Patent Application
20210210331
The invention relates generally to mass spectrometry, and more particularly to an apparatus and method for measurement of the mass-to-charge ratio and charge of a single ion.
Charge detection mass spectrometry (CDMS) is a technique where the masses of individual ions are determined from concurrent measurement of each ion's mass-to-charge ratio (m/z) and charge. One technique used in academic laboratories for CDMS, referred to 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”, Rapid Communications in Mass Spectrometry 9:15 (1995), 1528-1538. In such instruments, an ion's m/z is determined by its oscillation frequency between the mirrors, while its charge is determined based upon the amplitude of the signal on the inductive detector. Separate and direct measurement of the charge thus overcomes a common challenge for large and/or heterogeneous analytes investigated with conventional electrospray mass spectrometry, where it may not be possible to separate incrementally charged ion species and thereby infer charge state.
Existing ion trap CDMS instrumentation presents several significant technical challenges. First, because the potential generated by opposing mirrors is generally anharmonic, the measured frequency is dependent on the initial kinetic energy of the ion. This may lead to poor m/z measurement accuracy for single particles, which also results in poor resolution when assembling a histogram of measured mases. In addition, the signal generated by the inductive detector is not sinusoidal, but processing of the signal is performed using Fourier transform analysis. The resultant signal is distributed among numerous harmonics, which significantly reduces overall system sensitivity. This imposes an additional restriction where only a single ion species can be analyzed at a time, leading to very long acquisition cycles. Finally, ions are moved directly in existing CDMS instrumentation from the source to the mirrors, without proper desolvation. The lack of desolvation may result in the observation of mass shifts during the measurement period as the ion loses solvent.
Against this background, there is a need in the art for an apparatus and method for concurrent determination of an ion's m/z and charge that avoids the problems arising in prior art devices associated with anharmonic ion motion and incomplete desolvation.
Roughly described, an apparatus and corresponding method are disclosed for measurement of the m/z and charge of an ion, and consequently its mass, by detection of the frequency and amplitude of an image current signal induced by the ion's oscillatory movement within an electrostatic trap. The electrostatic trap includes a plurality of electrodes to which non-oscillatory voltages are applied. The electrodes are shaped and arranged to establish an electrostatic trapping field that has causes the ion to undergo harmonic motion with respect to a longitudinal axis of the trap. The apparatus further includes a detector that generates a time-varying signal representative of the current induced on the detector by the harmonic longitudinal motion of the ion. A data system receives the time-varying signal from the detector, and processes the signal to derive its frequency and amplitude. The data system is further configured to determine the ion's m/z from the derived frequency, and to determine the ion's charge from the derived amplitude.
In more specific embodiments, the electrostatic trap is formed from coaxially arranged inner and outer electrodes, each elongated along a longitudinal axis, and the ion is trapped in the annular space between the electrodes. The inner and outer electrodes may be shaped and arranged to establish a quadro-logarithmic field in the annular space, such that the restorative force exerted by the field along the central axis is proportional to the position of the ion along the central axis relative to a transverse plane of symmetry. The outer electrode may be split in half along the transverse plane of symmetry into first and second parts, and the detector may comprise a differential amplifier connected across the first and second parts. The data system may derive the frequency and amplitude of the detector signal by applying a fast Fourier transform (FFT) routine to convert the signal from the time to the frequency domain. The ion may be trapped in an ion store prior to release to the electrostatic trap to reduce its kinetic energy and promote complete desolvation. Analysis of two or more ion species may be performed simultaneously within the electrostatic trap, such that the data system processes the signal produced by the motion of the two or more ion species to derive multiple peaks, each peak having an associated frequency and amplitude and corresponding to a different one of the ion species.
In accordance with another embodiment, a method is provided for determining the m/z and charge of an ion of interest. The method includes injecting an ion population including the ion of interest into a trapping region, and establishing an electrostatic trapping field within the region that causes the ion population to undergo harmonic motion along a central axis. A time-varying signal, representative of the current induced on a detector by the harmonic motion of the ion population, is processed to derive a frequency and an amplitude associated with the ion of interest. The m/z and charge of the ion of interest may then be determined respectively from the derived frequency and amplitude.
In more specific implementations of the foregoing method, the electrostatic field may be established in an annular region between an inner and outer electrode, and the electrostatic trapping field may be a quadro-logarithmic field. The ion of interest may be a protein, protein complex, viral capsid, or high molecular weight polymer. The ion population may include two (or more) ions of interest, and the method may include deriving, from the time-varying signal, frequencies and amplitudes associated with each of the two ions of interest, and determining the m/z and charge state for each ion of interest from their respective derived frequencies and amplitudes.
In the accompanying drawings:
Ions generated by source 105 are directed and focused through a series of ion optics disposed in vacuum chambers of progressively reduced pressures. As depicted in
Apparatus 100 may additionally include a quadrupole mass filter (QMF) 110 that transmits only those ions within a selected range of values of m/z. 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 the selectively transmitted ions is set by appropriate adjustment of the amplitudes of the RF and resolving direct current (DC) voltages applied to the electrodes of QMF 110 to establish an electric field that causes ions having m/z's outside of the selected range to develop unstable trajectories. The transmitted ions may thereafter traverse additional ion optics (e.g., lenses and RF multipoles) and enter ion store 115. As is known in the art, ion store 115 employs a combination of oscillatory and static fields to confine the ions to its interior. In a specific implementation, ion store 115 may take the form of a curved trap (referred to colloquially as a “c-trap”) of the type utilized in Orbitrap mass spectrometers sold by Thermo Fisher Scientific. The curved trap is composed of a set of generally parallel rod electrodes that are curved concavely toward the ion exit. Radial confinement of ions within ion store 115 may be achieved by applying oscillatory voltages in a prescribed phase relationship to opposed pairs of the rod electrodes, while axial confinement may be effected by applying static voltages to end lenses positioned axially outwardly of the rod electrodes.
Ions entering ion store 115 may be confined therein for a prescribed cooling period in order to reduce their kinetic energies prior to introduction of the ions into electrostatic trap. Confinement of the ions within the ion store for a prescribed period may also assist in desolvation of the ions, i.e., removal of any residual solvent moieties from the analyte ion. As discussed hereinabove, the presence of residual solvent may result in mass shifts during analysis which interfere with the ability to accurately measure m/z and charge. To facilitate kinetic cooling and desolvation of the ions, an inert gas such as argon or helium may be added to the ion store internal volume; however, the cooling gas pressure should be regulated to avoid unintended fragmentation of the analyte ions and/or excessive leakage of the gas into electrostatic trap 120. The duration of the cooling period will depend on a number of factors, including the kinetic energy of ions entering ion store 115, the inert gas pressure, and the desired kinetic energy profile of ions injected into electrostatic trap 120. After the cooling period has been completed, ions confined in ion store 115 may be radially ejected from ion store toward entrance lenses 125, which act to focus and direct ions into inlet 130 of electrostatic trap 120. Rapid ejection of ions from ion store 115 toward the electrostatic trap inlet may be performed by rapidly collapsing the oscillatory field within the ion store interior and applying a DC pulse to the rod electrodes positioned away from the direction of ejection.
To accurately measure ion charge using the CDMS technique, only individual ions of a particular ion species can be present in electrostatic trap 120 during a measurement event. As used herein, the term “ion species” refers to an ion of a given elemental/isotopic composition and charge state; ions of different elemental compositions are considered to be different ion species, as well as are ions of the same elemental composition but different charge states. The term “ion of interest” is also used herein to designate a particular ion species. If multiple ions of the same ion species are present during a measurement event, then 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 an individual ion. To avoid this type of mismeasurement, the ion population within ion store 115 should be kept sufficiently small such that the likelihood that two ions of the same ion species are confined within the ion store is maintained at an acceptable minimum. This may be accomplished by attenuation of the ion beam generated by source 105 (more specifically, by “detuning” ion optics located in the upstream ion path such that high losses of ions occur) and/or via regulation of the fill time (the period during which ions are accepted into ion store 115). To control the fill time, one or more ion optic components located upstream in the ion path of ion store may be operated as a gate to selectively allow or block passage of ions into the internal volume of ion store 115.
Electrostatic trap 120 may take the form of an orbital electrostatic trap, of the type commercially available from Thermo Fisher Scientific under the trademark “Orbitrap” and depicted in cross-section in
where r and z are cylindrical coordinates (r=0 being the central longitudinal axis and z=0 being the transverse plane of symmetry), C is a constant, k is field curvature, and Rm is the characteristic radius. This field is sometimes referred to as a quadro-logarithmic field.
Outer electrode 140 is split along the transverse plane of symmetry into first and second parts 150 and 155, which are separated from each other by a narrow insulating gap. This arrangement enables the use of outer electrode 140, together with differential amplifier 160, as an image current detector. The presence of an ion proximal to the outer electrode induces a charge (of a polarity opposite to that of the ion) in the electrode having a magnitude proportional to the charge of the ion. The oscillatory back-and-forth movement of an ion along the z-axis between the first 150 and second 155 parts of outer electrode 140 causes image current detector 132 to output a time varying signal (referred to as a “transient”) having a frequency equal to the frequency of the ion's longitudinal oscillation and an amplitude proportional to the ion's charge.
Ions may be introduced tangentially into trapping region 145 through inlet aperture 130 formed in outer electrode 240. Inlet aperture 130 is axially offset (along the z-axis) from the transverse plane of symmetry, such that, upon introduction into trapping region 145, the ions experience a restorative force in the direction of the plane of symmetry, causing the ions to initiate longitudinal oscillation along the z-axis while orbiting inner electrode 135, as illustrated in
Measurement of charge state and m/z, and consequent calculation of the product mass, proceeds by the acquisition and processing of the transient. Transient acquisition by detector 132 is initiated promptly after injection of the analyte ion(s), and continued for a predetermined transient length. The transient length required for accurate measurement of m/z and charge state will vary according to the analyte, as well as the physical and operational parameters of electrostatic ion trap 120. In general, the transient will need to be of adequate duration to allow the signal to be reliably distinguished from noise. For a typical analyte ion, it is anticipated that a satisfactory signal-to-noise ratio may be achieved using a commercially-available orbital trapping mass analyzer at a transient length of 500 milliseconds. It will be understood that the maximum transient length will be limited by the duration for which the analyte ion is stably trapped within 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 detector 132 is processed by data system 165, the functions of which will be described below in connection with
As noted above, the motion along the z-axis of an analyte ion trapped within the field generated in trapping region 145 is harmonic and may be represented as a simple sinusoidal function. The output of FFT module 210 will thereby yield a frequency spectrum that has a strong peak of amplitude A at the fundamental frequency of oscillation w of the ion being analyzed. When multiple ion species are present within the electrostatic trap during the measurement event (i.e., during acquisition of a 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 a trapped ion is anharmonic and non-sinusoidal (for which the FFT output will include numerous peaks distributed among various components), the signal for each ion species in the electrostatic trap 120 will be concentrated into a single peak appearing at the fundamental frequency of oscillation, thereby improving sensitivity and enabling charge measurement for lower-charge ions relative to prior art CDMS devices.
The frequency spectrum generated by FFT module 210 is provided as input to m/z determination module 215 and charge determination module 220, which process the frequency spectrum to respectively determine the m/z and charge of the analyte ion(s). M/z determination module 215 is configured to identify, for the or each analyte ion species present in the spectrum, the fundamental frequency of oscillation of the analyte ion. This frequency is then converted to a value of m/z. As noted above, the frequency of oscillatory ion motion along the z-axis is inversely proportional to the square root of the ion's m/z in accordance with the relation:
Thus, the m/z may be determined from the measured ion frequency using an empirically established frequency vs. m/z calibration curve generated by fitting an inverse square-root curve to data points acquired for analyte ions of known m/z, as is known in the art.
Charge determination module 220 is configured to process the frequency spectrum and provide as output, for the or each analyte ion species present in the spectrum, a value of the ion's charge. This operation is performed by determining the amplitude of the peak corresponding to the analyte ion's fundamental frequency of oscillation and converting the amplitude to a charge value in accordance with a predetermined relation between measured amplitude and charge. This relation may be established empirically using a curve fit to amplitude measurements acquired for calibrant ions of known m/z and charge. To a rough approximation, the relation between the peak amplitude and charge may be linear, since the charge induced on the detection electrodes by the motion of an ion will be equal and opposite to the ion's charge. However, for certain implementations of the invention, the relation between charge and peak amplitude may also be influenced, for a particular analyte ion, by the ion's m/z and initial kinetic energy. Referring to
In other implementations of the invention, charge determination module 220 may calculate charge based on a more complex relationship that takes into account instrument parameters, such as electrode voltages, that affect the initial kinetic energy of the analyte ion and may influence the relation between peak amplitude and ion charge (via changing the ion trajectory with respect to the detector electrodes). For this reason, it may be necessary to empirically construct calibration curves at a range of instrument parameters.
Once the m/z and charge of the analyte ion has been determined, the mass of the ion may be calculated simply via the product of the determined m/z and charge. If the spectrum contains multiple ion species, the mass for each ion species is calculated by the product of the m/z and charge determined for that species.
In certain implementations, the transient acquisition and m/z and charge determination steps will be performed repeatedly for an ion population (initially stored in the c-trap) that includes the analyte ion species. The resultant calculated masses may be binned to obtain a mass histogram, with the peak of the histogram representing the most likely mass. Generally, the width of the histogram will depend on the accuracy of the image charge determination, with narrower widths being indicative of high accuracy. Other techniques, including averaging, may be employed to improve the reliability of mass determination.
While the invention has been described above and depicted in the drawings in connection with its implementation in an orbital electrostatic trap having a quadro-logarithmic trapping field, it should be understood that this implementation is described by way of an illustrative rather than a limiting example. The invention may be implemented in any electrostatic trap or equivalent structure in which the confined ions undergo harmonic motion along a longitudinal axis, including traps in which the ions do not undergo orbital motion. An example of a non-orbital electrostatic trap that may be suitable for implementation of the present invention is the Cassinian trap described in Köster, “The Concept of Electrostatic Non-Orbital Harmonic Ion Trapping”, International Journal of Mass Spectrometry, V. 287, pp. 114-118 (2009), which is incorporated herein by reference.
Those skilled in the art will further recognize that the term “harmonic motion”, as used herein, includes motion that includes small deviations from purely harmonic motion, but where such deviations are operationally insubstantial such that the motion is predominately harmonic (i.e., that it can be substantially accurately modeled as an oscillatory function having a single frequency). In any “real-world” electrostatic trap, the electric field will include faults arising from (for example), dimensional and alignment errors and electrode truncation, which cause the restoring force to depart slightly from being a linear function of the ion's position relative to the central plane of symmetry, in turn causing the ion's motion to deviate by a small amount from purely harmonic. Such motion should be construed as being within the scope of “harmonic motion”, as set forth in the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/34009 | 5/24/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62679287 | Jun 2018 | US |
