Patent Description:
Spectral congestion in top down and native mass spectrometry analysis is a major drawback that hinders efficient data interpretation. This occurs when several (or many) different fragment ions in a mass spectrometry measurement possess comparable mass-to-charge ratios (m/z). The isotopic distributions for the different fragment ions with the same m/z will overlap when viewed on a m/z spectral graph and cannot be distinguished one from another. <CIT> describes a mass spectrometer comprising a first ion trap or ion guide, a single ion mobility spectrometer or separator stage and a second ion trap or ion guide arranged downstream of the ion mobility spectrometer or separator. <CIT> describes a mass spectrometer comprising a first quadrupole rod set mass filter, a collision cell, an ion mobility spectrometer or separator, an ion guide or collision cell arranged downstream of the ion mobility spectrometer or separator, a second quadrupole rod set mass filter and an ion detector. <CIT> describes an ion separation instrument including an ion source coupled to at least a first ion mobility spectrometer having an ion outlet coupled to a mass spectrometer. A further example of relevant prior art is known from <CIT>.

At its most general, the invention is to use differences in the ion mobility amongst a plurality of different ion species within an ion sample having comparable m/z, to spatially separate the different ion species according to their ion mobility. Once spatially separated, the individual ion species may be individually and separately trapped, moved and controlled as desired for analysis (e.g. to produce separate or fractional m/z spectra).

The velocity vd with which an ion drifts through a buffer gas in response to a uniform applied electric field E, is determined by ion mobility K of the ion in the form: vd = KE. The ion mobility can be calculated via the Mason-Schamp equation:<MAT>.

Here z is the ion charge and µ is the reduced mass of the ion (m<NUM>) and the drift gas molecules (m<NUM>): µ = (m<NUM> m<NUM>)/ (m<NUM> + m<NUM>).

The constant term "const. " is determined by the drift gas number density, the drift gas temperature, and the collision cross section between the ion and the drift gas molecules. Thus, ion mobility differs between ions of the same mass-to-charge ratio.

In so far, different versions of ion mobility spectrometers combined with mass spectrometry have been disclosed and are utilized to separate or disperse precursor ions in mobility space. Example devices are described in <CIT>; <CIT>; and <CIT>. In such devices the fragmentation cell is disposed downstream from the ion mobility spectrometer, such that the ion mobility spectrometer does not receive fragment ions and any fragment ions generated from precursor ions in the gas phase are formed after the ion mobility separation step is performed on un-fragmented ions. The inventors have realized that ion mobility technology can be employed to distinguish between different ions having the same mass-to-charge ratio, to separate or disperse fragment ions in mobility space and to reduce congestion of complex fragmentation (m/z) spectra, particularly, complex fragmentation spectra produced by mass analysis of fragment ion populations produced in RF ion traps utilizing electron capture dissociation, photo-dissociation or other types of ion activation-dissociation methods applied in the analysis of intact macromolecular ions. Specifically, the invention is concerned with the simplification of complex fragmentation spectra produced in the analysis of intact proteins, polypeptide chains (top down and middle down) and native mass spectrometry including the analysis of protein complexes, protein-drug conjugates, and antibodies in a middle-down or top down approach, facilitating peak identification and precise peak annotation using both manual and automated data post-processing methods.

The invention may be implemented using an ion drift cell containing a buffer gas within which a homogeneous (e.g. DC) electrical field is applied to impose a drift force to ions in the drift cell in a direction along the longitudinal axis of the drift cell. The electrical field drives ions through the drift cell where they interact with the neutral molecules of the buffer gas contained within the drift cell. The buffer gas contained in the drift cell is most preferably a gas in an equilibrium state. That is to say, the buffer gas contained in the drift cell is preferably a non-flowing gas. The different species of ions are separated based on ion mobility. Different species of ions traverse different distances within the drift cell in a given time interval. The spatially separated ions may be trapped, stored or confined in separate respective locations axially along the drift cell and ejected from the drift cell separately, or simply transmitted through the cell, without trapping, for direct mass analysis.

The drift cell may be tubular e.g., with a series of ring-shaped electrodes arranged in a stack. Other drift cell designs can be used, for example a drift cell formed using separated, planar conductive plates or boards opposed/facing each other, e.g. two gold-plated printed circuit boards, for example, such as is described in: <NPL>; or <NPL>.

The invention provides a new method that utilizes a high performance ion mobility drift cell combined with an ion trap, specifically a radio-frequency (RF) ion trap, preferably a segmented RF linear ion trap. Fragment ions, which in a typical top-down fragmentation experiment may comprise of ><NUM> or even ><NUM> distinct isotopic distributions simultaneously confined in a RF field of an ion trap, and originating from one or more precursor ions mass selected in an ion trap or in a quadrupole mass filter, are injected and separated inside a pressurized drift cell at pressures in the range of <NUM>. 1mbar or higher (e.g. <15mbar). Fragment ions will separate according to mass, shape and charge state. Fragment ions may be simply transmitted toward a mass analyser or divided and stored in trapping regions of the drift cell provided by applying pulsed or switched DC fields superimposed on the drift electric field (E). The static DC gradient of the drift electric field (E) may be combined with the pulsed or switched DC electric fields to form discrete mobility-separated ion groups, which may be stored in trapping regions within the drift cell.

An RF field is applied to the electrodes of the ion mobility spectrometer to store the mobility-separated ion groups in the trapping regions of the drift cell. The RF field may be generated by two anti-phase sinusoidal RF waveforms each applied to the odd and even numbered electrodes of the stack or system of electrodes of the drift cell, respectively. Reversing of the drift electric field (E), by reversing the static DC gradient applied across the drift cell may be implemented to move/transfer discrete mobility-separated ion groups separately into an ion trap and/or to a mass analyser. Preferably an orbitrap or a time-of-flight (TOF) mass analyser may be used for this purpose. The above-described fragment ion transfer method may be implemented in cycles, each being preferably of <<NUM> per transferred ion group.

The apparatus and method according to the present invention may greatly reduce spectral density since ions with different charge states appearing at the same position in the m/z scale may now be spatially separated, and/or trapped and ultimately mass analysed separately. The new method permits the simplification of complex m/z spectra and associated improvements in peak assignment to allow de-novo sequencing of proteins, for example.

Provided is an apparatus and a method as set out in the accompanying claims herein. In an aspect, the invention provides a mass spectrometer for analysing fragment ions according to claim <NUM>.

In a second aspect the invention provides a method for separating fragment ions according to claim <NUM>.

The mass spectrometer may provide at least one region along the ion optical path of the mass spectrometer for storing at least a fraction of the mobility separated fragment ions. The mass spectrometer may provide a control system of electrodes, or a system of electrodes and associated DC potentials switched, or switchable, for transferring the selected fraction to a mass analyser to create a m/z spectrum, where the generated m/z spectrum contains information from only a fraction of the initial population of the fragment ions (e.g. produced by the ion trap) to reduce spectral complexity. The control system may comprise the system of electrodes, and may be configured to drive the electrodes by applying DC electrical potentials to those electrodes. It may be configured to drive the electrodes by applying one or more switched DC signals and/or one or more RF signals. A switched DC signal has a DC level that is switchable between a plurality of separate and different DC levels. The control system of the mass spectrometer may also comprise electronics (e.g. one or more electronics units) configured for generating one or more RF electrical potentials, and/or one or more static DC electrical potentials (i.e. non-switched) and/or one or more switched DC electrical potentials. The control system may be arranged to distribute the generated one or more electrical potentials to the system of electrodes of the mass spectrometer, and may be configured to apply the distributed one or more electrical potentials to electrodes of the mass spectrometer apparatus in a synchronized manner. Synchronisation of the application of the distributed one or more electrical potentials to electrodes of the mass spectrometer apparatus, may be achieved by switching different electrodes of the system of electrodes, so as to permit the trapping and/or transfer of the ions through the system using the electrical potentials applied to the ions by the system of driven electrodes. In this way, the control system may control the manner in which the system of electrodes transfers ions, e.g. to a mass analyser.

The ion trap, the ion mobility spectrometer and the mass analyser units including all intermediate ion optical components for transferring ions between the separate units are all comprised of individual sets of electrodes collectively forming a system of electrodes for processing ions. Processing involves activating, fragmenting, separating and mass analysing ions in a mass spectrometer that incorporates the unique features outlined in the present invention.

The mass spectrometer may provide a series of trapping regions established toward the exit end of the ion mobility spectrometer and switched during the separation step for splitting fragment ions into separate groups or fractions. The mass spectrometer may provide a set of switches, e.g. floating to the level set by the drift electric field E of the ion mobility drift cell, for switching at least the DC potentials applied to the ion mobility spectrometer to release separated groups of ions in consecutive steps to produce individual m/z spectra in each step of the process. RF signals must be applied to all electrodes forming the trapping regions in the drift cell. Preferably, electrodes are spaced apart by a gap of <<NUM> and electrode pitch is preferably set to <<NUM>. Desirably, the number of spectra produced in a single cycle equals the number of groups separated in the ion mobility spectrometer.

The mass spectrometer may be arranged such that the switching of the DC potentials applied to the ion mobility cell also includes reversing the DC gradient established across the stack of electrodes. In yet another aspect, the invention may provide a method for separating fragment ions in an ion mobility spectrometer, the method comprising generating fragment ions in a RF linear ion trap (e.g. a quadrupole ion trap), injecting and separating ions inside a pressurized ion mobility cell, isolating or transmitting at least one group of fragment ions from the initial ion population of fragments, and transferring the at least one isolated or transmitted group of ions towards a mass analyser to produce a m/z spectrum containing reduced information compared to the m/z spectrum that includes all fragment ions.

The following features may be provided: at least one region along an ion optical path of the ion mobility spectrometer for storing at least a fraction of the mobility-separated fragment ions; a control system comprising a system of electrodes and arranged for applying associated DC electrical potentials to the electrodes and for switching or changing at least one of the DC electrical potentials for transferring the stored fraction of the mobility-separated fragment ions to a mass analyser for generating a m/z spectrum therefrom; wherein the generated m/z spectrum contains only a fraction of the initial population of the fragment ions received from said ion source, thereby to reduce spectral complexity and facilitate peak assignment and data interpretation using a variety of post processing methods.

The mass spectrometer comprises an ion trap as said ion source for producing fragment ions. The mass spectrometer may comprise the mass analyser for producing a m/z spectrum from said stored fraction of the mobility-separated fragment ions transferred to it by said control system. The control system may be arranged to apply to the ion mobility spectrometer a series of radio-frequency (RF) and DC electrical potentials for generating electric fields defining ion trapping regions disposed toward an exit end of the ion mobility spectrometer, for trapping the mobility-separated fragment ions thereat. The control system may be arranged to switch electrical potentials from amongst said series of electrical potentials, for separating, partitioning or splitting the mobility-separated fragment ions into spatially separate groups. The control system may be arranged for switching at least a portion of the DC electrical potentials applied to the ion mobility spectrometer to release said spatially separate groups of ions in temporally consecutive steps to permit said m/z spectra to be generated therefrom. The control system may comprise a set of switches arranged for switching said electrical potentials from amongst said series of electrical potentials. Switching and synchronization of all electrical potentials is performed by the control system, which includes the system of electrodes arranged to guide, separate and/or trap (and desirably process) ions in a mass spectrometer of the present invention. The control system also comprises a set of electronics units for generating RF, static and switched DC electrical potentials that are distributed and applied to the system of electrodes of the mass spectrometer apparatus.

In another aspect, the following is provided. a series of RF and DC trapping regions established toward the exit end of the ion mobility spectrometer and switched during the separation step for splitting the fragment ions into separate groups; a set of switches for switching at least one of the DC potentials applied to the ion mobility spectrometer to release separated groups of ions in consecutive steps to produce a number of m/z spectra in each step; where the number of spectra produced in a single cycle equals the number of groups separated in the ion mobility spectrometer.

The switching of the DC potentials applied to the ion mobility cell may also include reversing the DC gradient established across the stack of electrodes.

In another aspect, the invention may provide a method comprising: generating a population of fragment ions in an ion trap; injecting the generated population of fragment ions into a pressurized ion mobility drift cell and separating the injected population of fragment ions inside the pressurized ion mobility drift cell; trapping at least a fraction of the separated population of fragment ions; and, transferring the at least a fraction of trapped fragment ions towards a mass analyser to produce at least one m/z spectrum containing reduced information compared to a m/z spectrum that includes the population of fragment ions. The ion trap, in this method, may be an RF linear ion trap.

The method includes providing a system of electrodes, and driving the electrodes by applying DC electrical potentials to those electrodes. It may include driving the electrodes by applying one or more switched DC signals and/or one or more RF signals. A switched DC signal has a DC level that is switchable between a plurality of separate and different DC levels. The method may comprise generating one or more RF electrical potentials, and/or one or more static DC electrical potentials (i.e. non-switched) and/or one or more switched DC electrical potentials. The method may include distributing the generated one or more electrical potentials to the system of electrodes, and may include applying the distributed one or more electrical potentials to electrodes in a synchronized manner. Synchronisation of the application of the distributed one or more electrical potentials to electrodes, may include switching different electrodes of the system of electrodes, so as to permit the trapping and/or transfer of the ions through the system using the electrical potentials applied to the ions by the system of driven electrodes. In this way, the control method may control the manner in which the system of electrodes transfers ions, e.g. to a mass analyser.

<FIG> shows an ion mobility drift cell (hereafter also referred to as ion mobility spectrometer - IMS) <NUM> connected to a RF linear ion trap <NUM> (e.g. a quadrupole ion trap), and used for receiving, separating and returning separated packets of ions into the a mass analyser <NUM> which may be disposed upstream of the linear ion trap <NUM> for reducing spectral complexity and facilitating spectral interpretation with both automated and manual processing methods. Also shown are RF multipole (e.g. hexapole) ion guides <NUM> for transferring fragment ions from the segmented RF linear ion trap <NUM> to the ion mobility drift cell <NUM> and operated at an intermediate pressure to bridge the large operating pressure differences that may be established between the RF linear ion trap and the ion mobility cell. Fragment ions can be stored in the RF multipole ion guide <NUM> and gated into the ion mobility drift cell <NUM>. The RF multipole ion guide <NUM> is used to gate ions into the ion mobility drift cell and is segmented and arranged to apply a weak DC gradient <NUM> for forcing ions to accumulate near the exit-end of the device where thermalisation may be performed, e.g. at ><NUM>-<NUM> mbar pressure. An additional voltage applied to a differential lens electrode <NUM>, located between the ion mobility drift cell <NUM> and the RF multipole ion guide <NUM>. This additional voltage can be switched to inject ions from the RF multipole ion guide <NUM> into the pressurized cell (~<NUM> mbar) of the ion mobility drift cell <NUM>. The RF multipole ion guide <NUM> is operated in pulsed DC mode or switched DC mode <NUM>, and ion potential energy is thereby raised, as denoted by the arrow <NUM>, to the appropriate level prior to injection into the ion mobility drift cell <NUM>. In accordance with the invention, the pulsed high-voltage DC level <NUM> of the RF multipole ion guide <NUM> is reached simultaneously with the pulsed high-voltage DC level <NUM> of the ion mobility drift cell <NUM>. Therefore, different ion optical components <NUM> and <NUM> disposed in different vacuum regions are pulsed simultaneously.

<FIG> also shows a schematic diagram of the pulsed or switched DC potential profile <NUM> created across the cell by the application of DC potentials to the electrodes of the mass spectrometer, individual trapping regions <NUM> for storing and separating ions and the ability to reverse the DC gradient <NUM> to return separated packets of fragment ions <NUM> towards the mass analyser <NUM>. An RF field <NUM> is preferably applied to the electrodes of the drift cell <NUM> to confine ions radially and minimize losses due to diffusion. The ion trap <NUM>, the ion mobility spectrometer <NUM> and the mass analyser <NUM> units including the RF hexapole ion guide <NUM> and all differential aperture lenses, for example lens <NUM>, for transferring ions between the separate units are all comprised of individual sets of electrode(s) collectively forming a system of electrodes for processing ions.

<FIG> shows an example of a m/z spectrum with three overlapping isotopic distributions of fragment ions <NUM> having different charge states, and the <NUM>+ and <NUM>+ charge state isotopic distributions buried beneath the <NUM>+ distribution complicating peak assignment and identification. The ion mobility system will allow for separating the different charges states and generating individual spectra <NUM>, <NUM> and <NUM>, where each contains only one of the three original isotopic distributions, which would otherwise appear in the same m/z spectrum and would have been indistinguishable. Similarly, <FIG> shows the separation of three different ion groups, for example the different charge state fragment ions presented in <FIG>, and sequential transfer of the separated groups to a mass analyser for producing fractional m/z spectra with reduced (i.e. less 'cluttered' and more clear) information following trapping and sequential release.

It is emphasized that the mass analyser can also be positioned beyond the drift cell and linear ion trap in the forward direction (i.e. left-hand side of <FIG>), in which case, ions will be released in the forward direction and there will be no need to reverse the DC gradient or even trap the mobility separated fractions in different regions of the drift cell prior to mass analysis. A more detailed description of this preferred embodiment is provided with reference to <FIG>.

<FIG> shows a linear ion mobility drift cell <NUM> with a wire-free injection gate <NUM> incorporated at the front end of the system and an additional Bradbury-Nielsen (BN) gate <NUM> installed at the back end of the cell for selecting a narrow mobility range prior to trapping or prior to mass measurement in the mass analyser. The ion mobility drift cell <NUM> is enclosed in a pressurized chamber <NUM>. Preferentially, the ion mobility drift cell <NUM> is differentially pumped by a mechanical vacuum pump and actively maintained at ><NUM> mbar pressure, e.g. by admitting the buffer gas through a leak valve, for example. The buffer gas or drift gas where ions are pulled through by the application of an external electrical field is a gas in a state of equilibrium.

Two guarding hexapoles (e.g. RF hexapole ion guides) <NUM> and <NUM> are disposed on either side of the ion mobility drift cell <NUM> and operated at a pressure of e.g. <<NUM> mbar, or preferably within a pressure range of ><NUM>-<NUM> mbar and <<NUM> mbar. The entrance RF hexapole <NUM> is used for receiving the ions from an ion source or an ion trap, (e.g. preferably an omnitrap ion trap platform, for example such as is described in <CIT>) and storing ions at a first potential energy level <NUM>. Subsequently ion potential energy is lifted or raised to a second potential energy <NUM> level by fast switching of all DC voltages applied to the segments simultaneously. Ions are gently transferred (or gated) <NUM> through a differential aperture <NUM> into the ion mobility drift cell <NUM> and transferred to (e.g. and preferably re-trapped at) the injection gate region <NUM> (e.g. a wire-free injection gate, for example), where an additional voltage pulse, or a switched DC level <NUM> is applied to release ions and initiate the ion mobility separation step. Separated ions can be trapped in trapping regions as described in <FIG>, or only a narrow mobility range can be transmitted beyond this point, either stored inside the pressurized cell, or transferred to the exit hexapole.

In accordance with the embodiments of the invention, ions are stored inside the ion mobility drift cell <NUM> by switching at least two of the superimposed DC signals in a synchronized manner to select ions with a certain mobility range, or by using the BN gate <NUM> to select and transfer ions to the exit RF hexapole or to yet another trapping region within the mobility cell disposed further downstream from the BN gate. At least a portion of the mobility separated ions, e.g. whether trapped or transmitted through the ion mobility drift cell <NUM> for example, is subsequently transferred to a mass analyser for mass analysis or back to the RF linear ion trap for further processing.

Ions introduced at the exit RF hexapole can be stored and their potential energy can be raised at an appropriate level <NUM> to facilitate transferring back to the linear ion trap or to a mass analyser. Both the linear ion trap and the mass analyser can be disposed either upstream or downstream from the ion mobility cell. Alternatively, the RF hexapole ion guide can be used as a simple transmission device.

The switching and synchronization of all electrical potentials is performed by the control system, which includes the system of electrodes arranged to guide, separate and/or trap the ions (i.e. to control them) in the mass spectrometer and also the DC-RF signals applied to the electrodes of the mass spectrometer.

<FIG> shows the ion mobility drift cell <NUM> where the positions of the trapping regions inside the drift cell are highlighted <NUM>, <NUM> and <NUM>. In this preferred design the length of each of the trapping regions is optimized with the third trapping region designed with extended length to accommodate the faster mobility ions. The length of the trapping regions is optimized to disperse the information of a single m/z spectrum containing all fragment ions to multiple spectra, each of similar spectral density. Alternatively, a BN gate and preferably at least one trapping region utilizing at least one DC signal superimposed on the drift electric field E of the ion mobility drift cell is used to select a narrow mobility range, which is subsequently transmitted to the mass analyser producing a single m/z spectrum with reduced complexity. The signals applied to drive the BN gate and/or the trapping regions within the drift cell are preferably scanned to cover the entire range of ion mobilities of interest producing sequential m/z spectra of reduced complexity.

In yet another mode of operation, and preferably in cases where very dense and highly complex fragmentation patterns are to be produced in a single cycle of a typical experiment, the ion mobility spectrometer disclosed herein is designed with extended length or produced in a cyclic or folded configuration to enhance mobility separation of the ion population. Separated fragment ion populations can be subsequently transmitted to the mass analyser or stored in multiple trapping regions and finally separated fractions can be injected in a step wise manner without losses to the mass analyser or the linear ion trap for further processing.

<FIG> shows an example of a separation of a fragment ion population in two-dimensions (m/z and drift time) <NUM>, highlighting the dispersion patterns for different charges states, n+ <NUM>, (n+<NUM>)+ <NUM> and (n+<NUM>)+ <NUM> where n=<NUM>,<NUM>,<NUM> and the overlap typically observed in the m/z scale <NUM>. In this example, injection of ions in the drift cell disclosed here allows for separating the ion population in three fractions <NUM>, and subsequently detecting these fractions independently. Spectral congestion is therefore significantly reduced. Consequently, the efficiency of automated peak processing is increased by reducing the number of false positive identifications while the confidence of the true positive fragments identified by algorithms developed for protein sequencing is considerably improved, especially de-novo sequencing algorithms. In <FIG>, fractions A, B and C are separated in the ion mobility cell and released independently towards the mass analyser. Consequently, the detection of the entire population is reduced by a factor of 3x.

In yet another preferred embodiment, a BN gate is used in transmission mode to select at least one desired fraction of the initial fragment ion population in order to resolve overlapping isotopic distributions of fragment ions. In this approach trapping is not performed. Desirably, the mass analyser is disposed downstream from the drift cell, although an upstream arrangement is also envisaged with the DC gradient applied across the drift cell reversed immediately after the selection step performed with the BN gate or with the fast switching DC signals superimposed on the electric field E of the drift cell.

<FIG> shows a preferred embodiment of the present invention comprised of a RF linear ion trap <NUM> with hyperbolic electrodes <NUM> and further segmented in the axial direction for fragmenting ions in different regions designed to apply different methods of ion activation-dissociation, an ion mobility drift cell <NUM> for separating or selectively transmitting fragment ions according to ion mobility and a TOF mass analyser <NUM> disposed further downstream for producing m/z spectra.

The different regions where ions can be trapped are also highlighted using the axial DC profile and corresponding transitions <NUM> established across the different ion optical components (trapping regions within the drift cell are not shown). In this preferred configuration field reversal of the drift electric field E is exercised for redirecting mobility selected ions back to the RF linear ion trap <NUM> for further processing.

In another mode of operation, rich populations of fragment ions produced in any of the different trapping regions of the ion trap <NUM> are injected in the drift cell <NUM>, separated and sampled continuously by an orthogonal pulser <NUM> of an orthogonal TOF analyser <NUM> with the extraction voltage pulse applied at the highest possible repetition rate to produce a high quality ion mobility spectrum. No trapping in the ion mobility cell is exercised in this mode of operation. In yet another mode of operation, mobility separated ions are stored in the consecutive trapping regions of the drift cell <NUM>, similarly to the examples disclosed with reference to <FIG> and <FIG>, and ejected separately and in a sequential manner to the TOF analyser (<NUM>) where the arrival time of the mobility separated fraction in the effective region of the orthogonal pulser of the TOF analyser <NUM> is synchronized with the TOF extraction voltage pulse. Sequential storage of the different fractions of the fragment ion population at the RF hexapole trap <NUM> following the separation process in the drift cell <NUM> may also be desirable to enhance the duty cycle of the experiment. The RF hexapole trap or ion guide <NUM> at the exit of the ion mobility drift cell <NUM> is also operated in the pressure range of ><NUM>-<NUM>mbar and <<NUM> mbar.

The control system of the mass spectrometer shown in <FIG> also comprises a set of electronics units (not shown) for generating RF, static and switched DC electrical potentials that are distributed and applied to the electrodes of the mass spectrometer apparatus in a synchronized manner. The control system is configured to drive the electrodes by applying one or more switched DC signals and/or one or more RF signals. The switched DC signals each have a DC level that is switchable between multiple separate and different DC levels. The electronics of the electronics units is configured for generating RF electrical potentials, and/or static DC electrical potentials (i.e. non-switched) and/or switched DC electrical potentials as desired. The control system is arranged to distribute the generated one or more electrical potentials to the system of electrodes of the mass spectrometer, and is configured to apply the distributed electrical potentials to electrodes of the mass spectrometer apparatus as indicated schematically in <FIG>. This distribution is done in a synchronized manner whereby the application of the distributed electrical potentials to electrodes of the mass spectrometer is achieved by switching different electrodes of the system of electrodes in synchrony, so as to permit the control (e.g. trapping and/or transfer) of the ions through the system using the electrical potentials imposed on the ions by the driven electrodes.

In yet another mode of operation, a narrow range in m/z can be isolated in the RF linear ion trap first and subsequently injected and separated in the ion mobility drift cell. This method allows for the simplification of the m/z spectrum prior to ion mobility separation. Concentrating in a highly congested region of the m/z spectrum only by injecting a narrow m/z band will enhance spectral interpretation further. Injection of selected mass ranges in the ion mobility spectrometer may include one or more fractions of the complete m/z spectrum.

Overall, separation in ion mobility space and detection of separated fractions will not only resolve overlapping isotopic distributions of different charge state, but it will also reduce chemical noise which interferes with sequence specific fragments that must be identified and assigned with high confidence. These hardware advancements are inherently connected with methods and algorithms developed to process such complex data sets in an automated manner. The identification of monoisotopic peaks in higher charge state fragments becomes particularly problematic, if not impossible, in congested m/z spectra. Fitting entire isotopic distributions is therefore highly advantageous, eliminating the need for performing m/z spectra deconvolution and avoiding substantial errors associated with such an approach.

The hardware solutions disclosed herein for reducing spectral complexity is are also associated with advances in fragment ion matching algorithms. In particular, a new method is disclosed for peak assignment where theoretical isotopic distributions from a known peptide or protein sequence are calculated and superimposed to experimental m/z spectra. A score function is created by subtracting the theoretical from the experimental m/z spectrum. Preferably, an interpolation algorithm is applied to the theoretical isotopic distributions to match the m/z step values of the experimental spectrum. Least square differences of the matched theoretical and experimental m/z spectra are subsequently calculated (score values or calculated quantities are raised in the power of <NUM> prior to subtraction of the two spectra). Minimization of this quantity, the value of the score function, will allow excluding false positives more accurately whilst enhancing the number of true positive identifications. This method involves all isotopic peaks present in the mass spectrum and does not require deconvolution typically performed in existing peak assignment algorithms. The algorithm is preferably applied in a sequential manner across the m/z spectrum, that is, fitting of the theoretical isotopic distributions is performed for specific or selected windows in the m/z scale to accelerate processing, preferably starting from the lowest m/. z values of interest. Entire spectra can be processed, starting from the low end to the high end in the m/z scale.

<FIG> shows a flow chart diagram highlighting the key elements of the software algorithm disclosed herein for annotating fragment ion m/z spectra. A known protein sequence <NUM> is imported into a fragment calculator <NUM> producing a list of fragment ions, for example a list containing primary fragments, internal fragments and side chain losses, also providing the chemical formula for each of the fragments that is used to produce isotopic distributions using an isotopic distribution calculator <NUM>. A theoretical spectrum can therefore be constructed <NUM> using the theoretical isotopic distribution calculator <NUM> which is subsequently matched to the experimental spectrum <NUM> using the methods described above.

De-novo sequencing algorithms can also be integrated with this method. Different sections of an unknown peptide of protein sequence can be ascertained by matching the m/z difference of isotopic distributions to particular amino acids (AA). Suggested AA sequences can then be aligned and subjected to the post-processing method disclosed with reference to <FIG>. The same approach can be extended to lipids, DNA-RNA molecular ions and other biologically relevant species.

Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein without departing from the extent of the invention determined by the appended claims. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well, without departing from the extent of the invention determined by the appended claims. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

The foregoing description of preferred embodiments and best mode of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. It is not intended to be exhaustive or limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application and to enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

Claim 1:
A mass spectrometer for analysing fragment ions, the mass spectrometer comprising:
an ion source for producing fragment ions, wherein the ion source for producing fragment ions comprises an RF ion trap (<NUM>, <NUM>);
an RF multipole ion guide (<NUM>, <NUM>, <NUM>, <NUM>);
an ion mobility spectrometer (<NUM>, <NUM>, <NUM>) for receiving fragment ions produced by the ion source and for separating at least a fraction of the received fragment ions according to their ion mobility into mobility-separated fragment ions; and
a system of electrodes (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) configured to receive DC electrical potentials to transfer at least a fraction of the mobility-separated fragment ions to a mass analyser (<NUM>, <NUM>) for generating a mass-to-charge (m/z) spectrum exhibiting reduced spectral complexity;
wherein the mass spectrometer is arranged to eject ions from the RF ion trap (<NUM>, <NUM>), to re-trap the ejected ions within the RF multipole ion guide (<NUM>, <NUM>, <NUM>, <NUM>), and subsequently to gate the re-trapped ions into the ion mobility spectrometer (<NUM>, <NUM>, <NUM>);
characterised by:
wherein the system of electrodes (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) is configured to receive DC electrical potentials so as to raise the potential energy level of the ions stored in the RF multipole ion guide (<NUM>, <NUM>, <NUM>, <NUM>) and simultaneously to raise the DC voltage gradient established across the ion mobility spectrometer (<NUM>, <NUM>, <NUM>) that induces separation.