阅读说明：本技术 利用谐波振荡和共振离子选择性时间概览（stori）图的电荷检测质谱法 (Charge detection mass spectrometry with harmonic oscillation and resonant ion Selective Time Overview (STORI) plots ) 是由 M·W·森柯 P·F·叶 D·E·格林菲尔德 S·C·博 于 2020-04-22 设计创作，主要内容包括：公开了用于执行电荷检测质谱法以测量单个所关注离子的质量的设备和方法。使所述所关注离子在静电阱的俘获场中经历谐波振荡移动,使得图像电流检测器生成表示所述离子的振荡移动的时变信号。处理(例如,通过傅里叶变换)此时变信号(瞬态)以得出所述离子的频率,并且因此确定所述离子的质荷比(m/z)。通过构建共振离子选择性时间概览(STORI)图来确定离子电荷,所述STORI图跟踪可归因于所述所关注离子的信号的时间演变,并且其中所述STORI图的斜率与所述电荷有关。所述STORI图还可以用于标识瞬态采集期间的离子衰减事件和/或相同质量的多个离子或不可分辨离子的存在。(Apparatus and methods for performing charge detection mass spectrometry to measure the mass of a single ion of interest are disclosed. The ions of interest are subjected to harmonic oscillatory movement in a trapping field of an electrostatic trap such that an image current detector generates a time-varying signal representative of the oscillatory movement of the ions. This time varying signal (transient) is processed (e.g. by fourier transform) to derive the frequency of the ions and hence determine the mass to charge ratio (m/z) of the ions. Ion charge is determined by constructing a resonant ion-Selective Time Overview (STORI) map that tracks the time evolution of a signal attributable to the ion of interest, and wherein the slope of the STORI map is related to the charge. The STORI map can also be used to identify ion decay events and/or the presence of multiple ions of the same mass or non-resolvable ions during transient acquisition.)

1. An apparatus for determining the mass-to-charge ratio (m/z) and charge of ions, the apparatus comprising:

an electrostatic trap having a plurality of electrodes shaped and arranged to establish an electrostatic trapping field within the electrostatic trap that subjects the ions to harmonic motion along a longitudinal axis and a voltage source for applying a set of non-oscillating voltages to the plurality of electrodes;

a detector that generates a time-varying signal in response to a current induced on the detector by the harmonic motion of the ions; and

a data system having logic to:

processing the time-varying signal to derive harmonic motion frequencies and determining the m/z from the derived frequencies;

STORI is generated according to the following equation_MAGTime overview of resonant ion Selectivity (STORI) versus time:

STORI_MAG(t_n)＝((STORI_REAL(t_n))²+(STORI_IMAG(t_n))²)^1/2

STORI_REAL(t_n)＝S(t_n)*sin(ω*t_n)+STORI_REAL(t_n-1)

and

STORI_IMAG(t_n)＝-S(t_n)*cos(ω*t_n)+STORI_IMAG(t_n-1)，

wherein S (t)_n) Is discretizing the time-varying signal at a point in time t_nAnd ω is the resulting harmonic motion frequency; and

the charge of an ion is determined from a stored relationship between the ion charge and the slope of the STORI map.

2. The apparatus of claim 1, wherein the plurality of electrodes includes an inner electrode elongated along the axis and an outer electrode radially surrounding the inner electrode, and wherein the electrostatic field is established in an annular space between the inner electrode and the outer electrode.

3. The apparatus of claim 2, wherein the inner and outer electrodes are shaped and arranged such that the electrostatic field has a potential distribution U (r, z) approximating the relationship:

where r is the position of the ion along a radial axis, z is the position of the ion along a central axis, k is the field curvature, C is a constant, and Rm is the characteristic field radius.

4. The apparatus of claim 2, wherein the outer electrode is divided into a first portion and a second portion along a transverse plane of symmetry of the electrostatic trap, and the detector comprises a differential amplifier connected between the first portion and the second portion.

5. The apparatus of claim 1, further comprising an ion store in which the ions are trapped and thereafter released in an ion path toward an entrance of the electrostatic trap.

6. The apparatus of claim 1, wherein the data system is configured to apply a fourier transform to the time-varying signal to construct a frequency spectrum.

7. The apparatus of claim 1, wherein the data system further comprises logic for visually displaying the STORI graph.

8. The apparatus of claim 1, wherein the data system further comprises logic for analyzing the STORI map to identify ion decay events.

9. A method for determining a mass-to-charge ratio (m/z) and a charge of an ion of interest, the method comprising:

(a) injecting a population of ions comprising the ions of interest into a trapping region and establishing an electrostatic trapping field within the region, the electrostatic trapping field causing the population of ions to undergo harmonic motion along a central axis;

(b) generating a time-varying signal representative of a current induced on a detector by the harmonic motion of the ion packets;

(c) processing the time-varying signal to derive a frequency of the induced current;

(d) determining the m/z of the ion of interest from the resulting frequency;

(e) STORI is generated according to the following equation_MAG(i) Time overview of resonant ion Selectivity (STORI) versus time:

STORI_MAG(t_n)＝((STORI_REAL(t_n))²+(STORI_IMAG(t_n))²)^1/2

STORI_REAL(t_n)＝S(t_n)*sin(ω*t_n)+STORI_REAL(t_n-1)

and

STORI_IMAG(t_n)＝-S(t_n)*cos(ω*t_n)+STORI_IMAG(t_n-1)，

wherein S (t)_n) Is that the time-varying signal is at a point in time t_nAnd ω is the resulting harmonic motion frequency; and

(f) the charge of an ion is determined from a stored relationship between the ion charge and the slope of the STORI map.

10. The method of claim 9, wherein the electrostatic field is established in an annular region between an inner electrode and an outer electrode radially surrounding the inner electrode, and wherein the electrostatic trapping field has a potential distribution U (r, z) approximating the relationship:

where r is the position of the ion along a radial axis, z is the position of the ion along a central axis, k is the field curvature, C is a constant, and Rm is the characteristic field radius.

11. The method of claim 9, wherein the step of processing comprises applying a fourier transform to the time-varying signal.

12. The method of claim 9, wherein the ion of interest is one of: proteins, protein complexes, and viral capsids.

13. The method of claim 9, wherein the ion of interest is a high molecular weight polymer.

14. The method of claim 9, further comprising performing repeated cycles of steps (a) - (f) and collecting the determined m/z and charge of the ion of interest for each cycle.

15. The method of claim 14, further comprising the step of constructing a histogram of calculated masses of the ions of interest from the collected determined m/z and charge of the ions of interest.

16. The method of claim 9, wherein the ion population includes a second ion of interest, and further wherein the step of processing the time-varying signal yields a first frequency for the ion of interest and a second frequency for the second ion of interest, and further comprising:

determining the m/z of the second ion of interest from the second frequency;

constructing a second STORI map of the second ion of interest; and

determining the charge of the second ion of interest from a slope of the second STORI map.

17. The apparatus of claim 1, further comprising ion optics located in an ion path upstream of the electrostatic trap, the ion optics configured to attenuate an ion beam directed toward the electrostatic trap.

18. The method of claim 9, further comprising the step of attenuating an ion beam directed toward the trapping region.

19. The method of claim 9, wherein the ion packets are confined in an ion reservoir prior to injection into the trapping region.

20. The method of claim 9, further comprising the step of evaluating the STORI map to evaluate whether an ion decay event has occurred.

21. The method of claim 9, further comprising the step of displaying the STORI map.

22. The apparatus of claim 1, wherein the logic for generating the STORI map includes instructions for pre-computing and caching a function G (ω, t) for a sequence of target time points, wherein G (ω, t) is a Fourier transform of a Heavyside function (HeavySyde function) H (t, s),

23. the method of claim 9, wherein the step of generating the STORI map includes pre-computing and caching a function G (ω, t) for a sequence of target time points, wherein G (ω, t) is a Fourier transform of a Hervesseld function H (t, s),

Technical Field

The present invention relates generally to mass spectrometry, and more particularly to apparatus and methods for measuring the mass-to-charge ratio and charge of individual ions.

Background

Charge Detection Mass Spectrometry (CDMS) is a technique for determining the mass of an individual ion by simultaneously measuring the mass-to-charge ratio (m/z) and the charge of each ion. One technique used in the academic laboratory of CDMS (known as ion trap CDMS) employs an inductive detector positioned between two opposing electrostatic mirrors, as described in Fuerstenau and Benner, "Molecular weight determination of megadalton DNA electrospray ions using charge detection time of flight Mass Spectrometry-of-flight Mass Spectrometry", Rapid communication in Mass Spectrometry (Rapid Communications in Mass Spectrometry) 9:15 (1995), 8 — 1538. In such instruments, the m/z of an ion is determined by its oscillation frequency between mirrors, while the charge of the ion is determined based on the amplitude of the signal on the inductive detector. The separation and direct measurement of charge thus overcomes the common challenges of large and/or heterogeneous analytes studied with conventional electrospray mass spectrometry, where it is not possible to separate the incrementally charged ion species and infer the charge state therefrom.

Existing ion trap CDMS instruments present several significant technical challenges. First, because the electrical potential generated by the opposing mirror is typically non-harmonic, the measured frequency depends on the initial kinetic energy of the ion. This may result in a poor accuracy of the m/z measurement of the individual particles, which may also result in poor resolution in assembling the histogram of the measured mass. In addition, the signal generated by the inductive detector is not sinusoidal, but is processed using fourier transform analysis. The generated signal is distributed among many harmonics, which significantly reduces the overall system sensitivity. This introduces an additional limitation in that only a single ion species can be analysed at a time, resulting in a very long acquisition period. Finally, the ions move directly from the source to the mirror in existing CDMS instruments without the need for proper desolvation. The lack of desolvation may result in mass shifts being observed during the measurement period due to the loss of solvent by the ions.

PCT publication No. WO2019/231,854 to Senko et al describes an apparatus and method that aims to address the shortcomings of existing CDMS instruments and techniques. This publication discloses the use of an electrostatic trap to create a trapping field that subjects the trapped ions to harmonic motion along a longitudinal axis, and an image current detector to generate a time-varying signal (also referred to as a transient signal) in response to the longitudinal motion of the ions. The time-varying signal is subjected to a fourier transform to determine the frequency and associated amplitude of at least one of the trapped ion species, and from the determined frequency and amplitude the m/z and charge of the trapped ion species are derived accordingly. While this approach has been successfully used to measure m/z and charge of high mass ion species under certain conditions, it can be susceptible to errors when the ions decay during the transient acquisition period or when there are multiple ions of the same ion species. Accordingly, there remains a need in the art for a CDMS apparatus and method that avoids or minimizes the errors that can occur when using the techniques described in the Senko et al publication.

Disclosure of Invention

Broadly described, an apparatus is disclosed for measuring the m/z and charge, and hence the mass, of ions by processing the image current signals caused by the oscillatory movement of the ions within an electrostatic trap to generate a resonant ion Selective Time Overview (STORI) diagram, defined below. The electrostatic trap includes a plurality of electrodes to which a non-oscillating voltage is applied. The electrodes are shaped and arranged to establish an electrostatic trapping field that subjects ions to harmonic motion relative to a longitudinal axis of the trap. The apparatus further includes a detector that generates a time-varying signal representative of a current induced on the detector by the harmonic longitudinal motion of the ions. A data system receives the time-varying signal from the detector and processes the signal to determine the m/z and charge of the ions. The determination of m/z is done by applying a discrete fourier transform to the time-varying signal to accurately identify the frequency ω of harmonic motion of the ions. Determination of ion charge can be achieved by constructing a resonant ion-selective time-overview (STORI) map, which constitutes the STORI_MAGA graph of the value of (a) against time. Each point in the STORI diagram is a time t_nThe product of the discretized time-varying signal S at (co) with the sine wave (equation 1 below) or cosine wave (equation 2 below) at the frequency of interest (ω), and the previous point in time t_n-1The previous STORI points obtained at (a) are summed as represented in the following equation.

STORI_REAL(t_n)＝S(t_n)*sin(ω*t_n)+STORI_REAL(t_n-1) (1)

STORI_IMAG(t_n)＝-S(t_n)*cos(ω*t_n)+STORI_IMAG(t_n-1) (2)

And

STORI_MAG(t_n)＝((STORI_REAL(t_n))²+(STORI_IMAG(t_n))²)^1/2 (3)

the charge of the ions is determined from the measured slope of the STORI map and calibration data that relates the slope of the STORI map to the charge of the ions. In addition to determining the charge in the trapped ion species, the STORI map can be used to identify and characterize ion decay events (in which ion species are dissociated during acquisition of a time-varying signal), as well as to identify and evaluate signals produced by two or more simultaneously trapped ions.

Once the m/z and charge of an ion are determined using these processing methods, the mass of the ion can be readily calculated from the product of these two values.

In a more specific embodiment, the electrostatic trap is formed by coaxially arranged inner and outer electrodes, each elongated along a longitudinal axis, and trapping ions in an annular space between the electrodes. The inner and outer electrodes may be shaped and arranged to establish a quadrupole logarithmic field in the annular space such that a restoring force applied by the field along the central axis is proportional to the position of the ions along the central axis relative to the transverse plane of symmetry. The outer electrode may be split in half into a first portion and a second portion along a transverse plane of symmetry, and the detector may comprise a differential amplifier connected across the first portion and the second portion. Ions may be trapped in an ion store prior to release to the electrostatic trap to reduce their kinetic energy and facilitate complete desolvation. Analysis of two or more ion species may be performed simultaneously within the electrostatic trap such that the data system constructs multiple STORI maps, where each STORI map is calculated using the frequency of motion of a different individual ion species, such that the charge state may be determined for each of the plurality of trapped ion species. The STORI map can be evaluated to determine whether two or more ions of the same ion species are present in the mass analyzer.

Embodiments of the invention further include methods for measuring m/z and charge of ions. According to such a method, ion packets containing ions of interest are injected into a trapping region, in which an electrostatic trapping field is established that subjects the ion packets to harmonic motion along a central axis. A time-varying signal representative of the current induced on the detector by said harmonic motion is generated. The time-varying signal is processed to derive harmonic motion frequencies of the ions of interest, which are in turn used to determine m/z of the ions. The time-varying signal is also processed in the manner described above to generate a STORI map of the ions of interest, and the charge state of the ions is determined from the slope of the STORI map.

Drawings

In the drawings:

FIG. 1 is a symbolic diagram of an apparatus for simultaneously measuring m/z and charge of ions according to an embodiment of the present invention;

FIG. 2 is a block diagram depicting logical components of the data system of FIG. 1;

FIG. 3 is a depiction of a STORI diagram for a single ion;

FIG. 4 is a depiction of a STORI graph of a single ion decaying during a signal acquisition period; and

fig. 5 is a depiction of a STORI graph of two ions that decay during a signal acquisition period.

Detailed Description

Specific embodiments of the present invention are described below, which are intended to be illustrative, not limiting. Those of skill in the art will recognize that the various features, structures, steps, and limitations disclosed in connection with the discrete embodiments may be combined or altered without departing from the scope of the invention.

FIG. 1 symbolically depicts a mass spectrometry apparatus 100 arranged in accordance with one embodiment of the present invention. The apparatus 100 includes an ionization source 105 that generates ions from a sample to be analyzed. As used herein, the term "ion" refers to any charged molecule or assembly of molecules, and is specifically intended to encompass high molecular weight entities sometimes referred to in the art as large ions, charged particles, and charged aerosols. Without limiting the scope of the invention, ions that may be analyzed by the apparatus 100 include proteins, protein complexes, antibodies, viral capsids, oligonucleotides, and high molecular weight polymers. The source 105 may take the form of an electrospray ionization (ESI) source in which ions are formed by ejecting charged droplets of a sample solution from a capillary tube to which an electrical potential is applied. The sample may be delivered to the source 105 as a continuous flow, for example as eluent from a chromatography column.

Ions generated by the source 105 are directed and focused through a series of ion optics disposed in a vacuum chamber of progressively decreasing pressure. As depicted in fig. 1, the ion optics may include an ion transfer tube, a stacked ring ion guide, a Radio Frequency (RF) multipole, and an electrostatic lens. The vacuum chamber containing the ion optics may be evacuated by any suitable pump or combination of pumps operable to maintain the pressure at a desired value.

Apparatus 100 may additionally include a Quadrupole Mass Filter (QMF)110 that transmits only those ions within a selected range of m/z values. The operation of quadrupole mass filters is well known in the art and need not be discussed in detail herein. Generally described, the m/z range of selectively transmitted ions is set by appropriately adjusting the amplitude of the RF and resolving the Direct Current (DC) voltage applied to the electrodes of QMF 110 to create an electric field that causes ions with m/z outside the selected range to develop unstable trajectories. The transmitted ions may thereafter pass through additional ion optics (e.g., lenses and RF multipole rods) and enter the ion repository 115. As is known in the art, the ion store 115 employs a combination of oscillating and static fields to confine ions within it. In a particular embodiment, the ion repository 115 may take the form of a curved trap (colloquially referred to as a "c-trap") of the type utilized in an Orbitrap mass spectrometer (Orbitrap mass spectrometer) sold by seemer Fisher Scientific. The curved trap is made up of a set of substantially parallel rod electrodes that are concavely curved towards the ion outlet. Radial confinement of ions within the ion reservoir 115 may be achieved by applying oscillating voltages to opposing pairs of rod electrodes in a prescribed phase relationship, while axial confinement may be achieved by applying static voltages to a tip lens positioned axially outside the rod electrodes.

Ions entering the ion store 115 may be confined therein for a prescribed cooling period in order to reduce their kinetic energy prior to introduction into the electrostatic trap. Confining the ions within the ion reservoir for a prescribed period of time may also assist in desolvation of the ions, i.e., removal of any residual solvent portion from the analyte ions. As discussed above, the presence of residual solvent can lead to mass shifts during analysis that can interfere with the ability to accurately measure m/z and charge. To promote kinetic cooling and desolvation of the ions, an inert gas such as argon or helium may be added to the interior volume of the ion reservoir; however, the cooling gas pressure should be adjusted to avoid unintended fragmentation of analyte ions and/or excessive leakage of gas into the electrostatic trap 120. The duration of the cooling period will depend on a number of factors including the kinetic energy of the ions entering the ion reservoir 115, the inert gas pressure, and the desired kinetic energy distribution of the ions injected into the electrostatic trap 120. After the cooling period ends, ions confined in the ion reservoir 115 may be ejected radially from the ion reservoir towards an entrance lens 125 for focusing and directing the ions into the entrance 130 of the electrostatic trap 120. Rapid ejection of ions from the ion store 115 may be performed by rapidly contracting an oscillating field within the interior of the ion store and applying a DC pulse to a rod electrode positioned away from the ejection direction.

To reliably measure ion charge using CDMS techniques, only a single ion of a particular ion species may be present in the electrostatic trap 120 during a measurement event. As used herein, the term "ionic species" refers to an ion having a given elemental/isotopic composition and charge state; ions having different elemental/isotopic compositions are considered to be different ion species, as well as ions having the same elemental composition but different charge states. The term "ionic species" is used herein interchangeably with the terms "analyte ion" and "ion of interest". If multiple ions of the same ion species are present during the measurement event, the measured charge state (determined from the amplitude of the signal generated by image current detector 132, as described below) will be a multiple of the actual charge state of the individual ions. To avoid this type of erroneous measurement, the ion population within the ion reservoir 115 should be kept small enough that the probability that two ions of the same ion species are confined within the ion reservoir is kept to an acceptable minimum. This may be achieved by attenuating the ion beam generated by the source 105 (more specifically, by "detuning" ion optics located in the upstream ion path such that high ion losses occur) and/or by adjusting the fill time (the period of time during which ions are accepted into the ion reservoir 115). To control the fill time, one or more ion optical components located in the ion path upstream of the ion store may operate as a gate to selectively allow or block ions from entering the interior volume of the ion store 115.

The electrostatic trap 120 may take the form of an orbital electrostatic trap of the type commercially available from seimer femoris technologies under the trademark "orbital trap" and depicted in cross-section in fig. 1. This orbitrap includes an inner mandrel-type electrode 135 defining a central longitudinal axis, designated as the z-axis in a cylindrical coordinate system. The outer barrel electrode 140 is coaxially positioned relative to the inner electrode 135, defining therebetween a generally annular trapping region 145 into which ions are implanted. The inner electrode 135 and the outer electrode 140 are each symmetric about a transverse plane (designated as z-0, and alternatively referred to as the "equator"), with the inner electrode 135 having a maximum outer radius R at the transverse plane of symmetry₁And the outer electrode 140 has a maximum inner radius R at the transverse plane of symmetry₂. As has been widely discussed in the scientific literature (see, e.g., Makarov, "Electrostatic axial Harmonic Orbital Capture: high Performance techniques of Mass Analysis (Electrical axial Harmonic tracking: AHigh-Performance technology of Mass Analysis)", "Analytical Chemistry (Analytical Chemistry)", volume 72, phase 6, phase 1156-Page 62 (2000), incorporated herein by reference), the inner and outer electrodes may be shaped (upon application of an electrostatic voltage to one or both of the electrodes) to establish an electrostatic potential U (r, z) within the trapping region 145 that approximates the relationship:

where r and z are cylindrical coordinates (r-0 is the central longitudinal axis and z-0 is the transverse plane of symmetry), C is a constant, k is the field curvature, and Rm is the characteristic radius. This field is sometimes referred to as a log quadrupole field.

The outer electrode 140 is divided along a transverse plane of symmetry into a first portion 150 and a second portion 155, which are separated from each other by a narrow insulating gap. This arrangement enables the use of the outer electrode 140 together with the differential amplifier 160 as an image current detector. The presence of ions near the outer electrode induces a charge in the electrode that is proportional in magnitude to the charge of the ions (opposite polarity to the polarity of the ions). The oscillating back and forth movement of ions along the z-axis between the first and second portions 150, 155 of the outer electrode 140 causes the image current detector 132 to output a time-varying signal (referred to as a "transient") having a frequency equal to the frequency of the longitudinal oscillations of the ions and an amplitude representative of the charge of the ions.

Ions may be introduced tangentially into the trapping region 145 through an entrance aperture 130 formed in the outer electrode 240. The entrance aperture 130 is axially offset (along the z-axis) from a transverse plane of symmetry such that upon introduction into the trapping region 145, the ions experience a restoring force in the direction of the plane of symmetry, causing the ions to begin to oscillate longitudinally along the z-axis as they orbit the inner electrode 135, as illustrated in fig. 1. The salient feature of a quadrupole logarithmic field is that its potential distribution contains no cross terms in r and z, and the potential in the z dimension is uniquely quadratic. Thus, ion motion along the z-axis can be described as a harmonic oscillator (since the force along the z-dimension exerted on the ion by the field is proportional to the displacement of the ion along the z-axis from the transverse plane of symmetry) and is completely independent of orbital motion. In this way, the frequency of oscillation of the ion, ω, along the z-axis is simply related to the mass-to-charge ratio (m/z) of the ion according to the following relationship:

the measurement of the state of charge and m/z and the consequent calculation of the product mass is performed by collecting and processing transients. Transient acquisition by detector 132 begins immediately after injection of analyte ions and continues for a predetermined transient length. The length of the transient required to accurately measure m/z and charge state will vary depending on the analyte and the physical and operating parameters of the electrostatic ion trap 120. Typically, the transients will need to be of sufficient duration to allow the signal to be reliably distinguished from noise. For typical analyte ions, it is expected that a satisfactory signal-to-noise ratio can be achieved at a transient length of 500 milliseconds using a commercially available orbital trapping mass analyzer. It will be appreciated that the maximum transient length will be limited by the duration of time that the analyte ions are stably trapped within the trapping region 145 without colliding with background gas atoms/molecules or other ions, which is in part a function of the trapping region pressure.

The transient signal produced by the detector 132 is processed by a data system 165, the function of which will be described below in connection with fig. 2. Although the data system 165 is depicted as a single block, the functionality of the data system may be distributed among several interconnected devices. The data system 165 will typically include a combination of special purpose and general purpose processors, special purpose circuitry, memory, storage, and input/output devices. The data system 165 is configured with logic, e.g., using executable software code, to perform a set of calculations to determine the fundamental frequency of motion of the analyte ions, and is configured to construct a STORI map corresponding to the ions, which in turn is used to derive m/z and charge states.

FIG. 2 depicts components of a data system 165. An analog-to-digital converter (ADC) module 205 receives the analog signal generated by the detector 132 and samples the signal at a prescribed sampling rate to generate a sequence of discrete time-intensity data values. The ADC block 205 may also perform a filtering function to attenuate extraneous noise and improve the signal-to-noise ratio. The time domain data is then passed to a Fast Fourier Transform (FFT) module 210 for conversion of the data into the frequency domain. FFT algorithms are well known in the art and therefore need not be discussed in detail herein. Generally described, an FFT algorithm rapidly computes the DFT of a sequence by factoring a Discrete Fourier Transform (DFT) matrix into a product of sparse factors. The FFT module 210 generates as an output a frequency spectrum representing a decomposition of a time domain data sequence into one or more frequency components, each frequency component comprising a single sinusoidal oscillation having its own amplitude.

As described above, the motion of analyte ions along the z-axis trapped within the field generated in the trapping region 145 is harmonic and can be expressed as a simple sinusoidal function. The output of the FFT module 210 will thus produce a spectrum with a strong peak at the fundamental frequency ω of oscillation of the ion of interest. When multiple ion species are present within the electrostatic trap during a measurement event (i.e., during an acquisition transient), then each ion species will exhibit a corresponding peak in the frequency spectrum. In contrast to prior art CDMS systems in which the oscillatory motion of the trapped ions is non-harmonic and non-sinusoidal (the FFT output will contain many peaks distributed among the various harmonics), the signal of each ion species in the electrostatic trap 120 will be concentrated into a single peak where the fundamental frequency of oscillation occurs, thereby increasing sensitivity relative to prior art CDMS devices and enabling charge measurements on lower charge ions.

The spectrum generated by the FFT module is provided as an input to the m/z determination module 215, which processes the spectrum to determine the m/z of the analyte ions. The M/z determination module 215 is configured to identify a fundamental frequency of oscillation of the analyte ions for the or each analyte ion species present in the spectrum. This frequency is then converted to a value of m/z. As described above, the frequency of the oscillating ion motion along the z-axis is inversely proportional to the square root of the m/z of the ion according to the following relationship:

thus, as is known in the art, m/z can be determined from measured ion frequencies using an empirically established frequency versus m/z calibration curve generated by fitting an inverse square root curve to data points collected for analyte ions of known m/z.

As described in further detail below, the charge determination module 220 is configured to process the STORI map constructed by module 217 and to provide as an output a value of the charge of the ions for the or each analyte ion species present in the spectrum.

Once the m/z and charge of the analyte ion have been determined, the mass of the ion can be calculated simply by multiplying the determined m/z and charge. If the spectrum contains multiple ion species, the mass of each ion species is calculated by the product of m/z and the charge determined for that species.

In certain embodiments, the transient acquisition and m/z and charge determination operations will be repeated for the analyte ions. The resulting calculated masses may be binned to obtain a mass histogram, where the peak of the histogram represents the most likely mass. In general, the width of the histogram will depend on the accuracy of the image charge determination, with narrower widths indicating high accuracy. Other techniques including averaging may be employed to improve the reliability of the quality determination.

Charge determination in CDMS using STORI maps

In CDMS, the ability to accurately assign charge and therefore mass depends on the ability to determine the amplitude of the signal corresponding to the ion of interest. In the case where the ions generate a signal during the entire signal acquisition period, the determination of the signal amplitude is simply done by the amplitude of the peak produced in the frequency domain, as described in the aforementioned Senko et al publication.

However, ions may "decay" (disintegrate) during the acquisition period, resulting in unstable ion trajectories. This may be due to collisions with background gas molecules, or simply because the ions are metastable. If the ions decay during the acquisition period, less signal will be generated, where the signal is proportional to the lifetime of the ions. Therefore, in order to convert the frequency domain amplitude back to an unattenuated time domain amplitude, it is necessary to be able to accurately determine the ion lifetime.

A conventional method for checking for temporal variations in time domain data is to use a short-term fourier transform (STFT). In this process, a portion of the total data set is converted to the frequency domain in an iterative manner, with a window of data sliding or stepping through the entire time domain data set. STFT has several disadvantages, including reduced sensitivity due to the use of smaller time domain data sets, along with temporal resolution limited by the size of the time domain data set and the size of the steps taken during processing.

An alternative technique for evaluating temporal variations in time domain data is described below and involves computing (using the charge determination module described above, or such other data system components as may be suitable for the purpose) a resonant ion Selective Time Overview (STORI) map, alternatively referred to as a correlation integration Curve (CIP) process. The computation resembles a discrete fourier transform, where the time domain data is multiplied by a sine wave of the frequency of interest, and the output is the dot product of the two. Each point in the STORI diagram is a time t_nThe product of the discretized time-varying signal S at (which can be determined from the fourier transform of the time-domain signal data) and the sine wave (equation 1 below) or cosine wave (equation 2 below) at the frequency of movement ω of the ion of interest, and the previous point in time t_n-1The previous STORI points obtained at (a) are summed as represented in the following equation.

STORI_REAL(t_n)＝S(t_n)*sin(ω*t_n)+STORI_REAL(t_n-1) (1)

STORI_IMAG(t_n)＝-S(t_n)*cos(ω*t_n)+STORI_IMAG(t_n-1) (2)

The above components each depend on the initial phase of the signal, and therefore none of the components alone can provide quantitative information about the amplitude of the signal. The phase correlation of the signal can be removed by calculating the magnitude of the real and imaginary STORI components, as shown in equation (3) below:

STORI_MAG(t_n)＝((STORI_REAL(t_n))²+(STORI_IMAG(t_n))²)^1/2 (3)

in the data system 165 depicted in fig. 2, the foregoing calculations are performed by operation of the STORI graph construction module 217 (e.g., by executing a set of software instructions) that receives as input the discretized time-domain signal data and outputs a STORI_MAGRepresentation of the graph with respect to time. In the case where there are multiple analyte ions of different masses and it is desired to determine the mass of each of the multiple analyte ions separately, a STORI map is then constructed for each analyte ion according to its individual moving frequency (which varies with respect to its m/z as described above). The charge determination/decay evaluation module 220 may then utilize the STORI map to determine the charge state and to identify and characterize ion decay events that occurred during the acquisition of the time-varying signal. STORI map building module 217 may also contain logic for causing the calculated STORI map to be visually displayed to an instrument operator on a monitor forming part of data system 165.

An example of a STORI plot for a single ion is depicted in fig. 4, where the ion of interest generates a signal throughout the signal acquisition period. In this STORI diagram, STORI_MAGThe change over time approximates a straight line with a constant slope. The STORI graph slope is a measure of the ion charge, where ions of higher charge exhibit a steeper slope relative to ions of lower charge. Thus, the charge state of the ions can be determined based on the slope of this line. In the configuration of fig. 2, the charge state determination is performed by module 220 using a stored set of empirically derived calibrations relating to the STORI map slope and charge state obtained using an analyte of known charge. Since the slope of the STORI map versus charge can be a function of the operating conditions of the mass analyzer (e.g., the electricity applied to inner electrode 135)Pressure) and thus the calibration data may be multidimensional where the slope versus charge relationship is empirically established for different instrument operating parameter values within an expected range.

The shape of the STORI diagram is also useful for revealing the occurrence of ion decay events. In fig. 4, a STORI plot is shown where a single ion decays at approximately 1.2 seconds. In a standard fourier transform, the peaks produced by such decaying ions will have a reduced intensity relative to the peaks derived from the fourier transform of the ions of fig. 3. This may be thought of as the charge ratio of the ions of figure 4 being lower than practical. However, examination of the STORI plots of fig. 3 and 4 shows that the slopes of the plots before the time point of about 1.2 seconds are the same, and thus the two ions have the same charge state. In certain embodiments, the STORI map construction module 217 or the charge determination module 220 may contain logic for evaluating the STORI map and providing an indication to an operator that a decay event has occurred (i.e., in response to detection of a change in slope across the acquisition period), or may contain logic for ignoring the decayed portion of the STORI map when determining charge. In other embodiments, as described above, where the transient acquisition and m/z and charge determination steps are repeatedly performed for the ions of interest in order to generate a histogram of the distribution of measured masses, the STORI map construction or charge determination module may discard (i.e., not included in the histogram construction) any transients in which a change in slope in the STORI map is observed over the acquisition period.

Visual inspection of the STORI diagram depicted in fig. 3 shows an initial "wiggle" (i.e., slight oscillation about a straight line) portion, which substantially disappears after about 1.2 seconds. This oscillatory behavior is actually due to the simultaneous presence of ions corresponding to the STORI diagram of fig. 4 in the trapping region of the electrostatic trap, which results in a repeating pattern of constructive and destructive interference. This may result in an incorrect estimate of the state of charge from the point at which the slope is measured during the period of constructive and destructive interference. This situation may be addressed by considering (i.e., by operation of the charge determination module 220) only the slope of the portion of the map corresponding to after the second ions have decayed, where the slope of the STORI map in fig. 3 is measured over the entire interference period, or by a more complex process.

One potential problem with CDMS in electrostatic or other harmonic trapping devices is the possibility of seeing two ions in the same signal, either because the two ions have the same mass or because the m/z of the two ions are close enough that they are not resolved during the acquisition period. For the case where two ions have the same m/z, it is difficult to distinguish from the case where one ion has twice the charge. Fig. 5 shows a more complex STORI diagram showing this. There are initially two ions with the same frequency (or m/z), where the first ion decays after about 0.15 seconds and the second ion decays after about 0.95 seconds. Gathering such information from the examination or processing of STORI maps is fairly straightforward, but extracting such information from standard fourier transform techniques is very difficult. In certain embodiments, the charge determination module 220 may be configured to process the STORI map generated by the map construction module 217 to determine whether there are multiple ions of the same mass (or whether there are non-resolvable ions), as indicated by certain characteristic maps, such as a slope change, and to take appropriate action, such as adjusting the determined charge accordingly, or by discarding data from the acquisition when constructing a histogram of the distribution of the measured mass.

In summary, the STORI map can be used to determine the charge state (in the case where ions remain unattenuated throughout the acquisition period, and where ion decay does occur), evaluate ion decay time, and distinguish the signal generated by multiple ions from the signal generated by a single ion.

In another application of the STORI map technique, the ion lifetime distribution of ions of interest can be determined by repeating the transient acquisition and examining the resulting STORI map to identify when a decay event has occurred, as evidenced by a change in slope of the map. If it can be assumed that the main cause of ion loss is collisions with background neutrals and that one collision is sufficient to eliminate one ion, the lifetime distribution can be viewed and the ion collision cross-section estimated in a manner similar to ion mobility spectrometry.

Alternative method for STORI calculation

An alternative method for calculating and constructing a STORI map, such as by module 217, is described below. This approach may yield benefits in terms of reduced computational expense and increased computational speed.

For transient S and frequency ω₀The STORI diagram is defined as follows:

the plot tracks individual ions at frequency ω₀The accumulation curve over time. The graph can be used to determine the start and end of an ion, the modification of an ion (e.g., loss of charge), and most importantly, the charge of an ion by the slope of the linear region in the graph.

The calculation of the STORI map is straightforward by simple integration (summing in the discrete case). However, the direct method is time consuming, mainly because exp (-i ω) occurs at many (about 1,000,000) time points₀s) is expensive to calculate. Efficiency can be improved by integrating (i.e., by decimation) only over a subset of the points in time of integration. However, there is a limit to the degree of decimation, since integration as a cumulative sum will accumulate errors over time.

Disclosed herein is a new approach that will allow extreme decimation of the integration time range (and almost complete avoidance of exp (-i ω)₀s) evaluation). Since the relevant features from the STORI map, such as slope, start and stop, are slowly varying, the extraction, even extreme extraction, does not compromise the quality of those features. On the other hand, the efficiency gain will be significant.

For clarity, assume that the transient S has only a single frequency ω:

S(t)＝A(ω)(cos(ωt)+isin(ωt))，

where A (ω) is the amplitude of the single frequency transient.

STORI simplification

This can be analytically calculated as follows:

performing integration to obtain

The above equation can now be easily extended to where the signal is the sum of signals with different ω:

S(t)＝∫A(ω)(cos(ωt)+isin(ωt))dω，

where A (ω) is now only the Fourier transform of S (t). Then, the STORI becomes

Changing the integral variable from ω to ω - ω₀So as to obtain the compound with the structure,

for further simplification, define

Then obtain

STORI(t)＝∫A(ω+ω₀)G(ω,t)dω

It is conceptually interesting to recognize that the above is simply a convolution of A with G, and where G is simply a Fourier transform of the Hewyside function (Heavydide function) H (t, s),

for efficiency, the key thing to note is for ω₀Is completely limited to the fourier transform of the function a, i.e. S. Thus, the function G may be pre-computed and cached for a sequence of target time points (such as 1024 evenly spaced points throughout the time range of interest). For any frequency of interest ω₀The cached G function may be reused to compute the convolution integral.

Finally, it is known that at the peak frequency ω₀In the vicinity, A (ω) drops very sharply, and G disappears from 0 to 1/ω very quickly. Thus, the STORI convolution integral need only be calculated over a very small range of ω (typically less than +/-100); incidentally, this also means that G only needs to be calculated and cached for a small number of points in ω. Using the 1024 target time points and +/-100 frequency point examples, the computation of a complete STORI graph requires only 1024 x 200 complex multiplications, which can be done on a millisecond time scale on any modern CPU. Even more extreme decimation may be performed, such as 256 rather than 1024 time points, to perform faster without degrading the quality of the STORI map.

Alternative to orbital electrostatic traps

While the invention has been described above and depicted in the drawings in connection with its implementation in an orbital electrostatic trap with a quadrupole logarithmic trapping field, it is to be understood that this implementation is described by way of illustrative, but non-limiting, example. The invention may be implemented in any electrostatic trap or equivalent structure in which confined ions undergo harmonic motion along a longitudinal axis, including traps in which ions do not undergo orbital motion. Examples of non-orbital electrostatic traps that may be suitable for practicing the present invention are in"Concept of electrostatic non-orbital harmonic ion trap (The Concept of E)Illustrative Non-organic Nano-atomic Ion Trapping), "the Cassinian trap (Cassinian trap) described in the Journal of International Mass Spectrometry (International Journal of Mass Spectrometry), Vol 287, p 114-118 (2009), which is incorporated herein by reference.

Deviation from pure harmonic motion

Those of ordinary skill in the art will recognize that the motion of ions along the longitudinal axis of an electrostatic trap or equivalent structure may exhibit slight deviations from pure harmonic (e.g., single frequency sinusoidal) motion due to, for example, electrode machining tolerances, component misalignment, electrical noise, and small field failures caused by electrode truncation. However, such slight deviations from pure harmonicity that would occur in any real-world device would not significantly degrade the performance of the method outlined above for deriving the m/z and charge state of the ions. Accordingly, the term "harmonic" as recited in the claims below should be construed to cover the following: there is a small, operationally insubstantial deviation from pure harmonic motion.