Two dimensional MS/MS acquisition modes

Abstract

A method of mass spectrometry is disclosed comprising performing a plurality of experimental runs, wherein each experimental run comprises: periodically mass analysing fragment or product ions at a plurality of time intervals, wherein a delay time is provided between the start of the experimental run and the first time interval at which the fragment or product ions are mass analysed. Different delay times are provided in different ones of the experimental runs and fragment or product ions that have been analysed in the same time interval in at least one of said experimental runs and that have been analysed in different time intervals in at least one other of said experimental runs are identified as fragment or product ions of interest. These fragment or product ions are thus determined to relate to different precursor ions and are used to identify their respective precursor ions.

Claims

1. A method of mass spectrometry comprising: a) performing a plurality of experimental runs, wherein each experimental run comprises: i) either mass selectively transmitting precursor ions into a fragmentation or reaction device, wherein the mass to charge ratios of the precursor ions transmitted is varied as a function of time, or transmitting precursor ions into a fragmentation or reaction device, wherein a physicochemical property of the precursor ions transmitted is varied as a function of time, ii) fragmenting or reacting the precursor ions in the fragmentation or reaction device so as to produce fragment or product ions, iii) periodically mass analysing the fragment or product ions at a plurality of time intervals, wherein a delay time is provided between the start of the experimental run and the first time interval at which the fragment or product ions are mass analysed; b) providing different delay times in different ones of said experimental runs; c) determining if a fragment or product ion has been analysed in a first one of the time intervals in one of said experimental runs; d) determining if said fragment or product ion has also been analysed in a different numbered time interval in at least one other of said experimental runs, and if it is determined that said fragment or product ion has also been analysed in a different numbered time interval in at least one other of said experimental runs, selecting said fragment or product ion as an ion of interest; and e) if said fragment or product ion has been selected as an ion of interest, determining an average or centroid value of the timings of the first time interval and the different numbered time interval, and using the average or centroid value to identify the respective precursor ion of the fragment or product ion of interest.

2. The method of claim 1, wherein step d) comprises identifying fragment or product ions that have been analysed in the same time interval in at least one of said experimental runs and that have also been analysed in different time intervals in at least one other of said experimental runs as fragment or product ions of interest, and determining that these fragment or product ions relate to different precursor ions; and wherein step e) comprises using the timings of said different time intervals to identify the respective precursor ions of the fragment or product ions of interest.

3. The method of claim 2, comprising determining the duration of time between the start of an experimental run and the timing of the time interval at which each of said fragment or product ions of interest is detected, and using each said duration of time to determine the mass to charge ratio of the respective precursor ion of the ion of interest.

4. The method of claim 2, wherein a first fragment or product ion of interest is analysed at a first time interval and is determined to relate to a first precursor ion, wherein the timing of the first time interval is used to determine the time at which the first precursor ion was transmitted into the fragmentation or reaction device, and wherein the time at which the first precursor ion was transmitted is used to determine the mass to charge ratio of the first precursor ion; and/or wherein a second, different fragment or product ion of interest is analysed at a second time interval and is determined to relate to a second, different precursor ion, wherein the timing of the second time interval is used to determine the time at which the second precursor ion was transmitted into the fragmentation or reaction device, and wherein the time at which the second precursor ion was transmitted is used to determine the mass to charge ratio of the second precursor ion.

5. The method of claim 1, comprising summing the mass spectral data from said plurality of experimental runs and/or wherein each experimental run comprises analysing ions at a plurality of N time intervals after the start of the experimental run, and wherein spectral data from the plurality of experimental runs is summed to provide composite spectral data having N time intervals, and wherein the nth time interval of the composite spectral data includes the spectral data from the nth time interval of each of the experimental runs.

6. The method of claim 5, wherein said fragment or product ions of interest are determined to be ions having spectral data in different time intervals of said composite spectral data.

7. The method of claim 6, wherein said fragment or product ions of interest also have spectral data in the same time interval of said composite spectral data.

8. The method of claim 1, wherein the fragment or product ions are analysed by a time of flight mass analyser that periodically pulses the fragment or product ions into a time of flight region, and wherein the durations between subsequent ones of said pulses correspond to said plurality of time intervals.

9. The method of claim 1, wherein the step of providing different delay times in different ones of said experimental runs comprises providing either random delay times or predetermined different delay times.

10. The method of claim 1, wherein the precursor ions are transmitted to said fragmentation or reaction device by an ion mobility separator, and wherein said physicochemical property is ion mobility.

11. A mass spectrometer comprising: a device for selectively transmitting ions according to a physicochemical property; a fragmentation or reaction device; a mass analyser; and control means arranged and configured to cause the mass spectrometer to perform a plurality of experimental runs, wherein each experimental run comprises: i) transmitting precursor ions through said device and into the fragmentation or reaction device, wherein a physicochemical property of the precursor ions transmitted is varied as a function of time, ii) fragmenting or reacting the precursor ions in the fragmentation or reaction device so as to produce fragment or product ions, iii) periodically mass analysing the fragment or product ions in the mass analyser at a plurality of time intervals, wherein a delay time is provided between the start of the experimental run and the first time interval at which the fragment or product ions are mass analysed; said control means being further arranged and configured to: provide different delay times in different ones of said experimental runs; determine if a fragment or product ion has been analysed in a first one of the time intervals in one of said experimental runs; determine if said fragment or product ion has also been analysed in a different numbered time interval in at least one other of said experimental runs, and if it is determined that said fragment or product ion has been analysed in a different numbered time interval in at least one other of said experimental runs, select said fragment or product ion as an ion of interest; and if said fragment or product ion has been selected as an ion of interest, determine an average or centroid value of the timings of the first time interval and the different numbered time interval, and use the average or centroid value to identify the respective precursor ion of the fragment or product ion of interest.

12. The mass spectrometer of claim 11, wherein the physicochemical property is mass to charge ratio.

13. The method of claim 1, wherein step i) comprises mass selectively transmitting precursor ions into the fragmentation or reaction device with a mass filter having a mass transmission window that is varied as a function of time; and step e) comprises using the timings of said first time interval and said different numbered time interval to determine the timing at which said respective precursor ion of the fragment or product ion of interest was transmitted by the mass filter, and thereby determining the mass to charge ratio of said respective precursor ion of the fragment or product ion of interest.

14. The method of claim 1, comprising using the timings of said first time interval and said different numbered time interval to identify the respective precursor ion of the fragment or product ion of interest.

15. The method of claim 1, wherein the step of determining an average or centroid time value of the timings of the first time interval and the different numbered time interval comprises determining an ion signal intensity weighted average time value of the first time interval and the different time interval.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:

(2) FIG. 1 shows a schematic of a mass spectrometer in accordance with the present invention;

(3) FIGS. 2 and 3 illustrate a first method in accordance with the present invention wherein the delay time between the start of an experiment and the analysis is varied;

(4) FIG. 4 illustrates a conventional method wherein the delay time between the start of an experiment and the analysis is constant; and

(5) FIGS. 5 and 6 illustrate a second method in accordance with the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF INVENTION

(6) FIG. 1 shows a schematic of an embodiment of a mass spectrometer according to the present invention. The mass spectrometer comprises a quadrupole mass filter 4, a gas cell 6 and an orthogonal acceleration Time-of-Flight mass analyser 8. During operation, the quadrupole mass filter 4 is set so as to have a relatively low resolution. For example, the quadrupole 4 may transmit precursor ions 2 within a transmission window having a width of 25 Da. Precursor ions 2 that are transmitted by the quadrupole mass filter 4 are accelerated into the gas cell 6 such that they fragment to produce fragment ions. These fragment ions are then mass analysed in the Time-of-Flight mass analyser 8.

(7) A precursor experiment starts at T0 by transmitting precursor ions through the quadrupole mass filter 4. The quadrupole mass filter 4 is scanned with time during the experiment such that the range of mass to charge ratios transmitted in the transmission window of the quadrupole mass filter 4 changes with time. The quadrupole mass filter 4 scans in a non-biased, data independent manner so as to onwardly transmit precursor ions having a restricted range of mass to charge ratios. As described above, the precursor ions are then fragmented and the resulting fragment ions are mass analysed in the Time-of-Flight mass analyser 8. The Time-of-Flight mass analyser 8 operates by periodically pushing/pulsing fragment ions into a time of flight region. The fragments ions separate according to mass to charge ratio in the time of flight region and are then detected on a detector. The duration between an ion being pushed/pulsed and the ion being detected is determined and used to calculate the mass to charge ratio of the ion.

(8) The precursor ion experiment is then repeated a plurality of times by scanning the quadrupole mass filter 4 a corresponding plurality of times.

(9) The timing at which fragment ions are detected may be correlated to the timing of the transmission window in which their precursor ions 2 were transmitted by the mass filter 4. The gas cell 6 preferably maintains the fidelity of the temporally separated fragment ions by use of a travelling wave or a linear accelerating electric field.

(10) The Time-of-Flight acquisition system operates so that multiple Time-of-Flight spectra may be combined and tagged with effective first dimensional time or an increment relative to some other start event. In the preferred embodiment the start event is the start of the quadrupole mass-to-charge ratio scan.

(11) Method 1

(12) FIG. 2 illustrates a first method in accordance with present invention. This method may be particularly advantageous where the precursor ions are separated by mass to charge ratio on relatively fast timescales, for example 1 to 100 milliseconds. FIG. 2 shows three diagrams of the timings of the extraction pulses of the Time of Flight mass analyser for three experiments, relative to the start time T0 of each experiment, i.e. the time at which ions begin to be transmitted by the quadrupole mass filter 4 in each experimental run. As can be seen, the time delay between two subsequent extraction pulses is constant. In the first experiment there is a first time delay, dt1, between the start of the experiment and the next extraction pulse of the Time of Flight mass analyser 8. In the second experiment there is a second time delay, dt2, between the start of the experiment and the next extraction pulse of the Time of Flight mass analyser 8. Time delay dt2 is smaller than time delay dt1. In the third experiment there is a third time delay, dt3, between the start of the experiment and the next extraction pulse of the Time of Flight mass analyser 8. Time delay dt3 is smaller than time delay dt1 and time delay dt2. Although only three experiments are shown, more than three experiments may be performed.

(13) According to the method of FIG. 2, the first extraction pulse/push of the Time of Flight mass analyser after a precursor m/z separation experiment start time T0 is assigned a push/pulse number of n=1 and each push thereafter is assigned an increasing integer number up to N, where the integer N multiplied by the duration between subsequent pushes is greater than the precursor ion separation experiment time, i.e. greater than the time over which the quadrupole mass filter is scanned.

(14) The data obtained by the Time of Flight mass analyser in the different experiments is integrated. The data obtained from push n=1 in each experiment is combined, the data obtained from push n=2 in each experiment is combined, the data from push n=3 in each experiment is combined and so on, up to push N. In other words, the data obtained from the nth push of any given experiment is combined with the data from the nth push of the other experiments. This provides a two dimensional data set, wherein the push number n effectively represents a time within the precursor ion separation experiment (i.e. a first dimension) and at each push number n an entire fragment ion mass to charge ratio spectrum is accessible and made up of combined data from multiple precursor ion experiments.

(15) As described above, the Time of Flight acquisition timings are not synchronised with the precursor separation experiment start time T0, because push number n=1 is delayed from start time T0 by different amounts in different experiments. This means that it is likely that a particular push number, for example push number n=100, will sample slightly different parts of a mass peak in different experiments.

(16) FIG. 3 helps illustrate the advantage of the method described above and shows the experiments stacked vertically and aligned by the start time T0 of each experiment, rather than shown horizontally in time as in FIG. 2. Five experiments are shown in FIG. 3, wherein the delay time between the start of the experiment T0 and the first push n=1 is different in each experiment. The length of the time delay desirably varies randomly between the different experiments (although the time delay has a duration less than the duration between two subsequent pulses). A first component and second component are analysed by the Time of Flight mass analyser in each experiment. The two components are received at the Time of Flight mass analyser separated by a time that is less than the duration between two subsequent pusher periods. As such, in some experiments the two components are analysed by the Time of Flight mass analyser in the same push, as can be seen in the first, third and fifth experiments. However, because the delay time between the start of the experiment T0 and the first push differs in each experiment, a push time falls between the two components in some of the experiments and so the two components are analysed in different pushes in these experiments, as can be seen in the second and fourth experiments in FIG. 3. When the data from the different experiments is combined this allows separation of the two components in the final data; as in some of the experiments a first of the components is analysed in the Mth push and in other experiments the first component is analysed in the (M−1)th push, and in some of the experiments the second component is analysed in the Mth push and in other experiments the second component is analysed in the (M+1)th push. This is shown by the summed plot at the bottom of FIG. 3. Combining many of the experiments together allows an accurate determination of the precursor mass.

(17) The embodiment described above is in contrast to the conventional way of acquiring data.

(18) FIG. 4 shows plots corresponding to those in FIG. 3, except wherein the data is acquired in a conventional manner. As shown in FIG. 4, the Time of Flight acquisition system is synchronised with the experimental start time T0 such that the time delay between the start time T0 and the first push of the time of Flight mass analyser is constant in each of the different experiments. Consequently, the two components always fall in the same bin and are analysed by the same push number (push M) in each experiment. This renders the two components inseparable in the final combined data, as shown in the lowermost plot of FIG. 4.

(19) It is possible to improve the synchronised approach shown in FIG. 4 by synchronising the experimental start time T0 with the acquisition system, but such that the time delay between the start time T0 and the first push of the time of Flight mass analyser is different in different experiments. Varying the delay time between T0 and push number 1 by a known amount and taking account of this known amount during the combining process enables the two components to be assigned to separate bins in at least some of the experiments. This would result in greater than N total bins or push numbers, effectively improving the digitisation. However, this approach is less preferred than the described in relation to FIG. 3 as it requires additional instrument control and increases the two-dimensional data file sizes.

(20) The approach described in relation to FIG. 4 having different delay times differs from the approach described in relation to FIG. 3, in that in FIG. 3 the time delay between the start time T0 and the first push of the time of Flight mass analyser is random and unsynchronised with the acquisition system.

(21) Method 2

(22) FIG. 5 illustrates another method in accordance to the present invention. This method may be particularly advantageous where the precursor ions are separated by mass to charge ratio on relatively fast timescales, for example 50 to greater than 1000 milliseconds.

(23) FIG. 5 shows a plot of the Time of Flight mass analyser pushes relative to the start time T0 of a precursor ions experiment. The duration between any two subsequent pushes is constant. According to this method, the data obtained from multiple consecutive pushes is combined, summed, averaged or integrated so as to produce less frequent data points or bins. In the example shown in FIG. 5, data from the first six pushes is combined to form data at time T1, where time T1 corresponds to the time of the sixth push. Data from the next six pushes is combined to form data at time T2, where time T2 corresponds to the time of the twelfth push. Data from the next six pushes is combined to form data at time T3, where time T3 corresponds to the time of the eighteenth push. Data from the next six pushes is combined to form data at time T4, where time T4 corresponds to the time of the twenty-fourth push.

(24) It is noteworthy that adjacent pushes or ToF spectra may be combined into different final bins, unlike traditional acquisition systems where different combined spectra are separated by the many pushes associated with instrument interscan times or delays. This improves the duty cycle of the system as a whole.

(25) FIG. 6 illustrates how two components that populate the same time bins in FIG. 5 can be separated. The upper plot in FIG. 6 shows two partially overlapping rectangles that represent two equal intensity components being received over partially overlapping time periods. A first component begins to be received between the first and second pushes, and stops being received between the thirteenth and fourteenth pushes. A second component begins to be received between the fourth and fifth pushes, and stops being received between the fifteenth and sixteenth pushes. It is desired to identify the centroids or weighted average times of the two components.

(26) The lower plot in FIG. 6 illustrates how discrete times (e.g. centroids or weighted average times) for the two components can be determined even though the first and second components arrive within same six pushes that are summed to form data at time T1. The plotted points in the lower plot of FIG. 6 at each of time T0, T1, T2, T3 and T4 represent the summed responses for each component between the previous output time bin and the current one. For example, the response for each component at bin time T1 equates to the sum of the data from the upper plot in FIG. 6 between times T0 and T1 (i.e. from the first 6 pushes). The second component is only present for a short initial period between times T0 and T1, and so returns a relatively low value at T1. In contrast, the first component is present for a relatively long time between times T0 and T1, and so returns a relatively high value at T1. The response at bin time T2 equates to the sum of the data from the upper plot of FIG. 6 between times T1 and T2 (i.e. from the seventh to twelfth pushes). As both the first and second components are present for the full duration between T1 and T2 they return the same response. The response at bin time T3 equates to the sum of the data from the upper plot of FIG. 6 between times T2 and T3 (i.e. from the thirteenth to eighteenth pushes). The first component is only present for a short initial period between times T2 and T3, and so returns a relatively low value at T3. In contrast, the second component is present for a relatively long time between times T2 and T3, and so returns a relatively high value at T3. The response at bin time T4 equates to the sum of the data from the upper plot of FIG. 6 between times T3 and T4 (i.e. from the nineteenth to twenty-fourth pushes). Neither of the components is present between times T3 and T4 and so both components return a value of zero at T4.

(27) Once the peak for each component has been detected and its boundaries have been established, a discrete time (e.g. a centroid or weighted average time) can be determined for the component. For example, the weighted average time may be determined via the equation below, where T.sub.k is the time bin and I.sub.k is the intensity value in the corresponding bin. The intensity is just the sum of all the individual bin intensities across the detected peak.

(28) $\stackrel{\_}{T}=\frac{\underset{k=1}{\overset{n}{.Math.}}{T}_k\times I_k}{\underset{k=1}{\overset{n}{.Math.}}{I}_k}$

(29) The integrating/summing approach of the acquisition system described in relation to the lower plot in FIG. 6 provides peaks for the two components that have different profiles at the leading and trailing edges, because the components are detected over different (overlapping) time periods. A weighted average can be determined for each peak so as to determine a distinct and correct time measurement for each component (ignoring the systematic shift due to the time assignment in FIGS. 5 and 6), despite the fact that the two components populate the same time bins. The distinct time measurements are shown in the lower plot of FIG. 6 as vertical lines on either side of time T2. These distinct time measurements can be converted into mass to charge ratios for the components.

(30) The integrating/summing technique of the preferred embodiment is in contrast to simply sampling the data at less frequent intervals. If the data was merely measured and acquired the data at the four time points T1, T2, T3 and T4 then the response for each component would be the same in each bin, and it would not be possible to determine discrete times for each component.

(31) The technique described in relation to FIGS. 5 and 6 enables the amount of data acquired to be reduced. For example, ideally the data from each of the pushes would be kept separate by having a sampling rate that is the same as the pusher rate. However, such pusher rates can be over 20,000 times per second, which would result in a vast amount of data. The approach described in relation to FIGS. 5 and 6 enables a reduced number of data points and reduced file sizes, whilst retaining some of the benefits associated with a fast sampling rate corresponding to the pusher rate.

(32) As shown in FIG. 6, the approach is particularly useful for systems where the rise/fall time of the precursor profiles is less than the first dimensional bin width (i.e. time bin), which is a likely issue with devices such as lower resolution scanning quadrupoles.

(33) In the method described in relation to FIGS. 5 and 6, the ToF acquisition system may operate either asynchronously or synchronously with the start time T0 of the precursor separation experiment.

(34) Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.

(35) For example, the embodiments have been described in terms of scanning a low resolution quadrupole in order to separate the precursor ions according to mass to charge ratio (i.e. a first dimensional separator). However, it is contemplated that alternative mass to charge ratio separators may be used such as, for example, ion traps, magnetic sectors and Time of Flight separators. It is contemplated that ion separators other than mass to charge ratio separators may be used, such as an ion mobility separator.

(36) The separator for separating the fragment ions (second dimensional separator) has been described in terms of a Time of Flight mass analyser. However, although less preferred due to typically slower timescales, the separator may be a separator or mass analyser other than a ToF mass analyser.

(37) In both methods the acquisition system produces a two dimensional data set with both dimension being m/z, one dimension precursor m/z and the other dimension fragment ion m/z. The orthogonal relationship between precursor ion m/z & fragment ion m/z allows precursor ion mass spectra to be effectively reproduced from fragment ion data.

(38) The choice of which of the two methods is used may depend on the timescales associated with the precursor ion separation in the first dimension and the timescales associated with ToF separation.

(39) In both methods the approach can be combined with un-fragmented precursor ion measurements scans and/or ToFMS.