Systems and Methods for Scaling Injection Waveform Amplitude During Ion Isolation

Abstract

This disclosure describes a method of adjusting the amplitude of broadband waveforms for isolation, especially during injection to a multipole trapping device. Isolation during injection to a trapping device is known to be an effective way of accumulating a desired population of ions while rejecting unwanted species. The waveform amplitude required to eject unwanted species varies as a function of isolation time, but using automated gain control, the time required to accumulate a given population of ions may vary over several orders of magnitude. Thus, when the injection times are very long, precursor ions of interest are resonated for a long time and may be inadvertently ejected from the trap, using conventional methods. By setting the waveform amplitude lower for longer accumulation times, good isolation efficiency can be maintained for the precursor, while still rejecting unwanted ions.

Claims

1. A method for accumulating and isolating a pre-determined quantity of a pre-determined ion species comprising a pre-determined isolation mass-to-charge ratio, (m/z).sub.ISO, within a within a radio-frequency (RF) ion trap of a mass spectrometer, the method comprising: (a) determining an accumulation time duration, t.sub.A, required to accumulate the pre-determined quantity of the pre-determined ion species within the RF ion trap based on a prior measurement of a flux of said pre-determined ion species within a stream of ions including the pre-determined ion species and other ion species comprising other mass-to-charge ratio (m/z) values; and (b) introducing the stream of ions into the RF ion trap for an accumulation time period having duration, t.sub.A, while simultaneously applying a notched supplemental AC voltage waveform to electrodes of the RF ion trap, the supplemental AC voltage waveform having component frequencies chosen to resonantly eject only ion species for which m/z(m/z).sub.ISO, wherein a time-varying amplitude, A(t), of the applied supplemental AC voltage waveform is caused to decay with time, t, during at least a portion of the accumulation time period.

2. A method as recited in claim 1, wherein the time-varying amplitude, A(t), of the applied supplemental AC voltage waveform is caused to decay exponentially with time, t, during the portion of the accumulation time period.

3. A method as recited in claim 2, wherein the time-varying amplitude of the supplemental AC voltage waveform during the portion of the accumulation time period is given by: A(t)=B+A.sub.0e.sup.C|t-t.sup.REF.sup.|, where Band Care empirically determined constants, t.sub.REF is a reference time and A.sub.0 is a reference amplitude of the supplemental AC voltage waveform, said waveform comprising a frequency profile previously determined to eject all ions for which m/z(m/z).sub.ISO in a substantially similar amount of time.

4. A method as recited in claim 1, wherein the time-varying amplitude, A(t), of the applied supplemental AC voltage waveform is caused to decay with time, t, during the entirety of the accumulation time period.

5. A mass spectrometer system comprising: an ionization source; an RF ion trap configured to receive a continuous stream of ions from the ionization source; a mass analyzer and an ion detector configured to receive ions from the ion source and to measure an ion flux of each of a plurality of ion species comprising respective mass-to-charge (m/z) values; a power supply configured to apply trapping voltages and a supplemental AC voltage waveform to the RF ion trap and to supply voltages to the mass analyzer; and a computer processor or electronic controller comprising program instructions operable to: cause the mass analyzer and ion detector to measure the ion flux of a pre-determined ion species within an ion stream also comprising a plurality of other ion species, the pre-determined ion species having a pre-determined isolation mass-to-charge ratio, (m/z).sub.ISO and the plurality of other ion species having respective different m/z values; determine, from the measured ion flux of the pre-determined ion species, a time duration required to accumulate a pre-determined quantity of the pre-determined ion species; and cause the RF ion trap to receive therein the stream of ions for an accumulation time period having duration, t.sub.A, while simultaneously causing the power supply to apply a notched supplemental AC voltage waveform to electrodes of the RF ion trap, the supplemental AC voltage waveform consisting of component frequencies effective to resonantly eject only ion species for which m/z(m/z).sub.ISO, the applied supplemental AC voltage waveform further comprising a time-varying amplitude, A(t), that decays with time, t, during at least a portion of the accumulation time period.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above noted and various other aspects of the present invention will become further apparent from the following description which is given by way of example only and with reference to the accompanying drawings, not drawn to scale, in which:

[0022] FIG. 1A is a plot of the displacement of an ion in the x-direction within a linear ion trap assuming damped, driven, harmonic oscillator behavior using Eq. (1), for driving frequency .sub.0=100 kHz and damping constant v=0.5 ms.sup.1, and showing bounding lines as given by Eq. (2);

[0023] FIG. 1B is a plot of the displacement of an ion in the x-direction within a linear ion trap assuming damped, driven, harmonic oscillator behavior using Eq. (1), for driving frequency .sub.0=100 kHz and damping constant v=0.1 ms.sup.1, and showing bounding lines as given by Eq. (2);

[0024] FIG. 1C a plot of the displacement of an ion in the x-direction within a linear ion trap assuming damped, driven, harmonic oscillator behavior using Eq. (1), for driving frequency .sub.0=100 kHz and damping constant v=0.001 ms.sup.1, and showing bounding lines as given by Eq. (2);

[0025] FIG. 1D is a plot ion-ejection curves, each curve showing the locus of points at which ions are ejected from a linear ion trap, in terms of ion-trap residence times and driving-force amplitudes, each curve relating to the respectively indicated value of the ratio .sub.0/v;

[0026] FIG. 2 is a plot experimentally-determined ion-ejection curves, each curve showing the locus of points at which ions are ejected from a linear ion trap, in terms of ion-trap residence times and driving-waveform amplitudes, each curve relating to the ejection of Cs.sup.+m/z=132.9 Da at the respectively indicated value of helium pressure;

[0027] FIG. 3 is a set of plots relating to the abundances of precursor and fragment ions during isolation of [MRFA+H].sup.+ within a linear ion trap using an excitation time was 4 ms and helium pressure was 6.910.sup.5 Torr, where the plotted precursor and fragment abundances are normalized to the initial abundance of the precursor; and

[0028] FIG. 4 is a pair of plots of maximum fragment abundance normalized to initial precursor abundance as a function of isolation time, using the same experimental conditions as noted with regard to FIG. 3;

[0029] FIG. 5A is a plot of experimentally observed ion isolation efficiency versus accumulation time during a LC/MS experiment in which the isolation waveform amplitude is optimized for an excitation time of 4 ms;

[0030] FIG. 5B is a plot of experimentally observed ion isolation efficiency versus accumulation time during a LC/MS experiment in which the isolation waveform amplitude was varied as a function of accumulation time; and

[0031] FIG. 6 is a schematic depiction of a general mass spectrometer system.

DETAILED DESCRIPTION

[0032] The following description is presented to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Accordingly, the disclosed materials, methods, and examples are illustrative only and not intended to be limiting. Various modifications to the described embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiments and examples shown but is to be accorded the widest possible scope in accordance with the features and principles shown and described. The particular features and advantages of the invention will become more apparent with reference to the FIGS. 1-4, 5A and 5B taken in conjunction with the following description.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. It will be appreciated that there is an implied about prior to the quantitative terms mentioned in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of comprise, comprises, comprising, contain, contains, containing, include, includes, and including are not intended to be limiting.

[0034] As used herein, a or an also may refer to at least one or one or more. Also, the use of or is inclusive, such that the phrase A or B is true when A is true, B is true, or both A and B are true. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, and as commonly used in the art of mass spectrometry, the term DC does not specifically refer to or necessarily imply the flow of an electric current but, instead, refers to a non-oscillatory voltage which may be either constant or variable. Likewise, as used herein, and as commonly used in the art of mass spectrometry, the term AC does not specifically refer to or necessarily imply the existence of an alternating current but, instead, refers to an oscillatory voltage or oscillatory voltage waveform. The term RF refers to an oscillatory voltage or oscillatory voltage waveform for which the frequency of oscillation is in the radio-frequency range.

[0035] The regulation of ion populations in modern ion trapping instruments includes accumulating ions for a variable amount of time, based on feedback (as, for example, relating to ion flux rate at a given time) from prior acquisitions. At all other times, the ion beam is discarded through some gating mechanism, such as Senko U.S. Pat. No. 8,026,475. The length of the time period of ion accumulation can vary over many orders of magnitude, from about 10.sup.6 s, to 1 s. For ion trapping devices within which nominally quadrupolar potentials are generated, ion motion during isolation can be approximated, in many cases, as corresponding to the motion of a damped, driven, harmonic oscillator. When the ion is driven at resonance, the ion motion (in this instance, displacement x(t) parallel to the x-direction, as a function of time) and its amplitude with respect to time, a(t), are given by Eq. (1) and Eq. (2), as shown below, where E is the amplitude of the driving force, coo is the frequency of ion motion and excitation, and v (units of inverse time) is a damping constant.

[00001]$\begin{array}{cc}x\left(t\right)=E.Math.\frac{{}_0}{v}.Math.\left(1-e^{-\mathrm{vt}/2}\right).Math.\sin \left({}_0.Math.t\right)& \mathrm{Eq}..Math.\left(1\right)\\ a\left(t\right)=E.Math.\frac{{}_0}{v}.Math.\left(1-e^{-\mathrm{vt}/2}\right)& \mathrm{Eq}..Math.\left(2\right)\end{array}$

[0036] Examples of the trajectories of damped, driven oscillators at several different values of v are given in FIG. 1A-1C, where the illustrated oscillatory trajectories are calculated by Eq. (1), and the amplitudes, shown as outlining the trajectory envelope, are calculated using Eq. (2). The trajectory presented in FIG. 1A is calculated using the greatest damping (0.5 ms.sup.1) and the trajectory presented in FIG. 1D is calculated using the least damping (0.001 ms.sup.1). Upon rearranging Eq. (2), a relation is given in Eq. (3) for the magnitude of the driving force (supplemental voltage waveform amplitude) E that is necessary for the particle to have a certain displacement amplitude, a.sub.ej, which could be considered as the distance from the center of the trapping device to the trapping electrodes, i.e. the distance from the trap center or central axis at which the ion is ejected from the trap. Examples of ion

[00002]$\begin{array}{cc}E=\frac{a_{\mathrm{ej}}}{\frac{{}_0}{v}.Math.\left(1-e^{-\mathrm{vt}/2}\right)}& \mathrm{Eq}..Math.\left(3\right)\end{array}$

ejection curves calculated using Eq. (3) with the aforementioned damping constants are shown in FIG. 1D.

[0037] FIGS. 1A-1D demonstrate some of the fundamentals of resonance ejection in a quadrupolar device. For example, to eject an ion in a shorter amount of time, more excitation (greater supplemental voltage waveform amplitude, E) is needed, and the growth in displacement amplitude is nominally linear in the absence of collisions with neutral gas molecules. If the damping in the device is high, then there is a threshold excitation amplitude required to eject the ion, even for indefinitely long times. However, if there is little damping, then eventually an ion will be ejected, even with a small excitation. These conclusions are confirmed by the experimental results shown in FIG. 2, where the excitation amplitude required to eject Cesium ion (m/z 132) is plotted as a function of time. The data are fit to a generalization of Eq. (3), given in Eq. (4).

[00003]$\begin{array}{cc}y=\frac{a}{b-c.Math..Math.\exp \left(-\mathrm{dx}\right)}& \mathrm{Eq}..Math.\left(4\right)\end{array}$

Cesium ion was chosen for this experiment, because it is an monoatomic ion and, as such, does not dissociate into smaller particles when subjected to neutral gas collisions. Thus. in these data, it is not necessary to consider fragmentation processes, which are discussed below.

[0038] Typically, the amplitude of the isolation waveform required for efficient ejection of unwanted species and efficient retention of the species of interest is determined for one particular isolation time duration. A method for performing this calibration was described previously in co-pending and commonly owned U.S. patent application Ser. No. 14/709,387 (Attorney Docket No. 19679US1/NAT) filed on May 11, 2015 and titled Systems and Methods for Ion Isolation, the disclosure of which is incorporated herein by reference in its entirety. A method described in that co-pending application includes supplying an isolation waveform to a radio frequency ion trap, the isolation waveform having at least one notch at a target mass-to-charge ratio, the isolation waveform having a frequency profile determined to eject unwanted ions at a plurality of frequencies in a substantially similar amount of time. The isolation waveform may-have frequency-dependent amplitude that can apply an excitation force to unwanted ions at a plurality of frequencies such that they can be ejected in a substantially similar amount of time, such as substantially simultaneously. The isolation waveform may include a notch at a certain frequency corresponding to the oscillation of the ion species to be isolated such that an excitation force is not applied to the ions to be isolated and such that they are not removed from an RF ion trap. According to the above-noted co-pending application, the frequency profile may be determined by: (1) supplying an ion population from a calibrant to be injected into a radio frequency ion trap, the ion population having a plurality of ion species covering a range of mass-to-charge ratios; (2) applying a waveform having a flat frequency profile to the radio frequency ion trap; (3) identifying ions of the ion population remaining in the radio frequency ion trap; (4) repeating steps 1-3 at increasing amplitudes of the waveform to identify an amplitude at which all the ions of a given ion species are ejected from the radio frequency ion trap for each ion species of the ion population; and (5) characterizing the frequency profile for the radio frequency ion trap based on the amplitudes at which all the ions of a given ion species are ejected from the radio frequency ion trap. The steps 1-4 may be repeated at multiple trapping radio frequency amplitude levels so as to cover a range of possible frequencies. Unfortunately, the data in FIG. 2 demonstrate that the calibrated amplitude may be too high at longer excitation times. In the context of isolation during injection to the quadrupole ion trap, this excess of excitation can lead to inefficient collection of the ion of interest.

[0039] In addition to potential loss of ions (such as precursor ions) as a result of ejection from an ion trap, the ions of interest (precursor ions) can also be lost due to fragmentation within the trap. Even though the broadband excitation does not contain power in a range around the precursor oscillation frequency, the precursor kinetic energy is increased due to off-resonance excitation. Collisions with the neutral trapping gas start to transfer more energy to the precursor than they remove, and fragment ions will form when the accumulated precursor internal energy is sufficiently great.

[0040] In order to determine how unwanted fragmentation may contribute to ion loss, an experiment was performed to characterize the fragmentation of a peptide as it is being isolated. In the experiment, the singly charged peptide MRFA was infused, and isolated using a quadrupole mass filter to remove all other background species. Then a notched broadband isolation waveform was applied such that no excitation energy was applied at frequencies within a range about the characteristic frequency of the precursor ion, or within a range of the characteristic frequencies of the expected major fragments. The width of the isolation range was either 0 Da (no notch) or 2 Da for the precursor, and 10 Da for the fragments. The amplitude of the waveform was increased, and the abundances of the precursor and fragments were monitored relative to the initial precursor abundance (FIG. 3). The experimental results indicate that when the notch width about the precursor is 2 Da, more waveform amplitude is needed to completely eliminate the precursor, compared to when the excitation waveform has no notch (0 Da). This result is due to the precursor only receiving off-resonance excitation in the 2 Da case. In both cases, however, fragments are formed at the amplitude corresponding to the onset of precursor ejection. The amplitude of the waveform should be set, in this case, in the range between 30 and 40, so that the precursor isn't ejected with too much voltage, but unwanted ions near the notch are ejected. The data show that, in this range, about 10% of the precursor is actually lost due to fragmentation instead of from direct ejection. The experiment was repeated on the doubly charged version of MRFA which is much more fragile. However, in this latter experiment, the fragmentation was much less, because the ion was isolated at a higher Mathieu q value, where the rate of change of frequency with respect to mass is higher, and off-resonance excitation is reduced. Thus the fragmentation is expected to depend not only on the thermodynamic properties of the precursor, but on the parameters of the isolation.

[0041] The change in fragment formation with respect to excitation time duration during the above experiment was also characterized (FIG. 4). As expected, the fragment formation follows first order kinetics, and can be modeled with an exponential decay with time. At long times, the fraction of precursor lost due to fragmentation is about 0.3, which is significant. However, because the fragmentation occurs at the same waveform amplitude corresponding to cause the onset of precursor ejection, the optimum waveform amplitude exhibits the same general behavior as indicated by Eqs. (3) and (4), as time duration is increased. The amplitude given by this function reduces precursor losses due to both ejection and fragmentation at long times.

[0042] To confirm these assertions and test a novel method, the isolation efficiency was measured for peptide ions of a HeLa cell digest in a nano-LCMS/MS experiment. Isolation waveforms were applied during ion accumulation as well as for a 4 ms time period after isolation. Isolation efficiency was estimated as the flux of precursor ions as measured in a MS/MS experiment (with no applied collision energy) divided by the flux of precursor ions in a previous survey experiment. In a first experiment, the isolation waveform amplitude was determined via the method described previously (in the aforementioned U.S. patent application Ser. No. 14/709,387) for a 4 ms injection time. The results of this experiment (depicted in FIG. 5A) demonstrate that, under these conditions, isolation efficiency drops to nearly zero at injection times longer than 10 ms. However, when the amplitude of the isolation waveform during accumulation is varied as a function of the accumulation time period, as depicted in FIG. 5B, the results are dramatically better (FIG. 5B). For this latter experiment, an exponential decay function was used to set the waveform amplitude as a function of time, as shown in Eq. (5), prior to deriving the relations of Eq. (3) and Eq. (4).

A(t)=B+A.sub.0e.sup.C|t-t.sup.REF.sup.|Eq. (5)

In the above Eq. (5), the constant A.sub.0 represents the 4 ms excitation amplitude, the reference time, t.sub.REF, is 4 ms, and B and C are empirically determined constants. The values of the constants B and C were obtained by a fit to data (for isolation of an ion species at m/z 400) in the form of FIG. 2. Many similar functions can actually give an improvement, as long as they are generally decreasing as a function of time.

[0043] The discussion included in this application is intended to serve as a basic description. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Any patents, patent applications, patent application publications or other literature mentioned herein are hereby incorporated by reference herein in their respective entirety as if fully set forth herein, except that, in the event of any conflict between the incorporated reference and the present specification, the language of the present specification will control.