specifically, FIG. 7A includes three mass spectra 710, 720, and 730 respectively corresponding to the mass spectra of the first, second, and third EDD products generated by the conventional systems. A first EDD product may be generated from a precursor ion by a 1stelectron detachment. A 2nd EDD product, on the other hand, may be generated from a first CRS by a 2nd electron detachment, and a 3rd EDD product may be generated from a 2nd CRS by a 3rd electron detachment. Because of the decrease of the charge states from the precursor ions to higher order CRS, the products at the same dissociation site show mass difference by the mass of hydrogen. That is, the mass of the 2ndEDD product may be lower than the mass of the 1stEDD product by -H; and the mass of the 3rd EDD product may be lower than the mass of the 1stEDD products by -2H.

[0101] FIG. 7B, on the other hand, includes three mass spectra 760, 770, and 780 also respectively corresponding to the mass spectra of the first, second, and third EDD products but generated by systems utilizing the disclosed techniques according to various embodiments.

[0102] Mass spectra 710 and 760 are similar. They both include six peaks (pedes 711-716 in spectrum 710, and peaks 761-766 in spectrum 760) corresponding to the isotopes of the first EDD product, which is the desired product in this case. Therefore, spectra 710 and 760 show that the disclosed techniques preserve the intensity of the desired fragments.

[0103] Mass spectra 720 and 770, on the other hand, are different. Mass spectra 720 includes six discernible peaks 721-726, corresponding to the isotopes of the second EDD product, indicating that this type of undesired product is generated by the conventional systems. Mass spectra 770, on the other hand, shows very low intensity peaks 771-776 at the location of the isotopes for the second EDD product, showing that the disclosed techniques are able to reduce the production of this undesired product.

[0104] In a similar manner, mass spectra 730 shows that the conventional techniques generate a discernible amount of the undesirable third EDD (indicated by the discerniblepeaks 731-736) while such peaks are missing from mass spectra 780 (their expected m / z values on the X axis being marked by labels 781-786), indicating that those undesired products are not generated in a significant amount by the disclosed techniques of various embodiments.

[0105] Various embodiments utilize additional techniques to address a variability of the extraction voltage due to the accumulation of the space charge in the trap region. More specifically, in some embodiments, the optimum extraction voltage may depend on the amount of the total charge accumulated in the trap region. In particular, accumulation of the precursor ions in the trap region may create a repulsive electric force (or, equivalently, may lower the height of the potential energy barrier) applied to the charges to be extracted from the ion trap and thus reduce the magnitude of the optimum extraction voltage. Such a variability may pose a problem because it may require an adjustment of the extraction voltage based on the intensity of precursor ion current.

[0106] FIG. 8A illustrates the problem of variability of the optimum extraction voltage addressed by some embodiments. More specifically, FIG. 8 A includes five extraction intensity diagrams 810, 812, 814, 816, and 818. In all five cases, the system has loaded the trap region with the precursor ions for 40 ms. The five cases differ, however, by the intensity of the precursor ion current (ITC) during the loading. In particular, the ITC is set to 100% (full intensity produced by the ion source) for diagram 810, and is then divided by two for each consecutive diagram. That is, the ITC for the four diagrams 812-818 has been set to 50%, 25%, 12%, and 6%, respectively. As a result, the total amount of charge accumulated in the trap region in the five cases has decreased respectively from the first to the last case. Consequently, the optimum extraction voltage (indicated by the cross section of the dashed line with the X axis in each diagram) increases from around 0.5 v in extraction intensity diagram 810 (ITC= 100%) to around 1.3 v in extraction intensity diagram 850 (ITC= 6%).

[0107] Some embodiments address the above-described problem using one or more of the charge control techniques described below. FIG. 8B illustrates the results of utilizing the charge control techniques to address the problem of variability of the optimum extraction voltage according to some embodiments. More specifically, FIG. 8B includes extraction intensity diagrams 820, 822, 824, 826, and 828 derived for five different precursor ion currentintensities. In the embodiments corresponding to diagrams 820-828, similar to diagrams 810- 818 of FIG. 8A, the ITC has decreased by factors of 2 from 100% to 6%. Those embodiments, however, have utilized the charge control techniques to maintain the extraction voltage at a constant value, here around 0.8v in all 5 cases, as indicated by the dashed line.

[0108] Various embodiments address the problem of the dependence of the optimum extraction voltage to the accumulated charge by controlling the amount of the precursor ions accumulated in the trap region, using one or more of the charge control techniques detailed below. In various embodiments, the system may achieve such a control by determining the intensity of the precursor ion current during the loading step or determining the total amount of the charge accumulated in the trap region. Based on these determinations, the system may accordingly determine the loading duration such that the total accumulated charge is maintained at a predetermined amount based on which the optimum extraction voltage has been set. In some embodiments, for example, the loading duration may be inversely proportional to the intensity of the precursor ion current. In the embodiments shown in FIG. 8B, for example, the loading duration is set to 20 ms for the 100% ITC of the embodiment in diagram 820; and the loading duration has multiplied by 2 for each consecutive embodiment (that is, from 40 ms in diagram 822 to 320 ms in diagram 828) to compensate for the decrease in the ITC, which is divided by 2 in each consecutive embodiment.

[0109] Various charge control techniques may use various systems or methods to determine the intensity of the precursor ion current or the magnitude of the accumulated charge. For example, in some embodiments, the system may perform a preliminary operation. During the preliminary operation, the system may pass the precursor ions through the ion trap without trapping them, that is, without performing the loading or the irradiation steps. Instead, the system may directly send the precursor ions to the mass spectrometer to measure the intensity of the precursor ion current and, based on the intensity of the current, determine the loading duration. Afterwards, the system may switch to the main operation (that is, for example, the recursive method) starting from a loading step for which the duration is the determined loading duration.

[0110] Alternatively, or additionally, during the preliminary operation the system may perform the loading step for a pre-set loading duration. The pre-set loading duration may be ashort duration set at, for example, 1 ms. After this loading, the system may measure the precursor ion charge in the trap region by, for example, releasing the trapped ions to the mass spectrometer to measure their magnitude. In these embodiments, therefore, the preliminary operation may include the loading step and the analysis step, but not the iterations of the irradiation-extraction operations. Therefore, as explained above, these charge control techniques enable some embodiments to avoid variability of the optimum extraction voltage.

[0111] Those having ordinary skill will appreciate that various changes may be made to the above embodiments without departing from the scope of the disclosure.

[0112] Although some aspects have been described in the context of a system or an apparatus, it is clear that these aspects may also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0113] The foregoing description of the embodiments has been presented for purposes of illustration only. It is not exhaustive and does not limit the embodiments to the precise form disclosed. While several exemplary embodiments and features are described, modifications, adaptations, and other implementations may be possible, without departing from the spirit and scope of the embodiments. Accordingly, unless explicitly stated otherwise, the descriptions relate to one or more embodiments and should not be construed to limit the embodiments as a whole. This is true regardless of whether or not the disclosure states that a feature is related to “a,” “the,” “one,” “one or more,” “some,” or “various” embodiments. As used herein, the singular forms “a,” “an,” and “the” may include the plural forms unless the context clearly dictates otherwise. Further, the term “coupled” does not exclude the presence of intermediate elements between the coupled items. Also, stating that a feature may exist indicates that the feature may exist in one or more embodiments.

[0114] In this disclosure, the terms “include,” “comprise,” “contain,” and “have,” when used after a set or a system, mean an open inclusion and do not exclude addition of other, non-enumerated, members to the set or to the system. Further, unless stated otherwise ordeducted otherwise from the context, the conjunction “or,” if used, is not exclusive, but is instead inclusive to mean and / or.

[0115] Moreover, if these terms are used, a set may include one or more members, and a subset of a set may include one or more than one, including all, members of the set.

[0116] Further, if used in this disclosure, and unless stated or deducted otherwise, a first variable is an increasing function of a second variable if the first variable does not decrease and instead generally increases when the second variable increases. On the other hand, a first variable is a decreasing function of a second variable if the first variable does not increase and instead generally decreases when the second variable increases. In some embodiment, a first variable may be an increasing or a decreasing function of a second variable if, respectively, the first variable is directly or inversely proportional to the second variable.

[0117] The disclosed compositions, systems, methods, and apparatus are not limited to any specific aspect or feature or combinations thereof, nor do the disclosed compositions, systems, methods, and apparatus require that any one or more specific advantages be present or problems be solved. Any theories of operation are to facilitate explanation, but the disclosed compositions, systems, methods, and apparatus are not limited to such theories of operation.

[0118] Modifications and variations are possible in light of the above teachings or may be acquired from practicing the embodiments. For example, the described steps need not be performed in the same sequence discussed or with the same degree of separation. Likewise various steps may be omitted, repeated, combined, or performed in parallel, as necessary, to achieve the same or similar objectives. Similarly, the systems described need not necessarily include all parts described in the embodiments, and may also include other parts not described in the embodiments. Accordingly, the embodiments are not limited to the abovedescribed details, but instead are defined by the appended claims in light of their full scope of equivalents. Further, the present disclosure is directed toward all novel and non-obvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with one another.

[0119] While the present disclosure has been particularly described in conjunction with specific embodiments, many alternatives, modifications, and variations will be apparent in light of the foregoing description. It is therefore contemplated that the appended claims will embrace any such alternatives, modifications, and variations as falling within the true spirit and scope of the present disclosure.

Claims

CLAIMS1. A mass spectrometry method comprising: loading a plurality of precursor ions in a trap region; performing an iteration of irradiation-extraction operations, wherein each irradiationextraction operation in the iteration includes: applying an electron beam to the trap region to produce fragmented ions, and extracting, from the trap region, a high m / z subset of the fragmented ions with m / z ratios greater than a specific value; and performing mass spectrometry analysis on a subset of the fragmented ions remaining in the trap region, wherein the iteration includes at least two irradiation-extraction operations.

2. The method of claim 1, wherein the specific value is an m / z value of the precursor ion.

3. The method of claim 1, wherein: the loading lasts for a loading duration; and the loading duration depends on a magnitude of charge accumulated in the trap region.

4. The method of claim 3, wherein the magnitude of charge accumulated in the trap region is determined based on a mass spectrometry spectrum of accumulated charge during a preliminary operation.

5. The method of claim 3, wherein the loading duration is inversely related to an intensity of a precursor ion current.

6. The method of claim 5, wherein the loading duration is inversely proportional to the intensity of the precursor ion current.

7. The method of claim 5, wherein the loading duration is determined based on a mass spectrometry spectrum of the precursor ion current during a preliminary operation.

8. The method of any one of claims 1-7, further comprising a pausing after applying the electron beam and before extracting the high m / z subset of the fragmented ions.

9. The method of claim 8, wherein a duration of the pausing is less than 5 milliseconds.

10. The method of any one of claims 1-7, wherein after the extracting, a majority of fragmented ions with m / z values lower than the specific value remain in the trap region.

11. The method of any one of claims 1-7, further performing a nucleotide sequence determination of the precursor ions.

12. The method of any one of claims 1-7, wherein the precursor ions are polynucleotides.

13. The method of claim 12, wherein the polynucleotide is an oligonucleotide.

14. The method of claim 13, wherein a z value of the oligonucleotide is in a range of 5-15.

15. The method of claim 13, wherein the polynucleotide is an RNA.

16. The method of any one of claims 1-7, wherein: the electron beam is applied for an irradiation duration; and the irradiation duration depends on an intensity of second charge reduced ions in the trap region.

17. The method of claim 16, wherein the irradiation duration is determined so that after applying the electron beam, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

18. The method of claim 16, wherein the irradiation duration is in a range of 1-300 ms.

19. The method of claim 16, wherein: the electron beam is also applied at an irradiation intensity; and the irradiation intensity also depends on the intensity of the second charge reduced ions in the trap region.

20. The method of claim 19, wherein the irradiation duration and the irradiation intensity are determined so that after applying the electron beam, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

21. The method of any one of claims 1-7, wherein: the electron beam is applied at an irradiation intensity; and the irradiation intensity depends on an intensity of second charge reduced ions in the trap region.

22. The method of claim 21, wherein the irradiation intensity is determined so that after applying the electron beam, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

23. The method of claim 21, wherein the irradiation intensity is in a range of 0.01 pA to 10 pA.

24. A mass spectrometry system comprising: an ion trap configured to receive a plurality of precursor ions and to load the plurality of precursor ions in a trap region; an electron gun configured to perform an irradiation operation by applying an electron beam to the trap region to produce fragmented ions; an RF gate configured to perform an extraction operation by extracting, from the trap region, a high m / z subset of the fragmented ions with m / z ratios greater than a specific value; and a mass spectrometer configured to perform mass spectrometry analysis on a subset of the fragmented ions remaining in the trap region, wherein: an irradiation-extraction operation, including performing the irradiation operation and the extraction operation, is repeated at least twice before performing the mass spectrometry analysis on the subset of the fragmented ions remaining in the trap region.

25. The system of claim 24, wherein the specific value is an m / z value of the precursor ion.

26. The system of claim 24, wherein: the loading lasts for a loading duration; and the loading duration depends on a magnitude of charge accumulated in the trap region.

27. The system of claim 26, wherein the mass spectrometer is configured to determine the magnitude of charge accumulated in the trap region based on a mass spectrometry spectrum of accumulated charge during a preliminary operation.

28. The system of claim 26, wherein the loading duration is inversely related to an intensity of a precursor ion current.

29. The system of claim 28, wherein the loading duration is inversely proportional to the intensity of the precursor ion current.

30. The system of claim 28, wherein the loading duration is determined based on a mass spectrometry spectrum of the precursor ion current during a preliminary operation.

31. The system of any one of claims 24-30, wherein the RF gate is further configured to pause after the irradiation operation before performing the extraction operation.

32. The system of claim 31, wherein a duration of the pause is less than 5 milliseconds.

33. The system of any one of claims 24-30, wherein after the extraction operation a majority of fragmented ions with m / z values lower than the specific value remain in the trap region.

34. The system of any one of claims 24-30, wherein: the electron gun is configured to perform the irradiation operation for an irradiation duration; and the irradiation duration depends on an intensity of second charge reduced ions in the trap region.

35. The system of claim 34, wherein the irradiation duration is determined so that after the irradiation operation, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

36. The system of claim 34, wherein the irradiation duration is in a range of 1-300 ms.

37. The system of claim 34, wherein: the electron gun is further configured to perform the irradiation operation at an irradiation intensity; and the irradiation intensity also depends on the intensity of the second charge reduced ions in the trap region.

38. The system of claim 37, wherein the irradiation duration and the irradiation intensity are determined so that after applying the electron beam, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

39. The system of any one of claims 24-30, wherein:the electron gun is configured to perform the irradiation operation at an irradiation intensity; and the irradiation intensity depends on an intensity of second charge reduced ions in the trap region.

40. The system of claim 39, wherein the irradiation intensity is determined so that after the irradiation operation, the intensity of the second charge reduced ions in the trap region is lower than an intensity of first charge reduced ions in the trap region.

41. The system of claim 39, wherein the irradiation intensity is in a range of 0.01 pA to 10 pA.

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