Ion accumulation control for analytical instrument

Abstract

A method of operating an instrument which comprises a first and second ion stores, comprising determining whether a target accumulation time for the second ion store is greater than a threshold accumulation time. When the target accumulation time is less than the threshold accumulation time, ions are accumulated within the second ion store using an accumulation time that is based on the target accumulation time. When the target accumulation time is greater than the threshold accumulation time, ions are accumulated within the first ion store using a first accumulation time that is based on a difference between the target accumulation time and the threshold accumulation time, the ions accumulated in the first ion store are passed to the second ion store, and further ions are accumulated within the second ion store using a second accumulation time that is based on the threshold accumulation time.

Claims

1. A method of operating an analytical instrument that comprises a first ion store and a second ion store arranged downstream of the first ion store, the method comprising: determining whether a target accumulation time for the second ion store is greater than a threshold accumulation time; when it is determined that the target accumulation time is less than the threshold accumulation time: accumulating ions within the second ion store using an accumulation time based on the target accumulation time; and when it is determined that the target accumulation time is greater than the threshold accumulation time: accumulating ions within the first ion store using a first accumulation time based on a difference between the target accumulation time and the threshold accumulation time, passing the ions accumulated in the first ion store to the second ion store, and accumulating further ions within the second ion store using a second accumulation time based on the threshold accumulation time.

2. The method of claim 1, wherein the instrument comprises at least one first gate configured to control an accumulation time of ions in the first ion store, and at least one second gate configured to control an accumulation time of ions in the second ion store, wherein a response time of the at least one second gate is faster than a response time of the at least one first gate.

3. The method of claim 1, wherein the instrument comprises an ion source and one or more ion optical devices arranged between the ion source and the second ion store, wherein the one or more ion optical devices are configured to transmit ions from the ion source to the second ion store, and wherein the first ion store is arranged within the one or more ion optical devices.

4. The method of claim 3, wherein the first ion store is formed in a transfer ion guide of the one or more ion optical devices.

5. The method of claim 1, wherein the instrument includes a first mass filter arranged upstream of the second ion store, and wherein the first ion store is arranged upstream of the first mass filter.

6. The method of claim 5, wherein the instrument includes a second mass filter arranged upstream of the first mass filter, wherein a resolution of the second mass filter is less than a resolution of the first mass filter, and wherein the first ion store is arranged between the first mass filter and the second mass filter.

7. The method of claim 5, further comprising the first mass filter filtering ions according to their mass to charge ratio, wherein the first mass filter filters ions using an isolation window having a width>about 2 Da.

8. The method of claim 1, wherein the instrument comprises a mass analyser arranged downstream of the second ion store, and wherein the method comprises passing ions accumulated in the second ion store to the mass analyser, and analysing the ions using the mass analyser.

9. The method of claim 8, wherein the mass analyser analysing the ions produces a time-varying transient signal, and wherein the method further comprises producing a mass spectrum from the time-varying transient signal using a phase-constrained spectrum deconvolution method (DSDM).

10. The method of claim 9, wherein the time-varying transient signal has a duration <50 ms.

11. The method of claim 1, wherein the instrument is operated in a cyclical manner, and wherein the threshold accumulation time is based on a difference between a total cycle time for the instrument and a time per cycle in which the instrument is operated in a mode in which ions are other than accumulated in the second ion store.

12. The method of claim 11, wherein the time per cycle in which the instrument is operated in the mode in which ions are other than accumulated in the second ion store comprises a time per cycle in which the second ion store is operated in a non-accumulating mode of operation while ions accumulated in the second ion store are processed and/or passed to a mass analyser for analysis.

13. The method of claim 11, further comprising operating the instrument with a repetition rate>20 Hz, >40 Hz, >60 Hz, or >80 Hz.

14. The method of claim 1, wherein accumulating ions within the second ion store using an accumulation time based on the target accumulation time comprises: operating the first ion store in a transmissive mode of operation during the accumulation time, such that ions pass through the first ion store during the accumulation time, without being accumulated within the first ion store; and operating the second ion store in an accumulation mode during the accumulation time, such that ions are accumulated within the second ion store during the accumulation time.

15. The method of claim 1, wherein accumulating ions within the first ion store using the first accumulation time comprises: operating the first ion store in an accumulation mode during the first accumulation time, such that ions are accumulated within the first ion store during the first accumulation time.

16. The method of claim 1, wherein passing the ions accumulated in the first ion store to the second ion store comprises: operating the first ion store in transmissive mode such that ions accumulated in the first ion store are passed to the second ion store; and operating the second ion store in an accumulation mode, such that ions passed to the second ion store from the first ion store are accumulated within the second ion store.

17. The method of claim 1, wherein accumulating further ions within the second ion store using the second accumulation time comprises: operating the first ion store in a transmissive mode during the second accumulation time, such that ions pass through the first ion store during the second accumulation time, without being accumulated within the first ion store; and operating the second ion store in an accumulation mode during the second accumulation time, such that ions are accumulated within the second ion store during the second accumulation time.

18. The method of claim 1, wherein when it is determined that the target accumulation time is equal to the threshold accumulation time, the method comprises accumulating ions within the second ion store using an accumulation time based on the target accumulation time.

19. A non-transitory computer readable storage medium storing computer software code which when executed on a processor causes an analytical instrument that comprises a first ion store and a second ion store arranged downstream of the first ion store to perform the steps of: determining whether a target accumulation time for the second ion store is greater than a threshold accumulation time; when it is determined that the target accumulation time is less than the threshold accumulation time: accumulating ions within the second ion store using an accumulation time based on the target accumulation time; and when it is determined that the target accumulation time is greater than the threshold accumulation time: accumulating ions within the first ion store using a first accumulation time based on a difference between the target accumulation time and the threshold accumulation time, passing the ions accumulated in the first ion store to the second ion store, and accumulating further ions within the second ion store using a second accumulation time based on the threshold accumulation time.

20. An analytical instrument, such as a mass spectrometer, comprising: a first ion store; a second ion store, wherein the second ion store is arranged downstream of the first ion store; and a control system configured to: determine whether a target accumulation time for the second ion store is greater than a threshold accumulation time; when it is determined that the target accumulation time is less than the threshold accumulation time: cause ions to be accumulated within the second ion store using an accumulation time based on the target accumulation time; and when it is determined that the target accumulation time is greater than the threshold accumulation time: cause ions to be accumulated within the first ion store using a first accumulation time based on a difference between the target accumulation time and the threshold accumulation time, cause the ions accumulated in the first ion store to be passed to the second ion store, and cause further ions to be accumulated within the second ion store using a second accumulation time based on the threshold accumulation time.

Description

DESCRIPTION OF THE DRAWINGS

[0071] Various embodiments will now be described in more detail with reference to the accompanying Figures, in which:

[0072] FIG. 1 shows schematically a mass spectrometer that may be operated in accordance with embodiments;

[0073] FIG. 2 shows schematically a mass spectrometer that may be operated in accordance with embodiments;

[0074] FIG. 3 shows schematically a mass spectrometer that may be operated in accordance with embodiments;

[0075] FIG. 4 illustrates a known method of operating the mass spectrometer of FIG. 2 or FIG. 3;

[0076] FIG. 5A shows the method of FIG. 4, FIG. 5B illustrates a method of operating the mass spectrometer of FIG. 2 or FIG. 3 in accordance with embodiments, and FIG. 5C illustrates a method of operating the mass spectrometer of FIG. 2 or FIG. 3 in accordance with embodiments;

[0077] FIG. 6 illustrates a method of operating a mass spectrometer in accordance with embodiments;

[0078] FIG. 7 illustrates a method of operating a mass spectrometer in accordance with embodiments;

[0079] FIG. 8 shows voltages applied to the split lens, bent flatapole exit lens and C-Trap exit lens of the mass spectrometer of FIG. 2 or FIG. 3 when operated in accordance with embodiments;

[0080] FIG. 9 shows an estimation of the ion current for fluranthene when analysed using the mass spectrometer of FIG. 2 operated with and without the pre-accumulation mode enabled;

[0081] FIG. 10A shows a fragmentation spectrum of an isolated m/z 524 MRFA peptide obtained using the mass spectrometer of FIG. 2 operated with the pre-accumulation mode disabled, and FIG. 10B shows a fragmentation spectrum of the isolated m/z 524 MRFA peptide obtained using the mass spectrometer of FIG. 2 operated with the pre-accumulation mode enabled;

[0082] FIG. 11 shows a plot of ion current losses versus repetition rate obtained using the mass spectrometer of FIG. 2 operated with and without the pre-accumulation mode enabled;

[0083] FIGS. 12A-F show head-to-head comparisons of the number of peptides and protein groups identified for a one-hour chromatographic separation of 200 ng HeLa digest obtained using the mass spectrometer of FIG. 2 operated with and without the pre-accumulation mode enabled; and

[0084] FIG. 13A shows a fragmentation spectrum of the isolated m/z 524 MRFA peptide obtained using the mass spectrometer of FIG. 3 operated at 200 Hz with the pre-accumulation mode disabled, and FIG. 13B shows a fragmentation spectrum of the isolated m/z 524 MRFA peptide obtained using the mass spectrometer of FIG. 3 operated at 200 Hz with the pre-accumulation mode enabled.

DETAILED DESCRIPTION

[0085] FIG. 1 illustrates schematically a mass spectrometer that may be operated in accordance with embodiments. As shown in FIG. 1, the mass spectrometer includes an ion source 10, one or more ion transfer stages 20, an ion trap 30, and a mass analyser 40.

[0086] The ion source 10 is configured to generate ions from a sample. The ion source 10 can be any suitable continuous or pulsed ion source, such as an electrospray ionisation (ESI) ion source, a MALDI ion source, and atmospheric pressure ionisation (API) ion source, a plasma ion source, an electron ionisation ion source, a chemical ionisation ion source, and so on. More than one ion source may be provided and used. The ions may be any suitable type of ions to be analysed, e.g. small and large organic molecules, biomolecules, DNA, RNA, proteins, peptides, fragments thereof and the like.

[0087] The ion source 10 may coupled to a separation device such as a liquid chromatography separation device or a capillary electrophoresis separation device (not shown), such that the sample which is ionised in the ion source 10 comes from the separation device.

[0088] The ion transfer stage(s) 20 are arranged downstream of the ion source 10 and may include an atmospheric pressure interface and one or more ion guides, lenses and/or other ion optical devices configured such that some or all of the ions generated by the ion source can be transferred from the ion source 10 to the ion trap 30. The ion transfer stage(s) 20 may include any suitable number and configuration of ion optical devices, for example optionally including one or more RF and/or multipole ion guides, one or more ion guides for cooling ions, one or more mass selective ion guides, and so on.

[0089] The ion trap 30 is arranged downstream of the ion transfer stage(s) 20 and is configured to receive and accumulate ions from the ion source 10 (via the one or more ion transfer stages 20). The ion trap 30 can comprise any suitable type of ion trap, such as a multipole (e.g. quadrupole) ion trap.

[0090] In some embodiments, the ion trap 30 is elongated in an axial direction (thereby defining a trap axis) in which the ions enter the trap. Ions may be trapped radially in the trap by applying RF voltage(s) to trapping (e.g. rod) electrodes of the trap. The ion trap 30 may be or may include a curved linear ion trap (C-Trap), i.e. where the trapping rod electrodes are curved. However, the ion trap 30 may be or may include any other suitable type of ion trap, such as for example a linear ion trap.

[0091] The ion trap 30 includes an entrance lens or gate 32 and an exit lens or gate 34. The entrance lens 32 can be operated in an open mode, in which ions (from the ion source 10) can pass the entrance lens and enter the ion trap 30, or a closed mode in which ions (from the ion source 10) cannot pass the entrance lens 32 and do not enter the ion trap 30. When the entrance lens 32 is operated in its closed mode, ions already within the ion trap 30 will not be able to leave the ion trap via the entrance lens 32. Similarly, the exit lens 34 can be operated in an open mode, in which ions can pass the exit lens and leave the ion trap 30, or a closed mode in which ions cannot pass the entrance lens and do not leave the ion trap. The entrance lens 32 (the exit lens 34) can be closed or opened by applying a suitable voltage to the entrance lens 32 (to the exit lens 34).

[0092] Ions from the ion source 10 can be accumulated in the ion trap 30 by operating the exit lens 34 in its closed mode, while operating the entrance lens 32 in its open mode. After a desired ion fill time of ions into the ion trap 30, the entrance lens 32 can be closed (by altering the voltage applied to the entrance lens 32) such that ions cannot pass out of the trap 30 and such that ions from the ion source 10 can no longer enter the ion trap 30. Thus, the mass spectrometer is configured such that ions can be accumulated in the ion trap 30 with an adjustable accumulation time (fill time). By controlling the fill time of ions into the trap, where the flux of ions into the trap 30 is known or can be approximated, the total number of ions accumulated in the ion trap 30 can be controlled.

[0093] Once accumulated in the ion trap 30, ions within the trap can be ejected into the mass analyser 40. Ions may be ejected from the ion trap 30 in an axial direction, or the ions may be ejected from the trap 30 in a direction orthogonal to the axis of the trap (orthogonal ejection), for example by applying one or more suitable DC voltages to the ion trap 30.

[0094] The mass analyser 40 is arranged downstream of the ion trap 30 and is configured to receive ions from the ion trap 30. The mass analyser is configured to analyse the ions so as to determine their mass to charge ratio and/or mass, i.e. to produce a mass spectrum of the ions. The mass analyser 40 may be an ion trap mass analyser, such as an electrostatic orbital trap, and more specifically an Orbitrap™ FT mass analyser as made by Thermo Fisher Scientific. Alternatively, the mass analyser 40 may be a time-of-flight (ToF) mass analyser, such as a multi-reflecting time-of-flight (mr-ToF) mass analyser.

[0095] It should be noted that FIG. 1 is merely schematic, and that the mass spectrometer can, and in embodiments does, include any number of one or more additional components. For example, in particular embodiments, the mass spectrometer includes a collision or reaction cell. The instrument may include a single mass analyser, or more than one (e.g. two) mass analysers.

[0096] As also shown in FIG. 1, the mass spectrometer is under the control of a control unit 50, such as an appropriately programmed computer, which controls the operation of various components of the spectrometer and, for example, sets the voltages to be applied to the various components of the spectrometer. The control unit 50 may also receive and process data from various components including the detector(s), e.g. perform Fourier transformation on detected signals. The control unit 50 is configured, amongst other things, to determine the settings (e.g. ion trap 30 fill time, etc.) for the injection of ions into the mass analyser 40 for analytical scans.

[0097] The mass spectrometer may be operated such that successive batches of ions from the ion source 10 are each analysed by the mass analyser 40. Each batch of ions is firstly accumulated in the ion trap 30, and then the accumulated ions (or e.g. fragment ions derived from the accumulated ions) are injected into the mass analyser 40.

[0098] It can be desirable that each batch of ions analysed by the mass analyser 40 includes as many ions as possible, e.g. so as to improve the statistics of the mass spectrum. However, undesirable space charge effects can occur at relatively high ion concentrations and can limit mass resolution and mass accuracy. Therefore, the total number of ions accumulated in the ion trap 30 is controlled to optimise the number of ions injected into the mass analyser 40 to be below, but as close as possible to, a limit for the mass analyser 40 such as a space-charge limit for the mass analyser 40. The total number of ions accumulated in the ion trap 30 may also or instead be controlled to be below a limit for the ion trap 30 such as the space-charge limit for the ion trap 30. Typically, between 5×10.sup.3 and 1×10.sup.6 elementary charges should be stored, such as between 1×10.sup.4 and 1×10.sup.6, or between 1×10.sup.5 and 5×10.sup.5.

[0099] However, it may be the case that the flux of ions from the ion source 10 is highly variable. This is particularly the case where the ion source 10 is coupled to a separation device such as a liquid chromatography or capillary electrophoresis device, where the ion flux from the ion source 10 can vary over time by several order of magnitudes.

[0100] Therefore, embodiments use so-called automatic gain control (AGC) techniques to precisely control the total number of ions accumulated in the ion trap 30 despite a variable flux of ions into the trap 30. These techniques typically rely on an accurate and reliable real-time estimation of the present ion current or ion flux being received by the ion trap 30. Then, by controlling the filling time T of the ion trap 30, the total number of ions or the total amount of charge accumulated in the trap 30 (and injected into the mass analyser 40) can be suitably controlled.

[0101] Thus, for each batch of ions, a target accumulation time T may be determined based on an estimation of the present ion current or ion flux being received by the ion trap 30, and ions may be accumulated in the ion trap 30 for an amount of time equal to the target accumulation time T.

[0102] FIGS. 2 and 3 show in more detail two example mass spectrometers that may be operated in accordance with embodiments. It will be understood that the instruments shown in FIGS. 2 and 3 are non-limiting examples, and that numerous variations are possible.

[0103] In the embodiment depicted in FIG. 2, the instrument's ion source 10 is an electrospray ionisation (ESI) ion source. The instrument includes a vacuum interface, which includes a transfer tube 21, an ion funnel 22, a quadrupole pre-filter ion guide 23, and a “bent flatapole” ion guide 24. The bent flatapole ion guide 24 may be of the design described in U.S. Pat. No. 9,536,722.

[0104] The instrument also includes a mass filter in the form of a quadrupole mass filter 26, an ion trap 30a in the form of a curved linear ion trap (“C-Trap”), and a collision cell 30b in the form of an ion routing multipole collision cell (“IRM”). Ions from the ion source 10 can be accumulated in the C-Trap 30a and/or collision cell 30b by opening and closing a gating electrode located in a charge detector assembly 27, which is arranged between the C-Trap 30a and the mass filter 26.

[0105] The instrument also includes a mass analyser 40a in the form of an orbital ion trap mass analyser. As shown in FIG. 2, the orbital trap 40a comprises an inner electrode 41 elongated along the orbital trap axis and a split pair of outer electrodes 42, 43 which surround the inner electrode 41 and define therebetween a trapping volume in which ions are trapped and oscillate by orbiting around the inner electrode 41 to which is applied a trapping voltage whilst oscillating back and forth along the axis of the trap. The pair of outer electrodes 42, 43 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.

[0106] The outer electrodes 42, 43 typically function as a differential pair of detection electrodes and are coupled to respective inputs of a differential amplifier (not shown in FIG. 2), which in turn forms part of a digital data acquisition system to receive the detected signal. The detected signal can be processed using Fourier transformation to obtain a mass spectrum of ions within the trap.

[0107] Once accumulated in the ion trap 30a and/or collision cell 30b, ions can be ejected into the mass analyser 40a. To do this, the ions may be ejected from the trap 30a in a direction orthogonal to the axis of the trap (orthogonal ejection), for example by applying one or more suitable DC voltages to the ion trap 30a. The ions may be injected into the mass analyser 40a via one or more lenses and a deflector electrode. The mass analyser 40a is arranged downstream of the ion trap 30a and is configured to receive ions from the ion trap 30a (via the one or more lenses and the deflector electrode).

[0108] The collision or reaction cell 30b is arranged downstream of the ion trap 30a. Ions collected in the ion trap 30a can either be ejected orthogonally to the mass analyser 40a without entering the collision or reaction cell 30b, or the ions can be transmitted axially to the collision or reaction cell 30b for processing before returning the processed ions to the ion trap 30a for subsequent orthogonal ejection to the mass analyser 40a. The processing may comprise, for example, fragmenting the ions by collisions with a collision gas and/or a reagent in the collision cell 30b, or further cooling the ions by collisions with a gas at lower energies that do cause the ions to fragment.

[0109] Turning to FIG. 3, the mass spectrometer depicted in FIG. 3 is substantially similar to the mass spectrometer of FIG. 2. However, the mass spectrometer depicted in FIG. 3 includes an additional time-of-flight (ToF) mass analyser in the form of a multireflection time-of-flight (ToF) mass analyser 40b, which has been added to the rear of the instrument. This hybridized instrument is described in more detail in U.S. Pat. No. 10,699,888. In the instrument depicted in FIG. 3, the analyser is of the tilted-mirror type described in U.S. Pat. No. 9,136,101, but it will be understood that any type of ToF analyser could be used.

[0110] As shown in FIG. 3, the instrument includes a multipole ion guide 31 to allow ions to be transferred from the collision cell 30b to the time-of-flight mass analyser 40b. The time-of-flight mass analyser 40b includes an extraction trap 44, whereby ions are delivered from the collision cell 30b to the extraction trap 44 via the multipole ion guide 31. The ions are accumulated and cooled in the extraction trap 44.

[0111] The extraction trap 44 may incorporate two trapping regions, one at a relatively higher pressure for rapid ion cooling, and a second low pressure region for ion extraction. Ions are cooled in the high-pressure region and then transferred to the low-pressure region, where they are pulse ejected into the ToF analyser via a pair of deflectors 45. Ions oscillate between a pair of mirrors 46, which are tilted relative to one another so that the ion path is slowly deflected and redirected back to a detector 47. Correcting stripe electrodes 48 counter the loss of ion focus otherwise induced by the non-parallelism of the mirrors.

[0112] Modern mass spectrometers operate with ever faster repetition rates, allowing high performance over shorter experiments and a greater volume of samples to be processed. The main constraints on repetition rate are instrument sensitivity, as a certain accumulation time is required to gather sufficient sample ions for analysis, time required to process these ions for analysis, the analysis time itself, and the time required for electronics to switch between analyte targets.

[0113] In the instrument depicted in FIG. 2, ions generated by the electrospray ionisation (ESI) ion source 10 must traverse the vacuum interface, i.e. the transfer tube 21, ion funnel 22, quadrupole pre-filter ion guide 23, and bent flatapole ion guide 24, before being mass selected by the quadrupole mass filter 26, and being accumulated and/or fragmented in the ion trap 30a and/or collision cell 30b. Ions may then be passed back to the C-Trap 30a and pulse-ejected into the mass analyser 40a where they are analysed. In conventional operation, at the maximum allowed repetition rate of 40 Hz, or every 25 ms, the maximum accumulation time is only 10 ms, a duty cycle of only 40%.

[0114] The longer ions are measured in the mass analyser 40a the higher the resolution and the greater the sensitivity of the analyser. For the MS2 (ion fragmentation) measurements that typically dominate most applications, very high resolution is not required but high repetition rate and sensitivity are desirable. Thus, for these measurements, relatively short 16 ms mass analyser transients are often used, giving a resolution of around 7500 at m/z 200. Shorter transients remain viable, but at this point instrument operation overheads and the required ion accumulation time for well resolved spectra set the limit on the instrument's repetition rate to around 40 Hz.

[0115] The operation of the instrument may be parallelized to maximise efficiency. Notably, the measurement period of ions in the mass analyser 40a itself is very time consuming, and is typically decoupled from the process of loading and processing ions in the C-Trap 30a and collision cell 30b. A further parallelised stage is the switching of the voltages of the remaining ion optics and the transfer of ions across them to the ion gate.

[0116] FIG. 4 illustrates these main parallelised operations and their approximate timings. It is notable that the ion accumulation time is entirely coupled with the relatively slow operation of ion processing.

[0117] As described above, an important feature of commercial instruments is accurate control of the number of ions injected into the C-Trap 30a and mass analyser 40a, a process known as automatic gain control (AGC). This is performed by fine control of the fill time by a very fast beam deflecting ion gate within the charge detecting assembly 27 prior to the C-Trap 30a. This gate is typically accurate to around 30 μs (or less), although a more advanced dual gate design, e.g. described in U.S. Pat. No. 8,026,475, is accurate to around 1-2 μs. As described above, such precise control is required due to the vast variation in ion beam intensity, and the limited dynamic range of both the C-Trap 30a and mass analyser 40a.

[0118] The inventors now have recognised that a problem with existing instrument designs lies in the relatively poor (<50%) duty cycle when the instrument is operated at relatively high repetition rates. This can lower sensitivity for fast and/or low sample load experiments, and can prevent higher still repetition rates from being accessible. The primary reason for this problem can be seen in FIG. 4. The ion accumulation time is run in series with the C-Trap/IRM ion processing and reset, which are very time-consuming operations, and which block the C-Trap 30a for ion accumulation.

[0119] Although this problem can be less severe in a time-of-flight instrument, such as the instrument depicted in FIG. 3, these instruments have their own timing issues. In particular, the inventors have recognised that a problem lies in the potentially long time periods for switching the front-end electronics and quadrupole 26 to accommodate different m/z target ions and transfer them through to the C-Trap 30a. In the instrument depicted in FIG. 3, transfer and processing of ions in the extraction trap 44 block the trap for a relatively long period, around 3 ms. Combined with around 1 ms to prepare the quadrupole 26, this leaves very little time for ion accumulation at the desired 200 Hz/5 ms repetition rate.

[0120] Embodiments address the problem of sensitivity loss at high repetition rates due to restrictions on ion accumulation time imposed by non-parallelisable instrument operations. Specifically, embodiments address the problems associated with the time overhead created by the C-Trap/IRM ion processing and reset sequence, and the m/z target switching and ion transfer time for the instrument front end (i.e. ion funnel/pre-filter/bent flatapole/quadrupole).

[0121] In accordance with various embodiments, a parallel ion accumulation stage is added within the one or more ion transfer stages 20. The pre-accumulation stage may be provided within any suitable stage of the one or more ion transfer stages 20. For example, referring to FIGS. 2 and 3, the ion funnel, the pre-filter 23, the bent flatapole ion guide 24, or the mass filter 26 may be operated as a pre-accumulation ion trap. In other instrument designs, equivalent or similar ion transfer stage(s) (e.g. the second part of a dual stage ion funnel) may be used in this manner. The process of pre-accumulating ions may run in parallel to the slow ion processing operations of the C-Trap/IRM assembly. This allows significant additional effective fill time.

[0122] With reference to FIGS. 2 and 3, in particular embodiments, the parallel ion accumulation stage is provided within the bent flatapole ion guide 24. The bent flatapole ion guide 24 is particularly suitable for use as a large capacity trapping device, as it incorporates a quadrupole RF trapping field, and a superimposed DC gradient for guiding ions. The bent flatapole 24 also possesses an end-lens 25 that may be voltage switched to act as a crude ion gate.

[0123] Because this end lens 25 (or its equivalent in other instrument designs) is a relatively slow device compared to the dedicated ion gate in the charge detecting assembly 27, it can only crudely control ion timings and is thus unsuitable for performing precise AGC with short fill times. Thus, to preserve AGC accuracy, pre-accumulation using the bent flatapole end lens 25 may be disabled if the desired fill time falls below a threshold fill time, e.g. that corresponds to the maximum fill time that the desired repetition rate can support via the prior art accumulation method.

[0124] This may be done by defining the fill time through the open ion gate 27 as a primary fill time, and defining the additional fill time within the bent flatapole ion guide 24 as an auxiliary fill time. The total fill time is then allocated to the primary until a maximum (e.g. such as around 10 ms) is reached, and then the remaining time is allocated to the auxiliary. By this means, linearity is maintained, and absolute AGC accuracy is only lost for very long fill times, where this becomes a small proportional loss.

[0125] Thus, in embodiments ion accumulation is controlled at two independent places. For the mass spectrometer shown in FIGS. 2 and 3, ion accumulation is already performed in the C-Trap/IRM 30 and controlled by a gating electrode located within the charge detector assembly 27. An additional accumulation stage is implemented for ion accumulation within the bent flatapole 24, and controlled by a voltage applied to the bent flatapole exit lens 25. This extra trapping sequence may be run in parallel to a preceding ion packet being processed within the C-Trap/IRM 30, when ions would otherwise be thrown away and wasted.

[0126] FIGS. 5A-C show a comparison between the prior art operation sequence (FIG. 5A) and the present embodiment (FIG. 5B). Each arrow describes the approximate movement of ions through the instrument in a series of operations, and each parallelized operation series has a separate arrow. It will be understood that FIG. 5 is a simplified description, as the instrument is in reality extremely complex, but adequately shows the most relevant stages. In FIG. 5B, an auxiliary fill time has only been added to the bent flatapole 24. However, as it has been added to the short first stage, there is a lot of free time before this stage equals the other two stages in time and starts to dominate.

[0127] It should be noted that a limitation of the depiction of FIG. 5 is that it shows the primary and auxiliary fill times in parallel, when in-fact they draw ions from the same source and in total must be shorter than the overall repetition rate.

[0128] FIG. 5C shows alternative timings that become viable using the techniques descried herein. In FIG. 5C, the primary fill time is allowed to shrink down to 2 ms, and the repetition rate increased to around 75 Hz. The unacceptable loss of duty cycle that would occur using the prior art accumulation method is obviated by the relatively long auxiliary fill period.

[0129] Beneficially, the pre-accumulation scheme may be seamlessly disabled between scans. As described above, sufficiently accurate control of ion population requires control of fill times down to around 30 μs or less for intense ion currents. However, gating via a lens in accordance with embodiments is much slower than this (and so acts as a less accurate guillotine), typically taking around 100 μs to open/close. Therefore, for intense ion beams with relatively short target fill times, it is desirable not to have any pre-accumulation process at all. In embodiments, where the pre-accumulation is controlled by a secondary fill time preceding the primary fill time, a fill time shorter than the maximum for the primary fill time will give an auxiliary fill time of zero, whereupon the exit lens 25 of the bent flatapole 24 never closes. For fill times exceeding this maximum, an auxiliary fill time may then start to be introduced.

[0130] FIG. 6 shows this order of precedence for dividing the total ion accumulation time between the primary and auxiliary fills. Essentially, the primary fill should be at or near the maximum before the auxiliary fill is used. In this way, the inaccuracy of the auxiliary fill only affects the measurement of ion current for long accumulations, where the timing error is proportionately small and so will only be a relatively small contributor to the overall error.

[0131] Additionally, the linear response of ion load with changing fill time should be maximised with this method. It should be noted, however, that corrections for small errors around the switching point may be provided and used. For example, to take account of the switching time of the bent flatapole exit lens 25, there may need to be a small (e.g. around 100 μs) extra opening time added to its shortest fill times.

[0132] FIG. 7 illustrates a method in accordance with embodiments. As shown in FIG. 7, a desired accumulation time T is firstly compared to a threshold accumulation time Tt for the ion trap (step 101), and it is determined whether or not the desired accumulation time T is greater than the threshold accumulation time Tt (step 102). As described above, the threshold accumulation time Tt may be set to be equal to, approximately equal to or less than the difference between the total cycle time for the instrument and a time per cycle in which the instrument is operated in a mode (or modes) in which ions are not (are other than) accumulated in the ion trap 30.

[0133] If the desired accumulation time T is less than or equal to the threshold accumulation time Tt, then ions are accumulated in the primary ion trap using the desired accumulation time T (step 103), i.e. in a “normal” manner.

[0134] If, however, the desired accumulation time T is greater than the threshold accumulation time Tt, then ions are pre-accumulated in the auxiliary ion trap using an auxiliary accumulation time which is approximately equal to the difference between the desired accumulation time T and the threshold accumulation time Tt (i.e. T minus Tt) (step 104). These ions that are accumulated in the auxiliary ion trap are then passed to the primary ion trap (step 105). Finally, additional ions are accumulated in the primary ion trap (to supplement the ions that were accumulated in the auxiliary trap and passed to the primary trap), using an accumulation time approximately equal to the threshold accumulation time Tt (step 106). As such, the total accumulation time for ions is approximately equal to the desired accumulation time, i.e. (T−Tt)+Tt=T.

[0135] Returning to FIG. 5, a further advantage of providing the pre-accumulation in an early, pre-mass filter (e.g. bent flatapole 24) stage of the instrument can be seen in how this breaks up the relatively long “electronics switch+ion transfer” stage. Naturally, it takes less time for ions to travel from source 10 to bent flatapole 24 than from source 10 to IRM 30b. The electronics switching time may also be improved, if for example the quadrupole electronics are the slowest part, as is the case on existing commercial instruments. This may reduce a stage that commonly takes >4 ms to parallelised stages that only require 1 or 2 ms in total.

[0136] Furthermore, in embodiments, the pre accumulation stage is downstream of a pre-filter 23. Advantageously, the pre-filter 23 allows rough mass selection of ions entering the bent flatapole 24, reducing the space charge load presented by unwanted ions, e.g. by around 90%, thus preventing overfilling of the device when running in accumulation mode, which might otherwise also impede the action of the main mass filter 26.

[0137] A simple version of the pre-accumulation method was programmed and applied to an Orbitrap™ instrument of the type illustrated in FIG. 2. In this method for all fragmentation spectra, the primary fill time was set to a fixed 10 ms, and the auxiliary fill time set to utilise the remainder of the available time defined by the instrument's repetition rate.

[0138] FIG. 8 illustrates timings of opening and closing the split lens 27 (“Beam Control”) and the bent flatapole exit lens 25 which control the primary and secondary accumulation times, together with the timing of the operation of the C-Trap exit lens. As shown in FIG. 8, the moment the primary injection process (“Beam Control”) terminates, the bent flatapole exit lens 25 is set from a transmitting voltage of −10V to a trapping voltage of +10V. The voltages applied to the C-Trap exit lens moves from a small negative voltage as ions are loaded to the IRM 30b to a slight positive ramped voltage as ions are returned from IRM 30b to C-Trap 30a. The voltage is then pulsed to +250V at the start of the analyser 40 injection cycle and then set down for reset.

[0139] FIG. 9 shows an estimation of the ion current for fluranthene with the pre-accumulation mode being switched from disabled to enabled. In FIG. 9, the ion current is estimated from mass analysis measurements using the orbital ion trap mass analyser 40. It can be seen that the ion current roughly doubles, thanks to the doubling of the duty cycle.

[0140] FIGS. 10A-B show fragmentation spectra of an isolated m/z 524 MRFA peptide, with pre-accumulation being set as disabled (FIG. 10A) and enabled (FIG. 10B). It can be seen that both fragment spectra are very similar in terms of the fragment ions present and relative ion abundancies. However, the “normalised largest peak” values, a measurement of ion current for the largest peak, is more than doubled when pre-accumulation is activated due to the increase in duty cycle.

[0141] FIG. 11 shows a comparison of ion current losses (normalized to 48 Hz) with repetition rate when the pre-accumulation method is enabled or disabled. The instrument repetition rate was increased by lowering the primary fill time, as described above with respect to FIG. 5C, bringing the cycle time from 21 down to 13 ms. The current of background ions was measured via the estimated total fill time and ion peak signal to noise ratio, allowing an estimation of the change in signal with greater repetition rate.

[0142] It can be seen that without pre-accumulation, the sensitivity of the instrument collapses rapidly at high repetition rate, but with pre-accumulation enabled, the sensitivity only loses around 10% of signal above 70 Hz. It is thought that this may relate to the efficiency of purging trapped ions from the bent flatapole 24, and getting them through the quadrupole 26 when only short primary fill times (<3 ms) are used.

[0143] FIGS. 12A-F illustrate the pre-accumulation method being used in a proteomics application, in this case a one-hour chromatographic separation of a complex sample, 200 ng HeLa digest, at several different method parameters. FIG. 12 shows head-to-head comparisons of the number of identified peptides and protein groups for the pre-accumulation and standard methods. The data on the left is for the instrument operating with a 120K MS1 and 15K MS2 resolution, and 23 ms fill time. The data in the middle is for the instrument operating with a 60K MS1 and 7.5K MS2 resolution, and 10 ms fill time. The data on the right is for the instrument operating with a 120K MS1 and 7.5K MS2 resolution, and a 10 ms fill time.

[0144] Despite these long, high concentration separations being relatively unflattering for this technique (as signal and time are not such desperate limiting factors), the number of identified peptides and protein groups are clearly and consistently increased. In one example, where the MS1 resolution was 120K and MS2 resolution 7.5K, the improvement in peptide identifications was an enormous 29%.

[0145] The simple pre-accumulation method was ported to a time-of-flight mass spectrometer of the type shown in FIG. 3. The instrument was operated at 200 Hz with timing overheads of 3 ms, approximately the lower limit for fast precursor switching and ion transfer with a bent-flatapole trapping stage (the lower limit without is even higher, 3.5-4 ms). The remaining 2 ms was set as the ion injection time.

[0146] FIGS. 13A-B show a comparison of signal for fragmented MRFA with and without pre-accumulation. FIG. 13 demonstrates that for these timings, the ion signal is more than doubled when using pre-accumulation. For data independent accumulation, where the precursor selection moves in only small steps, these timing overheads may be reduced to ˜1.5 ms and the duty-cycle benefits will be more modest, but still considerable.

[0147] It will be appreciated that embodiments relate to the use of two fill times in sequence, an optionally inaccurate one for the pre-accumulation, and an accurate one for the main ion trap (C-Trap/IRM) accumulation. This allows seamless switchable operation where parallelized scans are intermingled with non-parallelized scans for short fill times or for AGC pre-scans, to preserve accurate ion population control and linearity.

[0148] Beneficially, this can provide a doubling of instrument sensitivity for fast (e.g. 40 Hz) experiments, and removes a major bottleneck to faster acquisition still, allowing sensitive measurements at up to 75 Hz. With optimization, higher rates still of 80-100 Hz may be attainable. The technique may be applied to existing instruments without hardware changes. The method is particularly suitable for fast, low sample load experiments.

[0149] Although various particular embodiments have been described above, various alternative embodiments are possible.

[0150] For example, although various embodiments above are described in terms of Obitrap™ instruments, embodiments are applicable to other instrument designs. As described above with reference to FIG. 1, a generalised instrument layout comprises an ion source, interface, gate, trap, and analyser. Pre-accumulation may occur in a part of the interface, or at least before the gate that controls accumulation. In these embodiments, instead of the bent flatapole 24 being used for pre-accumulation, interface ion optical devices may be used, which may include a wide range of multipole ions guides/traps, along with stacked ring, ion funnel or ion carpet type devices.

[0151] In these embodiments, the instrument may comprise a mass filter, e.g. between the interface and gate. The mass filter may come before or after the gate, but for the accurate gating to work there must be at least one ion gate separate to the pre-accumulation device. This is present in some q-ToF instruments, which may gate ions prior to accumulation in their collision cell, which then functions as the trap.

[0152] The gate may also be integrated into the trap, for example where gating is controlled by the exit lens of a collision cell prior to ToF extraction.

[0153] As described above, the “pre-accumulation” method of various embodiments can significantly improve the sensitivity of the analytical instrument. However, a related problem arises in the context of Orbitrap™ analysers, because the sensitivity benefits are most pronounced at relatively high repetition rates, i.e. at relatively short (e.g. 8 ms or 16 ms) Orbitrap™ analyser transients (although at 32 ms the method still provides around a ⅓.sup.rd increase in ion signal). With these transient lengths, the resolution of the Orbitrap™ analyser is relatively low, in particular 3750 and 7500 at m/z 200, and rapidly drops at higher m/z.

[0154] For the Data Dependent Acquisition (DDA) methods described above, the sensitivity gains outweigh the losses incurred from the falling resolution. However, Data Independent Acquisition (DIA) methods are becoming more prominent, particularly at high throughput where they show excellent results. DIA methods are almost never run with 16 ms transients on Orbitrap™ instruments, and instead usually use 32 ms or 64 ms, even for short LC gradients. Whilst sensitivity to low level species is certainly an important consideration in these methods, resolution is a key factor, and 7500 can be too low for some applications. Higher resolution can be necessary to differentiate interfering peaks in complex spectra, and fragment mass accuracy can also be an important factor in these methods at low signal to noise.

[0155] Thus, a problem arises due to the relative underperformance of Orbitrap™ analysers in high throughput DIA experiments caused by high resolution and sensitivity requirements limiting repetition rate.

[0156] A major advance in Orbitrap™ analyser signal processing has been the development of the so-called “phase-constrained spectrum deconvolution” method, or “ϕSDM”, e.g. as described in Grinfeld, et al., Phase-constrained spectrum deconvolution for Fourier transform mass spectrometry, Anal. Chem., 2017, 89, 1202-1211, and also European Patent Application No. EP 3,086,354 the entire contents of which is incorporated herein by reference. Whilst computationally more expensive than the standard “eFT” method, it has the property of multiplying the resolving power for a given transient length, giving more confident assignment of peak abundance and position, and reducing the impact of interfering peaks.

[0157] ϕSDM “super resolution” spectral processing has substantial beneficial effects on DIA proteomics experiments in isolation. However, a shortcoming of DSDM alone is that it does nothing to improve the sensitivity of the instrument, and so whilst it provides high resolution at shorter Orbitrap™ analyser transients, in conventional methods the sensitivity still drops off. It is thus poorly compatible with conventional 16 ms and 8 ms transients, where Orbitrap™ analyser duty cycle drops off in addition to the normal reduction in ion accumulation time and signal/noise demanded by the faster repetition rate (as described above). This limitation can be more onerous than the requirement for a large amount of processing power.

[0158] In embodiments, the pre-accumulation method described above and the ϕSDM process are combined, in particular for high throughput DIA methods. This combination may also be made for DIA-like DDA methods, e.g. where the isolation window is wide and challenges are similar. The pre-accumulation method allows maintenance of duty cycle at low transient lengths, mitigating sensitivity losses, whilst the ϕSDM technique recovers the resolution loss. Together, the two methods lower the transient floor from 32 ms to 16 ms or even 8 ms, enabling high repetition rates, to even >70 Hz, that are beneficial for high throughput and/or short LC-gradient analyses.

[0159] In these embodiments, an Orbitrap™ mass spectrometer such as that shown in FIG. 2 or 3 may be used, coupled to a liquid chromatography device to supply separated sample, typically tryptic digests of biological protein samples. As described above, these instruments typically have a configuration whereby ion accumulation is blocked by the operation of the extraction trap (C-Trap) 30a.

[0160] As also described above, FIGS. 5B and 5C shows the modified instrument operation sequence and timings for pre-accumulation with 16 ms and 8 ms transients. By injecting ions first into the bent flatapole 24, a second parallel ion accumulation stage is created that may run whilst the C-Trap/IRM 30 is busy. At 40 Hz operation the duty cycle more than doubles, and at 75 Hz increases by five times. Ion trapping and release is controlled by switching the voltage on the bent flatpole exit lens 25, e.g. from +10 to −10V.

[0161] Collected Orbitrap™ analyser transient data, either MS and/or MS/MS spectra, may then be analysed via the DSDM technique.

[0162] The instrument may operate an otherwise conventional DIA method, whereby a series of pre-accumulation MS/MS fragmentation spectra are taken in a pre-programmed sweep across the mass range, with an optional full MS scan for quantitation of precursors. The full MS scan need not utilise the pre-accumulation technique, but may do so.

[0163] The DSDM technique can be applied to MS and/or MS/MS spectra. DSDM using an external processor can slow down the entire system when using long transients, as can be the case for full MS scans. Thus, in some embodiments, the ϕSDM technique is used only for MS/MS scans (and not for MS scans), i.e. so as to maintain speed.

[0164] Experiments that can particularly benefit from the combined technique include those that use short LC-gradients, e.g. less than 30 minutes such as 3-15 minutes, where high repetition rates are needed, and those that use substantial sample quantities (e.g. −50-2000 ng). Very low sample loads (e.g. <10 ng or <1 ng) produce sensitivity issues that the short transients can exacerbate, and are thus often studied with wide isolation windows and long transients. The isolation window similarly should preferably not be very narrow, though down to 2 may be used for DIA. The Orbitrap™ analyser transient length may be around 8-32 ms for MS/MS, so as to provide particular improvements from the combination of pre-accumulation and DSDM. The full-MS Orbitrap™ analyser transient may be longer, e.g. 32-128 ms.

[0165] In some embodiments, the DSDM technique may be applied only to particular regions of a spectrum, such as particularly congested regions of the spectrum, e.g. the precursor region, or areas with low signal/noise peaks that would benefit from greater mass accuracy. In this case, a regular profile spectrum may first be generated, and interrogated for regions to apply ϕSDM to. Such a filter may help to reduce the computational load of the method.

[0166] It will be appreciated that the combination of the pre-accumulation method and the ϕSDM technique make Orbitrap™ instruments compatible with short transients and thus fast DIA experiments. This benefit is synergistic, as the two must be used together to work. Conventional Orbitrap™ instruments are typically unsuited to high scan rates, especially for DIA experiments, due to loss of duty cycle, signal and commensurate loss of resolution causing interferences between closely spaced peaks. By applying the pre-accumulation method, the duty cycle problem is solved, but the resolution is still reduced. However, applying the ϕSDM technique recovers this to normal, suitable, levels.

[0167] It will be understood that there may still be a loss of ion accumulation time due to the shorter transient, however for short LC-gradients this is less of a problem as ion current is normally considerably higher.

[0168] Although these embodiments are particularly suited to DIA methods, they may also be of benefit for high throughput DDA, where the isolation window is sufficiently wide that spectra are complex, and resolution becomes important.

[0169] Other similar high resolution deconvolution techniques may be used in the same way, such as for example, the Least Squares Fit method, i.e. as described in the article Kozhinov, et al., (2022), “Super-resolution mass spectrometry enables rapid, accurate, and highly-multiplexed proteomics at the MS2-level”, bioRxiv. This techniques reports some similarities in properties and performance to DSDM.

[0170] Although the present invention has been described with reference to various embodiments, it will be understood that various changes may be made without departing from the scope of the invention as set out in the accompanying claims.