Patent Application
20100301205
The specification relates generally to mass spectrometry, and specifically to a method and apparatus for acquiring time profiles of ion intensities of product ions in a mass spectrometer.
When performing mass spectrometry, multiple pre-cursor ions trapped in an ion trap are generally ejected in sequence of mass, to be fragmented and analyzed in a mass analyzer. In order to achieve a better signal at the mass analyzer, the fill time of the ion trap can be increased. However this leads to increased space charge within the ion trap, which has a detrimental effect on the mass resolution (i.e. separation of ions of different mass) of the mass spectrometer. Achieving a balance between these two effects can be challenging, especially when there is a limited amount of sample.
A first aspect of the specification provides a method of operating a mass spectrometer comprising an ion trap, a fragmentation module connected to the ion trap, and a mass analyzer module positioned to receive ions from the fragmentation module. The method comprises ejecting precursor ions, trapped in the ion trap, in order of m/z ratio. The method further comprises fragmenting at least of some the precursor ions to form product ions at the fragmentation module. The method further comprises acquiring time profiles of ion intensities of the product ions received at the mass analyzer module, by recording a plurality of product mass spectra for each respective precursor ion. The method further comprises processing the plurality of product mass spectra using the time profile intensities to associate respective product ions with the respective precursor ions.
The method can further comprise alternating between a low energy fragmentation mode and a high energy fragmentation mode, wherein during the low energy fragmentation mode a first set of the plurality of product mass spectra are associated with substantially unfragmented precursor ions, and during the higher energy fragmentation mode a second set of the plurality of product mass spectra are associated with substantially fragmented product ions, such that the respective product ions can be associated with the respective precursor ions via the first set and the second set. For every one of the first set acquired, a plurality of second sets can be acquired.
The mass analyzer module can comprise a time of flight mass analyzer.
The mass analyzer module can comprise a quadrupole mass filter operated in a multiple ion monitoring mode.
The method can further comprise identifying the respective precursor ions from a residual unfragmented precursor ion intensity in the plurality of product mass spectra.
Processing the plurality of product mass spectra can comprise deconvoluting the respective ion intensities. At least two of the time profiles of ion intensities can overlap.
Ejecting precursor ions, trapped in the ion trap, can comprise ejecting precursor ions in sequential order of m/z ratio.
The method can further comprise applying an axial field in the fragmentation module to reduce transit time of ions in the fragmentation module.
A second aspect of the specification provides a mass spectrometer. The mass spectrometer comprises an ion trap enabled to eject precursor ions, trapped in the ion trap, in order of m/z ratio. The mass spectrometer further comprises a fragmentation module, connected to the ion trap, enabled to fragment at least of some the precursor ions to form product ions. The mass spectrometer further comprises a mass analyzer module positioned to receive ions from the fragmentation module. The mass analyzer module is enabled to: acquire time profiles of ion intensities of the product ions received at the mass analyzer module, by recording a plurality of product mass spectra for respective precursor ions; and process the plurality of product mass spectra using the time profile intensities to associate respective product ions with the respective precursor ions.
The mass spectrometer can be enabled to alternate between a low energy fragmentation mode and a high energy fragmentation mode, wherein during the low energy fragmentation mode a first set of the plurality of product mass spectra are associated with substantially unfragmented precursor ions and during the higher energy fragmentation mode a second set of the plurality of product mass spectra are associated with substantially fragmented product ions, such that the respective product ions can be associated with the respective precursor ions via the first set and the second set. For every one of the first set acquired, a plurality of second sets can be acquired.
The mass analyzer module can comprise a time of flight mass analyzer.
The mass analyzer module can comprise a quadrupole mass filter operated in a multiple ion monitoring mode.
The mass analyzer module can be further enabled to identify the respective precursor ions from a residual unfragmented precursor ion intensity in the plurality of product mass spectra.
To process the plurality of product mass spectra, the mass analyzer module can enabled to deconvolute the respective ion intensities. At least two of the time profiles of ion intensities can overlap.
To eject precursor ions, the ion trap can be further enabled to eject precursor ions in sequential order of m/z ratio.
The fragmentation module can be further enabled to apply an axial field to reduce transit time there through.
Embodiments are described with reference to the following figures, in which:
In operation, ionisable materials are introduced into ion source 120. Ion source 120 generally ionises the ionisable materials to produce precursor ions which are transferred to ion optics 130 (also identified as Q0, indicative that ion optics 130 take no part in the mass analysis). Precursor ions are transferred from ion optics 130 to ion trap 140 (also identified as Q1) enabled to eject ions (e.g. precursor ions) in order of m/z (mass to charge) ratio, in a manner described below. Ejected precursor ions can then be transferred to fragmentation module 150 (also identified as q2) for fragmentation, to form product ions. Product ions are subsequently transferred to mass analyzer 160 for mass analysis, resulting in production of product ion spectra.
Furthermore, while not depicted, mass spectrometer 100 can comprise any suitable number of vacuum pumps to provide a suitable vacuum in ion source 120, ion optics 130, ion trap 140, fragmentation module 150 and/or mass analyzer 160. It is understood that in some embodiments a vacuum differential can be created between certain elements of mass spectrometer 100: for example a vacuum differential is generally applied between ion source 120 and ion optics 130, such that ion source 120 is at atmospheric pressure and ion optics 130 are under vacuum. While also not depicted, mass spectrometer 100 can further comprise any suitable number of connectors, power sources, RF (radio-frequency) power sources, DC (direct current) power sources, gas sources (e.g. for ion source 120 and/or fragmentation module 150), and any other suitable components for enabling operation of mass spectrometer 100.
Ion source 120 comprises any suitable ion source for ionising ionisable materials. Ion source 120 can include, but is not limited to, an electrospray ion source, an ion spray ion source, a corona discharge device, and the like. In these embodiments, ion source 120 can be connected to a mass separation system (not depicted), such as a liquid chromatography system, enabled to dispense (e.g. elute) ionisable to ion source 120 in any suitable manner.
In specific non-limiting embodiments, ion source 120 can comprise a matrix-assisted laser desorption/ionisation (MALDI) ion source, and samples of ionisable materials are first dispensed onto a MALDI plate, which can generally comprise a translation stage. Correspondingly, ion source 120 is enabled to receive the ionisable materials via the MALDI plate, which can be inserted into the MALDI ion source, and ionise the samples of ionisable materials in any suitable order. In these embodiments, any suitable number of MALDI plates with any suitable number of samples dispensed there upon can be prepared prior to inserting them into the MALDI ion source.
Precursor ions produced at ion source 120 are transferred to ion optics 130, for example via a vacuum differential and/or a suitable electric field(s). Ion optics 130 can generally comprise any suitable multipole or RF ion guide including, but not limited to, a quadrupole rod set. Ion optics 130 are generally enabled to cool and focus precursor ions, and can further serve as an interface between ion source 120, at atmospheric pressure, and subsequent lower pressure vacuum modules of mass spectrometer 100.
Precursor ions are then transferred to ion trap 140, for example via any suitable vacuum differential and/or a suitable electric field(s), ion trap 140 enabled to eject precursor ions in order of m/z ratio, which are transferred to fragmentation module 150. It is understood that while ion trap 140 is identified as Q1 in
In any event, as precursor ions are ejected from ions trap 140, they are transferred to fragmentation module 150 for fragmentation such that product ions are produced. In some embodiments, fragmentation module 150 can be operated in alternating low energy fragmentation and high energy fragmentation modes to first identify precursor (i.e. parent) ions and associated respective product ions of each mass range.
Once fragmentation of precursor ions occurs, product ions are transferred to mass analyzer 160 for analysis and production of product ion spectra (i.e. product mass spectra). Mass analyzer 160 can comprise any suitable mass spectrometer module including, but not limited to, a time of flight (TOF) mass spectrometry module, a quadrupole mass spectrometry module, a linear ion trap module and the like.
Returning now to ion trap 140, ion trap 140 is generally filled with precursor ions for a given period of time, which is understood to be the “fill time”. While longer fill times can lead to better signal to noise ratio (SNR) in spectra acquired at mass analyzer 160, as more precursor ions are available for analysis, the space charge of such degrades mass resolution as when there are too many ions in ion trap 140, the electric field within ion trap 140 becomes distorted. The relatively high space charge in ion trap 140 makes the mass selective ejection from ion trap 140 inefficient. In addition, the relatively high space charge in ion trap 140 reduces the ion selectivity of ion trap 140.
For example, in some embodiments, ion trap 140 can be operated to scan through a range of masses in steps of 1 m/z: e.g. precursor ions of m/z 300 can be first ejected, and then precursor ions of m/z 301, etc. However, space charge can limit the mass resolution such that ejection profiles of precursor ions overlap: i.e. multiple precursor ions are ejected, rather than a single precursor ion. For example, as depicted in the ejection profile of
To compensate for this effect mass analyzer 160 is enabled to acquire time profiles of ion intensities of product ions received at mass analyzer 160 by recording a plurality of product mass spectra for respective precursor ions, each mass spectrum comprising respective ion m/z and respective ion intensities of product ions. The plurality of product mass spectra are then processed using the time profiles intensities to associate respective product ions with respective precursor ions. Hence, the time-resolved signal from the mass analyzer 160 can be used to determine which product ions are formed from a particular precursor ion.
Returning to the previous example, as ion trap 140 is scanned to eject a range of ions, m/z 300 can be ejected before m/z 301, but the ejection profiles can overlap as in
The previous example can again be considered, where the scan speed of ion trap 140 is 1000 amu/s and hence each mass peak should be ejected in 1 ms. However, if the resolution of the ion trap is degraded by space charge or other effects that can reduce the mass resolution (non-limiting examples of effects that can degrade or reduce the resolution include fast scan speed, or non-optimum geometry of the trap), then the ejection profiles can be several ms wide, as depicted in
To illustrate this, attention is directed to
In any event, signals for species labelled A and B are recorded at t=298 ms by mass analyzer 160, along with residual unfragmented precursor signals at m/z 300 and 301 (it is assumed for the purpose of this example that there are no other precursor ions between m/z 290 and m/z 310). At t=298 ms, product ion peaks (signals) associated with species A with precursor m/z of 300 are increasing in intensity while product ion peaks (signals) associated with species B with precursor m/z of 301 are also increasing in intensity. Unfragmented (residual) precursor ions of m/z 300 and m/z 301 are present together, indicating that both precursor ions are being ejected from ion trap 140 at this time. In the scan recorded at t=300, product ion peaks associated with species A have reached a maximum intensity, while species labelled B continue to increase in intensity.
In the scan recorded at t=302 ms, intensities of product ion peaks associated with species
A and with species B are both decreasing.
In processing the spectra acquired by mass analyzer 160, of which those shown in
Such association can be performed by deconvoluting the time profiles of the product ions in the scans acquired at t=298, 300 and 302 ms. For example, in some embodiments, signals of product ions, associated with different precursor ions, can overlap; in these embodiments, deconvolution of such overlapping signals can be performed to distinguish between them. For example, product mass spectra time profiles of some of the product ions in the example of
In some embodiments, fragmentation module 150 comprises a collision induced dissociation (CID) collision cell. In these embodiments, the transit time through the collision cell can be relatively short, so that the signals/peaks are not further broadened in transit. For example, ions can move through the collision cell in approximately 1 ms. If the transit time is too long, then mobility separation of ions may cause the product ion profiles to fail to track one another. Ions of different mobility will cause the profiles to separate. To reduce the transit time, an axial field can be applied in the collision cell. Any further overlap of ion profiles from different precursor ions, caused by broadening during transit through the collision cell, can be deconvoluted using the method of aligning ion current profiles.
In embodiments, fragmentation can occur via a process that does not change the velocity of the ions, for example fragmentation module 1650 can comprise a photofragmentation module. In these embodiments, product ions move at the same speed as precursor ions, and no time-separation occurs.
In some embodiments, there may be no unfragmented or residual precursor ions remaining in the product ion spectra. This can happen if the collision energy or fragmentation efficiency is very high. In such cases, alternate scans at low collision energy and high collision energy (sufficient to fragment ions), can be acquired, such that one set of spectra will contain precursor ion peaks and one set of spectra will contain product ions with some residual precursors ion peaks. This can allow the time profiles of precursor ions to be acquired independently of the product ions so that they can be aligned in order to associate specific product ion m/z values with specific precursor ion ink values. In some of these embodiments, the time profiles may not exactly match up because the initial energy in the collision cell is higher at higher collision energies. Hence, the low and high energy spectra can appear to be shifted in time. This time shift can be compensated for by a calibration process (e.g. calibrating time vs. energy of ions travelling through fragmentation module 150) and/or by shifting and aligning the two set of spectra.
Furthermore, there can be a duty cycle advantage to operating mass spectrometer 100 in this manner as the fill time can be increased. Such an advantage can be shown via a non-limiting exemplary calculation: Assume that the scan time of ion trap 140 is 500 ms (e.g. ion trap 140 scans through 500 amu at 1000 amu/s). Further assume that the fill time of ion trap 140 is 10 ms. Then the duty cycle will be 10 ms/500 ms or 2%. In comparison with conventional operation of a mass spectrometer for performing MSMS of everything which would acquire MSMS of each precursor m/z value for 1 ms each, this represents an increase of a factor of 10 in duty cycle and sensitivity. By increasing the fill time further, the duty cycle can be further increased; however the maximum fill time can be dependent on the space charge limit of ion trap 140. Since space charge limits the mass resolution, the limit in fill time will be determined by the ion current and by a minimum mass resolution that can be deconvoluted in acquired product mass spectra.
Furthermore, scan rate can be increased to further increase the duty cycle as: for example a scan of 4000 amu/s represents a scan time of 125 ms for a mass range of 500 amu. Then the duty cycle can be 8% (e.g. 10 ms/125 ms).
In other embodiments, various methods can be used to limit the space charge and therefore enable higher duty cycles, including but not limited to eliminating low mass ions by using a low mass cut-off and selectively ejecting specific intense background ions prior scanning.
In yet further embodiments, operation of mass spectrometer 100 to acquire time profiles of product spectra can be used in combination with selective isolation of specific mass ranges in order to eliminate unwanted ions. For example, a Filtered Noise Field (FNF) can be used to isolate specific mass windows around mass peaks of interest. Then a fast scan of ions in ion trap 140 can be performed to sequentially eject ions. In some of these embodiments, some regions of the spectrum can be empty, simplifying the alignment of the product ions with the respective precursor ions.
In some embodiments, operation of mass spectrometer 100 to acquire time profiles of product spectra can be used to obtain very fast MSMS scans under conditions where a high scan speed may result in decreased mass resolution. For example, at a scan speed of 10000 amu/s, unit mass resolution may not be achieved, even at low ion concentrations and ejected product ions will overlap. Deconvolution of a plurality of product mass spectra for precursor ions (i.e. from an acquired time profile) can be used to associate product ions with respective precursor ions. In these embodiments, the transit time of ions through fragmentation module 140 can contribute to broadening of the ejection profiles. However, alignment of the time profiles of the product ions can still allow the product ions to be assigned to specific precursor m/z values.
In embodiments where it is desired to obtain high mass resolution in a particular mass range, the scan rate of ion trap 140 can be lowered. For example, if a narrow mass range is scanned slowly, for example at 10 amu/sec, peak widths of <0.1 amu can be achieved. This can increase the signal-to-noise ratio (S/N) in complex samples. Aligning the profiles of the product ions of the ejected precursor ions can further increase the resolution, which also leads to better specificity by recording the time profile of product ions, as well as the product ion mass value, when ions are scanned from ion trap 140. It is understood, however, that the time resolution of mass analyzer 160 is on the order of, or faster than, the ejection profile of ion trap 140. For high resolution multiple reaction monitoring (MRM) acquisitions using mass analyzer 160 as a mass filter as the ejection profile of product ions can be measured by rapidly peak hopping the m/z value of mass analyzer 160 among the target product ion m/z values. This is similar to conventional MRM analysis except that ion trap 140 is slowly scanned over a narrow mass range around a known precursor m/z value. If there are two isobaric compounds with slightly different exact m/z values, one of which is a target m/z of interest, and one of which is a background or interference ion, both of which form a product ion of exactly the same m/z value, then the time profiles of the two product ions will be different, following the time profiles of the two precursor ions of slightly different m/z. The time profile of the product ion m/z can be deconvoluted to reduce or eliminate the interference. For example
Attention is now directed to
At step 610, precursor ions are ejected from ion trap 140, in order of m/z ratio. It is understood that precursor ions have been produced at ion source 140, and transferred to ion trap 140, using a given fill time. In general precursor ions are ejected from ion trap 140 and transferred to fragmentation module 150.
At step 620, at least some of the precursor ions are fragmented, to form product ions, at fragmentation module 150. It is understood that product ions are then transferred to mass analyzer 160.
At step 630, time profiles of ion intensities of product ions received at mass analyzer 160 are acquired by recording a plurality of product mass spectra for respective precursor ions (e.g. spectra acquired at a rate of several KHz, as described above), product mass spectra comprising respective ion ink and respective ion intensities of product ions, for example as depicted in
At step 640, the plurality of product mass spectra are processed using the time profiles intensities to associate respective product ions with respective precursor ions as depicted in
In some embodiments, method 600 can further comprise alternating operation of mass spectrometer 100 between a low energy fragmentation mode and a high energy fragmentation mode. During the low energy fragmentation mode, a first set of the plurality of product mass spectra are associated with substantially unfragmented precursor ions. During the higher energy fragmentation mode, a second set of the plurality of product mass spectra are associated with substantially fragmented product ions, such that the respective product ions can be associated with the respective precursor ions via the first set and the second set. For example, at the beginning of each step of a mass ejection scan in ion trap 140 (i.e. for each precursor ion ejected), fragmentation module 150 can first be controlled to allow precursor ions to be transferred there through without substantial fragmentation. Once an initial product mass spectra is acquired at mass analyzer 160, the initial product mass spectra substantially comprising a signal of at least one precursor ion, fragmentation module 150 is then controlled to produce fragmented product ions. Subsequent product mass spectra acquired at mass analyser 160 can then be associated with the at least one precursor ion identified in the initial scan. Any suitable number of alternations between a low energy fragmentation mode and a high energy fragmentation mode are within the scope of present embodiments. Furthermore, each of the first set and the second set of the plurality of product mass spectra can comprise any suitable number of spectra. In some non-limiting embodiments, for every one of the first set acquired, a plurality of second sets are acquired.
In general, method 600 can further comprise identifying the respective precursor ion from a residual unfragmented precursor ion intensity in the plurality of product mass spectra, as described above with reference to
In any event, space charge or other limitations of ion trap 140 an be obviated by acquiring a time profile of ion intensities of product ions received at a mass analyzer, and specifically by recording a plurality of product mass spectra for respective precursor ions ejected from ion trap 140, each product mass spectra comprising respective ion m/z and respective ion intensities of the product ions. The plurality of product mass spectra can be processed using the time profile intensities to associate respective product ions with respective precursor ions. Hence, the fill time of the ion trap 140 can be increased, and slower mass ejection scan speeds used, both of which increase the duty cycle of mass spectrometer 100.
Those skilled in the art will appreciate that in some embodiments, the functionality of mass spectrometer 100 can be implemented using pre-programmed hardware or firmware elements (e.g., application specific integrated circuits (ASICs), electrically erasable programmable read-only memories (EEPROMs), etc.), or other related components. In other embodiments, the functionality of mass spectrometer 100 can be achieved using a computing apparatus that has access to a code memory (not shown) which stores computer-readable program code for operation of the computing apparatus. The computer-readable program code could be stored on a computer readable storage medium which is fixed, tangible and readable directly by these components, (e.g., removable diskette, CD-ROM, ROM, fixed disk, USB drive). Alternatively, the computer-readable program code could be stored remotely but transmittable to these components via a modem or other interface device connected to a network (including, without limitation, the Internet) over a transmission medium. The transmission medium can be either a non-wireless medium (e.g., optical and/or digital and/or analog communications lines) or a wireless medium (e.g., microwave, infrared, free-space optical or other transmission schemes) or a combination thereof.
Persons skilled in the art will appreciate that there are yet more alternative implementations and modifications possible for implementing the embodiments, and that the above implementations and examples are only illustrations of one or more embodiments. The scope, therefore, is only to be limited by the claims appended hereto.
| Number | Date | Country | |
|---|---|---|---|
| 61181393 | May 2009 | US |
