Method and apparatus for mass analysis utilizing ion charge feedback

Abstract

A method of mass analysis and a mass spectrometer are provided wherein a batch of ions is accumulated in a mass analyzer; the batch of ions accumulated in the mass analyzer is detected using image current detection to provide a detected signal; the number of ions in the batch of ions accumulated in the mass analyzer is controlled using an algorithm based on a previous detected signal obtained using image current detection from a previous batch of ions accumulated in the mass analyzer; wherein one or more parameters of the algorithm are adjusted based on a measurement of ion current or charge obtained using an independent detector located outside of the mass analyzer.

Claims

1. A method of controlling the accumulation of ions in a mass spectrometer comprising: measuring an ion current or charge using a charge measuring device located downstream of an ion injection device at the end of an axis along which the ions are axially transmitted through the ion injection device such that the ions are transmitted through the injection device to reach the charge measuring device, followed by using the measured ion current or charge for adjusting the charge of a batch of ions subsequently injected from the ion injection device into a mass analyser located on a different axis and detected in the mass analyser using image current detection; wherein the frequency of measurement of ion current or charge using the charge measuring device is less than the frequency of obtaining detected signals from batches of ions in the mass analyser and wherein, between measurements of ion current or charge using the charge measuring device, measurements of total ion content are obtained using image current detection from ions injected into the mass analyser and used to control ion injection times for accumulating ions in the mass analyser.

2. A method as in claim 1 wherein the axis is in the direction of elongation of the injection device.

3. A method as in claim 2 wherein the ions are ejected to the mass analyser orthogonally from the injection device.

4. A method as in claim 1 wherein the charge measuring device is located downstream of a collision cell which is downstream of the injection device.

5. A method as in claim 1 wherein a multipole mass selector is provided upstream of the injection device.

6. A method as in claim 1 wherein the charge measuring device comprises one of: a collector plate, a faraday cup, a dynode, a secondary electron multiplier (SEM), a channeltron SEM, a microchannel SEM, a microball SEM, a charge-coupled device, a charge-injection device, an avalanche diode, an SEM with conversion into photons followed by a photomultiplier.

7. A method as in claim 1 wherein the mass analyser is a Fourier transform mass analyser.

8. A method as in claim 1 wherein the mass analyser is selected from the group of: an FT-ICR cell, an electrostatic trap, an electrostatic orbital trap and an RF ion trap.

9. A method as in claim 1 wherein the injection device comprises a linear ion trap.

10. A method as in claim 1 wherein the injection device comprises a curved linear ion trap.

11. A method as in claim 1 wherein the charge measuring device is used every 1 to 10 seconds to measure the ion current or charge.

12. A method as in claim 1 wherein the measurement of ion current or charge using the charge measuring device is performed concurrently with detecting a batch of ions accumulated in the mass analyser using image current detection.

13. A mass spectrometer comprising: a charge measuring device for measuring an ion current of ions, the charge measuring device located downstream of an ion injection device at the end of an axis along which ions are axially transmitted through the ion injection device such that the ions are transmitted through the injection device to reach the charge measuring device; a mass analyser located on a different axis and comprising detection electrodes for detecting a signal from a batch of ions accumulated in the analyser using image current detection; and a control arrangement operable to measure an ion current or charge using the charge measuring device and to use the measured ion current or charge to adjust the charge of a batch of ions subsequently injected into the mass analyser from the ion injection device and detected in the mass analyser using image current detection; wherein the control arrangement is further operable to measure ion current or charge using the charge measuring device less frequently than it obtains detected signals from batches of ions in the mass analyser and wherein, between measurements of ion current or charge using the charge measuring device, the control arrangement is operable to obtain measurements of total ion content using image current detection from ions injected into the mass analyser and to use the measurements of total ion content using image current detection to control ion injection times for accumulating ions in the mass analyser.

14. A mass spectrometer as in claim 13 wherein the axis is in the direction of elongation of the injection device.

15. A mass spectrometer as in claim 13 wherein the injection device is configured to eject ions to the mass analyser orthogonally from the injection device.

16. A mass spectrometer as in claim 13 wherein the charge measuring device is located downstream of a collision cell which is downstream of the injection device.

17. A mass spectrometer as in claim 13 wherein a multipole mass selector is located upstream of the injection device.

18. A mass spectrometer as in claim 13 wherein the charge measuring device comprises one of: a collector plate, a faraday cup, a dynode, a secondary electron multiplier (SEM), a channeltron SEM, a microchannel SEM, a microball SEM, a charge-coupled device, a charge-injection device, an avalanche diode, an SEM with conversion into photons followed by a photomultiplier.

19. A mass spectrometer as in claim 13 wherein the mass analyser is a Fourier transform mass analyser.

20. A mass spectrometer as in claim 13 wherein the mass analyser is selected from the group of: an FT-ICR cell, an electrostatic trap, an electrostatic orbital trap and an RF ion trap.

21. A mass spectrometer as in claim 13 wherein the injection device comprises a linear ion trap.

22. A mass spectrometer as in claim 13 wherein the injection device comprises a curved linear ion trap.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) In order to more fully understand the invention, various embodiments will now be described in more detail by way of examples with reference to the accompanying Figures in which:

(2) FIG. 1 shows schematically an embodiment of a mass spectrometer for carrying out the method of the present invention; and

(3) FIG. 2 shows a schematic flow chart of steps in an exemplary method according to the present invention.

(4) FIG. 3 shows an LC-MS mass chromatogram of a HeLa sample obtained using a prior art method of automatic gain control (AGC).

(5) FIG. 4 shows an LC-MS mass chromatogram of a HeLa sample obtained using the method of the present invention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(6) Referring to FIG. 1, a mass spectrometer 2 is shown in which ions are generated from a sample in an ion source (not shown), which may be a conventional ion source such as an electrospray. Ions may be generated as a continuous stream in the ion source as in electrospray, or in a pulsed manner as in a MALDI source. The sample which is ionised in the ion source may come from an interfaced instrument such as a liquid chromatograph (not shown). The ions pass through a heated capillary 4 (typically held at 320 C.), are transferred by an RF only S-lens 6 (RF amplitude 0-350 Vpp, being set mass dependent), and pass the S-lens exit lens 8 (typically held at 25V). The ions in the ion beam are next transmitted through an injection flatapole 10 and a bent flatapole 12 which are RF only devices to transmit the ions, the RF amplitude being set mass dependent. The ions then pass through a pair of lenses (both mass dependent, with inner lens 14 typically at about 4.5V, and outer lens 16 typically at about 100V) and enter a mass resolving quadrupole 18.

(7) The quadrupole 18 DC offset is typically 4.5 V. The differential RF and DC voltages of the quadrupole 18 are controlled to either transmit ions (RF only mode) or select ions of particular m/z for transmission by applying RF and DC according to the Mathieu stability diagram. It will be appreciated that, in other embodiments, instead of the mass resolving quadrupole 18, an RF only quadrupole or multipole may be used as an ion guide but the spectrometer would lack the capability of mass selection before analysis. In still other embodiments, an alternative mass resolving device may be employed instead of quadrupole 18, such as a linear ion trap, magnetic sector or a time-of-flight analyser. Such a mass resolving device could be used for mass selection and/or ion fragmentation. Turning back to the shown embodiment, the ion beam which is transmitted through quadrupole 18 exits from the quadrupole through a quadrupole exit lens 20 (typically held at 35 to 0V, the voltage being set mass dependent) and is switched on and off by a split lens 22. Then the ions are transferred through a transfer multipole 24 (RF only, RF amplitude being set mass dependent) and collected in a curved linear ion trap (C-trap) 26. The C-trap is elongated in an axial direction (thereby defining a trap axis) in which the ions enter the trap. Voltage on the C-Trap exit lens 28 can be set in such a way that ions cannot pass and thereby get stored within the C-trap 26. Similarly, after the desired ion fill time (or number of ion pulses e.g. with MALDI) into the C-trap has been reached, the voltage on C-trap entrance lens 30 is set such that ions cannot pass out of the trap and ions are no longer injected into the C-trap. More accurate gating of the incoming ion beam is provided by the split lens 22. The ions are trapped radially in the C-trap by applying RF voltage to the curved rods of the trap in a known manner.

(8) Ions which are stored within the C-trap 26 can be ejected orthogonally to the axis of the trap (orthogonal ejection) by pulsing DC to the C-trap in order for the ions to be injected, in this case via Z-lens 32, and deflector 33 into a mass analyser 34, which in this case is an electrostatic orbital trap, and more specifically an Orbitrap FT mass analyser made by Thermo Fisher Scientific. The orbital trap 34 comprises an inner electrode 40 elongated along the orbital trap axis and a split pair of outer electrodes 42, 44 which surround the inner electrode 40 and define therebetween a trapping volume in which ions are trapped and oscillate by orbiting around the inner electrode 40 to which is applied a trapping voltage whilst oscillating back and forth along the axis of the trap. The pair of outer electrodes 42, 44 function as detection electrodes to detect an image current induced by the oscillation of the ions in the trapping volume and thereby provide a detected signal. The outer electrodes 42, 44 thus constitute a first detector of the system. The outer electrodes 42, 44 typically function as differential pair of detection electrodes and are coupled to respective inputs of a differential amplifier (not shown), which in turn forms part of a digital data acquisition system (not shown) to receive the detected signal. The detected signal can be processed using Fourier transformation to obtain a mass spectrum.

(9) The mass spectrometer 2 further comprises a collision or reaction cell 50 downstream of the C-trap 26. Ions collected in the C-trap 26 can be ejected orthogonally as a pulse to the mass analyser 34 without entering the collision or reaction cell 52 or the ions can be transmitted axially to the collision or reaction cell for processing before returning the processed ions to the C-trap for subsequent orthogonal ejection to the mass analyser. The C-trap exit lens 28 in that case is set to allow ions to enter the collision or reaction cell 50 and ions can be injected into the collision or reaction cell by an appropriate voltage gradient between the C-trap and the collision or reaction cell (e.g. the collision or reaction cell may be offset to negative potential for positive ions). The collision energy can be controlled by this voltage gradient. The collision or reaction cell 50 comprises a multipole 52 to contain the ions. The collision or reaction cell 50, for example, may be pressurised with a collision gas so as to enable fragmentation (collision induced dissociation) of ions therein, or may contain a source of reactive ions for electron transfer dissociation (ETD) of ions therein. The ions are prevented from leaving the collision or reaction cell 50 axially by setting an appropriate voltage to a collision cell exit lens 54. The C-trap exit lens 28 at the other end of the collision or reaction cell 50 also acts as an entrance lens to the collision or reaction cell 50 and can be set to prevent ions leaving whilst they undergo processing in the collision or reaction cell if need be. In other embodiments, the collision or reaction cell 50 may have its own separate entrance lens. After processing in the collision or reaction cell 50 the potential of the cell 50 may be offset so as to eject ions back into the C-trap (the C-trap exit lens 28 being set to allow the return of the ions to the C-trap) for storage, for example the voltage offset of the cell 50 may be lifted to eject positive ions back to the C-trap. The ions thus stored in the C-trap may then be injected into the mass analyser 34 as described before.

(10) The mass spectrometer 2 further comprises an electrometer 60 which is situated downstream of the collision or reaction cell 50 and can be reached by the ion beam through an aperture 62 in the collisional cell exit lens 54. The electrometer 60 may be either a collector plate or Faraday cup and is connected to a high gain charge sensitive amplifier, typically with a gain of about 10.sup.11 V/Coulomb. It will be appreciated, however, that the electrometer 60 in other embodiments may be another type of charge measuring device. Preferably, the electrometer is of differential type which reduces noise pick-up from other electrical sources nearby. A first input of the electrometer is arranged to receive current or charge from the ion source while another input is arranged to have similar capacitance, dimensions and orientation to the first input but receives no ion current or charge at all. The electrometer 60 thus constitutes a second detector of the system, which is independent of the first detector, namely the image current detection electrodes 42, 44 of the mass analyser 34. In some embodiments the collision or reaction cell 50 may not be present, in which case the electrometer 60 is preferably located downstream of the C-trap behind C-trap exit lens 28.

(11) It will be appreciated that the path of the ion beam through the spectrometer and in the mass analyser is under appropriate evacuated conditions as known in the art, with different levels of vacuum appropriate for different parts of the spectrometer.

(12) The mass spectrometer 2 is under the control of a control unit, such as an appropriately programmed computer (not shown), which controls the operation of various components and, for example, sets the voltages to be applied to the various components and which receives and processes data from various components including the detectors. The computer is configured to use an algorithm in accordance with the present invention to determine the settings (e.g. injection time or number of ion pulses) for the injection of ions into the C-trap for analytical scans in order to achieve the desired ion content (i.e. number of ions) therein which avoids space charge effects whilst optimising the statistics of the collected data from the analytical scan.

(13) Alternatively to the arrangement shown in FIG. 1, the electrometer may be located off-axis, i.e. off the axis along which the ions are transmitted through the C-trap. In that case, the ions may be directed (e.g. deflected) off-axis by ion optics to reach the electrometer when required. The electrometer, or at least the deflecting ion optics, in such embodiments may be located upstream of the C-trap. As an example, one plate of the gating lens 22 could be used for this.

(14) Referring to FIG. 2 there is shown a schematic flow chart of steps in an exemplary method according to the present invention, which is hereinafter described in more detail and which may be carried out using the spectrometer shown in FIG. 1. In a step 101, ions are generated in the ion source. The generated ions are then transmitted, optionally with mass selection using the quadrupole 18 to select ions of a desired mass range, to the C-trap 26 where they are stored in step 102. The C-trap is typically filled with ions for a set time where a continuous ion source is used, such as an electrospray, or with a set number of ion pulses where a pulsed ion source is used, such as a MALDI source, i.e. the parameter IT.sub.Pro in the equations above. The filling conditions for the C-trap are set and controlled by the spectrometer's control arrangement.

(15) The stored ions are ejected from the C-trap 26 and injected as a pulse into the Orbitrap mass analyser 34. An Orbitrap mass analyser typically has a greater space charge capacity than the C-trap. Filling of the C-trap is therefore to be controlled to avoid overfilling the C-trap leading to space charge effects as described in more detail below.

(16) In step 103, the batch of ions accumulated in the mass analyser is detected using image current detection, i.e. on detection electrodes 42, 44, to obtain a detected signal, which is fed to the computer of the control arrangement. The detected signal may be used to produce a mass spectrum using Fourier transformation in a step 109, and this is done in the case where the image current detection in step 103 constitutes an analytical scan. Where the image current detection in step 103 is merely conducted for a short pre-scan then a mass spectrum may not be required from it.

(17) In step 104, the total charge of the ions in the mass analyser is determined from the detected signal obtained in step 103 by the computer, i.e. the parameter TIC.sub.Pre in the equations above is determined. In a preferred embodiment, this is done by summing together all signals above a (S/N) threshold and converting to charge using a conversion coefficient (determined during calibration or set a priori on the basis of properties of the preamplifier). In step 105 the computer uses the determined total ion charge in an algorithm to calculate a target injection time or number of pulses for a subsequent batch of ions into the C-trap thereafter to be accumulated in the mass analyser, i.e. the parameter IT.sub.Target in the equations above. The algorithm uses the thus determined total ion charge for the current batch of ions from step 104, TIC.sub.Pre, and the known set injection time or number of pulses into the C-trap that was used in step 102 for the current batch of ions (input 106), IT.sub.Pre, in order to determine settings for the C-trap such as a target injection time or target number of pulses into the C-trap for a subsequent batch of ions to be used for an analytical scan, IT.sub.Target. The settings are determined on the basis of achieving a desired or target total ion charge (hence number of ions) in the C-trap which avoids space charge effects (input 107), i.e. the parameter TIC.sub.Target in the equations above. The algorithm also uses an information input 108 which contains a measurement of integrated ion current (ion charge) from the independent detector, electrometer 60, i.e. the parameter I.sub.Ind. The measurement of integrated ion current from the independent electrometer adjusts the total ion charge determined from the image current detection by scaling it to the absolute total ion charge (integrated ion current) measured by the electrometer, i.e. by using the coefficient C in the equation above. The measurement of ion current or charge by the independent electrometer may be carried out periodically and typically less frequently than analytical scans. The measurement of ion current or charge by the independent electrometer is preferably performed during an analytical scan. For the electrometer measurement, e.g. after ions have been injected into the analyser for an analytical scan, the C-trap and collision cell 50 are set for transmission so that ions from the ion source are directed onto the electrometer 60 and an integrated ion current (ion charge) measured for a set time period or number of pulses (integrating period), e.g. the same period or number of pulses as the known injection time for the ion batch used to determine the total ion charge by image current detection. However, a different integrating period may be used as long as it is known, so that an integrated ion current (ion charge) corresponding to the known injection time or number of pulses into the C-trap can be obtained. The integrating period is typically of the order of about 10 to 200 ms, preferably 20 to 100 ms. The absolute total ion charge for the ion batch corresponding to the integrated ion current (ion charge) is thereby obtained from the electrometer measurement for input 108 in the algorithm.

(18) The method then uses the target injection time or number of pulses determined in step 105 for controlling injection of a subsequent batch of ions into the C-trap in step 110 thereby to store a desired or target number of ions in the C-trap which avoids space charge effects but optimizes data collection. Subsequently, the stored desired or target number of ions is ejected from the C-trap and injected into the mass analyser for detection in an analytical scan.

(19) In one preferred embodiment, the C-trap could transmit ions to the electrometer not in a continuous but in a pulsed manner. Although resulting in a longer measurement time for the same signal-to-noise ratio, it allows scanning simultaneously other devices of the instrument, such as RF on lens 6 or multipole 12 or quadrupole 18. It also could allow imitating any storage-related effects in the C-trap (e.g. decomposition of unwanted clusters).

(20) It will be appreciated that in the method batches of ions may be fragmented in the collision or reaction cell 50, in the manner described herein, as part of MS.sup.2 or MS.sup.n experiments.

(21) It will be appreciated that the spectrometer described with reference to FIG. 1 and the method described with reference to FIG. 2 are merely examples of implementations of the present invention. Numerous variations to the foregoing embodiments of the invention can be made while still falling within the scope of the invention.

(22) The electrometer 60 may also be useful in the following ways:

(23) 1. For optimization and characterization of the spectrometer prior to the injection device (e.g. C-trap), especially in combination with the mass filter 18, wherein the ion current or charge from the ion source is used as the criterion for optimisation. For example, in the shown embodiment, the C-trap 26 and the collision or reaction cell 50 can be set for axial transmission so that the ions are transmitted straight through the system to the electrometer 60 in order for the ion current or charge of the ion beam to be measured. The ion current or charge could, for example, be monitored using the electrometer 60 whilst optimising operating parameters of various components of the mass spectrometer, especially upstream of the C-trap.

(24) 2. For optimization and characterization of the spectrometer from the injection device (e.g. C-trap) to the mass analyser (e.g. Orbitrap) especially using well-defined calibration mixtures. The ratio between the measured ion current or charge (using the electrometer) from the ion source to the detected signal-to-noise ratios in the mass analyser Orbitrap analyzer can be used as the criterion for optimising and characterising. Also, the C-trap could transmit ions to the electrometer not in a continuous but in a pulsed manner, thus providing an indication of any storage-related effects such as fragmentation, ion losses or discriminations which might take place in a case of fault.

(25) 3. For estimation of the fractality of complex mixtures. Fractality is described as the property of the mixture to have a multiplicity of smaller peaks in vicinity of almost every intense mass peak, with each of the smaller peaks having in their turn a multitude of smaller peaks nearby. Such mixtures produce complicated interference effects in FTMS and therefore cannot be reliably quantified from FTMS detection alone. As the result, compensation of space charge effects cannot be carried out reliably thus resulting in the loss of external mass accuracy of the instrument. Fractality could be measured as a ratio of the total ion current or charge on electrometer and total ion current or charge as detected by image current detection. The higher the ratio, the more important is that factor for mass accuracy of the instrument.

(26) 4. For measuring the absolute ion numbers of mass-selected ions stored in the injection device (e.g. C-trap) and/or the collision or reaction cell for diagnostic purposes.

(27) The above methods may be implemented by means of a mass spectrometer comprising a mass analyser and an independent detector such as an electrometer.

(28) As described above, the present invention can enable full utilization of the analytical performance and space charge capacity of an Orbitrap system. In order to achieve this, in a typical Orbitrap instrument, the number of ions injected to the C-Trap needs to be controlled. The measurement of the ion current was previously either done via a dedicated AGC-prescan, which records a very short transient, or it could be done by using so-called Scan-to-Scan AGC which uses the first short section of the previous analytical scan. The resulting ion current from this short transient acquisition may be used to calculate the injection time for the next analytical scan. In some rare cases, however, the number of ions can be underestimated because of the low resolution and the lower signal response of this short transient acquisition. This is especially true for multiply charged ions and dense peaks below the noise threshold. To demonstrate this effect, an experiment was performed with the maximum inject time set untypically high. FIG. 3 shows a 60 minute gradient LC chromatogram of HeLa sample containing partially digested proteins and including the column wash part. Close to the end of the run, at retention times between 62 and 72 minutes, the signal is suppressed. A single spectrum from this section (middle trace) shows multiply charged species that won't be resolved in the short AGC-prescan and therefore will be underestimated. The second spectrum (lower trace) shows the average of three minutes, here partially digested proteins become visible showing ions that also cannot be seen by the short acquisition of the AGC-prescan which leads to further underestimation of the ion current. In this case the inject time for the analytical scan will be too long causing overfilling of the C-Trap, which leads to the suppression effect. A valid workaround formerly was to reduce the AGC target and to set the maximum inject time carefully to a dedicated level for each sample class.

(29) To improve the analytical robustness of the AGC control scheme, a C-Trap charge detection using the method of the present invention and an apparatus similar to that shown in FIG. 1 was used to monitor the AGC results every 5 to 10 seconds. In this method, during LC runs, the charge detection operation takes place in parallel to Orbitrap acquisition (i.e. concurrently). While the analytical scan in the Orbitrap was still being acquired, a few C-Trap injections were ejected to the charge collector (electrometer) to measure the C-Trap charge. From this the total ion current (TIC) was calculated and compared to the TIC observed by the short transient AGC-scan. If necessary, the injection time was regulated down to prevent the C-Trap from overfilling. This measure avoids the described signal suppression. Using the C-Trap charge detection the HeLa run was repeated and its chromatogram is shown in FIG. 4. To emphasize the effect by getting closer to the upper C-Trap space charge limit, the AGC target was set to 3e6 for this experiment. Now the suppression during the column wash process is eliminated. The spectrum shows now several analyte peaks which can be used for further confirmation.

(30) Herein the term mass means mass or mass-to charge ratio (m/z). It will also be appreciated that image current detection detects frequencies which correspond to masses or m/z values. Accordingly, references herein to mass, mass spectrum and the like also encompass the feature in frequency, which is representative of the mass term.

(31) As used herein, including in the claims, unless the context indicates otherwise, singular forms of the terms herein are to be construed as including the plural form and vice versa. For instance, unless the context indicates otherwise, a singular reference herein including in the claims, such as a or an means one or more.

(32) Throughout the description and claims of this specification, the words comprise, including, having and contain and variations of the words, for example comprising and comprises etc, mean including but not limited to, and are not intended to (and do not) exclude other components.

(33) It will be appreciated that variations to the foregoing embodiments of the invention can be made while still falling within the scope of the invention. Each feature disclosed in this specification, unless stated otherwise, may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

(34) The use of any and all examples, or exemplary language (for instance, such as, for example and like language) provided herein, is intended merely to better illustrate the invention and does not indicate a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(35) Any steps described in this specification may be performed in any order or simultaneously unless stated or the context requires otherwise.

(36) All of the features disclosed in this specification may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. Likewise, features described in non-essential combinations may be used separately (not in combination).