Patent Description:
A current focus of biological mass spectrometry is the identification, quantification, and structural elucidation of peptides, proteins, and related molecules. In the context of bottom-up proteomics experiments, proteins are subject to proteolytic digestion to break down into fragments of peptides which are then separated, usually with liquid chromatography (LC), before being introduced into an ion source of a mass spectrometer. Typically, the ion source for proteomics experiments implements electrospray ionization (ESI) to ionize the peptide. ESI of peptide-containing samples will typically produce both singly and multiply charged ions. That is, the ions include singly charged ions (e.g., +<NUM>), and multiply charged ions including doubly charged ions (e.g., +<NUM>), triply-charged ions (e.g., +<NUM>), and so forth of the peptide. Often, singly-charged ions will comprise a substantial portion or even a preponderance of the total.

Singly charged ions are generally of less interest in bottom-up proteomics experimentation as the singly charged ions are often the result of sample preparation, contamination, or other scenarios. The interesting information regarding the biologically significant peptides is obtained from analysis of the multiply charged ions.

In certain pulsed mass analyzers, such as an orbital electrostatic trap mass analyzer (commercially available from Thermo Fisher Scientific under the trademark "Orbitrap"), mass analysis is performed by storing ions produced by ESI in a storage trap and then transferred into the orbital electrostatic trap for mass analysis. The number of ions stored within the storage trap (or more specifically, the aggregate number of charges) is limited to a target that is determined in part by the storage trap geometry and dimensions. When the target number has been attained, the storage trap is switched to a closed state in which no additional ions are permitted to enter the storage trap. Because a significant portion of the total number of ions stored in the trap for subsequent mass analysis is represented by singly-charged ions, the number of (more analytically significant) multiply charged ions available for analysis is reduced, leading to decreased sensitivity. Furthermore, because the storage trap reaches capacity relatively quickly due to the presence of large numbers of singly-charged ions, much of the ions produced by the ion source are wasted. This reduces the duty cycle (i.e., the fraction of ions of interest produced by the ion source that are mass analyzed) of the mass spectrometer. <CIT> discloses systems and methods for integrating ion mobility and ion trap mass spectrometers. In certain examples, systems and methods for decoding double multiplexed data are described. The systems and methods can also perform multiple refining procedures in order to minimize the demultiplexing artifacts. The systems and methods can be used, for example, for the analysis of proteomic and petroleum samples, where the integration of IMS and high mass resolution are used for accurate assignment of molecular formulae. <CIT> discloses 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. In a mode of operation ions from the second ion trap or ion guide are passed from the second ion trap or ion guide back upstream to the ion mobility spectrometer or separator. <CIT> discloses an ion analysis apparatus for conducting differential ion mobility analysis and mass analysis. In embodiments, the apparatus comprises a differential ion mobility device in a vacuum enclosure of a mass spectrometer, located prior to the mass analyser, wherein the pumping system of the apparatus is configure to provide an operating pressure of <NUM>. 005kPa to 40kPa for the differential ion mobility device, and wherein the apparatus includes a digital asymmetric waveform generator that provides a waveform of frequency of 5OkHz to <NUM>. Examples demonstrate excellent resolving power and ion transmission. The ion mobility device can be a multipole, for example a <NUM>-pole and radial ion focusing can be achieved by applying a quadrupole field to the device in addition to a dipole field.

In a first aspect the present invention relates to an apparatus for analyzing a peptide-containing biological sample including: a chromatography device configured to temporally separate components of the biological sample; an electrospray ionization (ESI) source configured to receive a component separated from the biological sample and generate singly-charged ions and multiply-charged ions from the component; a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device configured to receive the singly-charged ions and the multiply-charged ions, and preferentially transmit multiply-charged ions; an ion accumulator arranged to receive and confine the ions transmitted by the FAIMS device; a storage trap configured to receive the ions released from the ion accumulator and store the released ions, the storage trap having a lower storage capacity than the ion accumulator; a mass analyzer configured to receive the ions stored in the storage trap for mass analysis; and a controller circuit configured to adjust operation of the accumulator to allow release a portion of the ions confined therein to the storage trap.

In some implementations, the storage trap is a curved linear ion trap, and the mass analyzer is an orbital electrostatic trap mass analyzer.

In some implementations, the ion accumulator is an ion funnel.

The mass spectrometer of the present invention is defined in claim <NUM>. The mass spectrometer includes: an ion source configured to receive a sample and generate singly-charged ions and multiply-charged ions from the sample; an ion-mobility spectrometer (IMS) configured to receive the singly-charged ions and the multiply-charged ions, and configured to allow transmission of more multiply-charged ions through an output of the IMS than transmission of the singly-charged ions through the output of the IMS; an ion accumulator configured to store the multiply-charged ions that drift through the output of the IMS; a storage trap configured to receive a portion of the multiply-charged ions stored by the ion accumulator; a mass analyzer configured to receive the portion of multiply-charged ions stored in the storage trap for mass analysis; and a controller circuit configured to determine an operational state of the mass analyzer and adjust operation of the ion accumulator to allow the portion of the multiply-charged ions to transmit from the ion storage to the storage trap.

In some implementations, the IMS is a field asymmetric-waveform ion-mobility spectrometer (FAIMS), and the transmission of the multiply-charged ions through the output is based on an application of a range of compensation voltages (CVs) applied to an electrode of the FAIMS that causes the multiply-charged ions to drift through to the output without impacting an electrode of the FAIMS and causes the singly-charged ions to impact an electrode of the FAIMS without drifting through the output.

In some implementations, the operational state of the mass analyzer is one of: currently performing mass analysis, or available to perform mass analysis, and wherein the operation of the ion funnel is adjusted to store the multiply-charged ions without transmitting the multiply-charged ions from the ion funnel to the ion trap when the operational state of the mass analyzer is currently performing mass analysis, and the operation of the ion funnel is adjusted to store the multiply-charged ions while allowing transmitting of the multiply-charged ions from the ion funnel to the ion trap when the operational state of the mass analyzer is available to perform mass analysis.

In some implementations, the controller circuit is configured to allow transmission of the portion of the multiply-charged ions stored in the ion accumulator to the storage trap based on a determination of the operational state of the mass analyzer indicating that the mass analyzer is available to perform mass analysis.

In some implementations, the mass spectrometer includes a separation device configured to separate the sample from a mixture, wherein the controller circuit is further configured to determine information related to how the sample is separated from the mixture, and wherein the controller is configured to adjust operational parameters of the IMS based on the determination of the information related to how the sample is separated from the mixture.

In some implementations, the IMS is a field asymmetric-waveform ion-mobility spectrometer (FAIMS), and the operational parameters are compensation voltages (CVs) applied to an electrode of the FAIMS.

In some implementations, the mass spectrometer includes a chromatography system configured to separate the sample from a mixture, wherein the controller circuit is further configured to determine a retention time of the sample, and wherein the controller is configured to adjust operational parameters of the IMS based on the determination of the retention time of the sample.

In some implementations, the chromatography system is a liquid chromatography (LC) system.

Another aspect of the present invention relates to a method of operating a mass spectrometer to analyze a biological sample. The method of the present invention is defined in claim <NUM>. The method includes: ionizing a sample to generate singly-charged ions and multiply-charged ions from the biological sample; transmitting more of the multiply-charged ions than the singly-charged ions; storing the multiply-charged ions in an ion accumulator, the ion accumulator storing more multiply-charged ions than singly-charged ions; determining that a mass analyzer is available to perform mass analysis; transmitting a portion of the multiply-charged ions from the ion accumulator to a storage trap based on the determination that the mass analyzer is available to perform mass analysis; injecting the portion of the multiply-charged ions from the storage trap to the mass analyzer; and performing a mass analysis of the portion of the multiply-charged ions.

In some implementations, transmitting more of the multiply-charged ions than the singly-charged ions includes: receiving, with a field asymmetric-waveform ion-mobility spectrometer (FAIMS), the singly-charged ions and the multiply-charged ions; and applying a range of compensation voltages (CVs) to an electrode of the FAIMS to cause the multiply-charged peptide ions to drift through to the output without impacting an electrode of the FAIMS and causes the singly-charged peptide ions to impact an electrode of the FAIMS without drifting through the output.

In some implementations, the biological sample is a mixture of peptides.

In some implementations, the mass analyzer is an orbital electrostatic trap mass analyzer.

In some implementations, the storage trap is a curved linear ion trap.

Some of the material described in this disclosure includes mass spectrometers and techniques for using ion mobility separation to improve the duty cycle of a mass spectrometer. As used herein, the term "ion mobility separation" and its variants include any device or technique in which ions are separated or filtered on the basis of their mobility properties, and is intended to embrace both conventional ion mobility separation devices such as a drift tube in which ions travel through a drift gas at a rate determined by their mobilities, as well as differential mobility devices (such as the FAIMS device described below), in which ions are separated or filtered in accordance with their ratios of high field to low field mobilities.

In one example, a mixture including peptides is introduced into a chromatography system such that different peptides in the mixture are separated and introduced into a mass spectrometer for analysis at different times. As a peptide is introduced into the mass spectrometer, the peptide and other co-eluting substances are ionized using electrospray ionization (ESI) to produce ions that are transported among the components of the mass spectrometer for mass analysis. Unfortunately, many of the ions produced using ESI with peptide-containing samples are singly charged ions that are less useful for proteomics experimentation than multiply charged ions.

As described later in this disclosure, ion mobility separation can be performed following the production of the ions by the ion source, but before storage of the ions in the storage trap. The ion mobility separation prevents or substantially reduces the transmission of the singly charged ions while allowing transmission of the multiply charged ions, resulting in the ions used for mass analysis to include more of the ions that are of analytical interest. For example, the ion mobility separation can be performed by a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device using a compensation voltage (CV) range that only or mostly allows the multiply charged ions to transmit through. By preventing, or reducing, the transmission of the singly charged ions to the storage trap, more of the ions accumulated in the storage trap are multiply charged ions. This improves the quality of the data acquired via the mass analysis. Moreover, this also improves the duty cycle of the mass spectrometer.

Additionally, ions can also be stored in an ion accumulator (storage, e.g., an ion funnel) disposed between the ion mobility separation device and the storage trap. That is, while the storage trap is closed, ions can be trapped and stored in the accumulator. When the storage trap is opened again, the ion accumulator allows a portion of the stored ions to transmit to storage within the storage trap. When the storage trap reaches capacity, the storage trap is closed again, the stored ions are transmitted from the storage trap to the orbital electrostatic trap, and the accumulator is closed to prevent transmission of ions, resulting in the ions being stored within the accumulator. This results in more of the ions available for mass analysis, which also improves the duty cycle and the quality of the data acquired.

Also described later in this disclosure is synchronizing the operation of the FAIMS device, another ion mobility spectrometry (IMS) device, and an orbital electrostatic trap. When the orbital electrostatic trap begins performing a mass analysis, this can trigger adjusting the CV of the FAIMS device and allow the IMS device to begin filtering ions.

Also described later in this disclosure, information regarding how the peptide is separated in a mixture using the LC system can also be determined and provided to the mass spectrometer. This information can be used to modify the CV of the FAIMS device, further improving the transmission of multiply charged ions.

In more detail, <FIG> illustrates an example of a mass spectrometer using ion mobility separation to increase the abundance of multiply charged ions used in mass analysis. <FIG> illustrates an example of a block diagram for operating the mass spectrometer of <FIG>. In the block diagram of <FIG>, a peptide is provided to a mass spectrometer (<NUM>). For example, in <FIG>, peptide <NUM> is a peptide separated from other peptides (and other components) in a mixture using liquid chromatography (LC), gas chromatography (GC), capillary electrophoresis (CE), or other type of system used to separate components of a mixture. In the example of proteins subject to digestion, the separate components of the mixture are peptides (e.g., fragments of the protein).

Returning to <FIG>, the peptide is then ionized to form ions (<NUM>). In <FIG>, this is depicted as peptide <NUM> being introduced into ion source <NUM> of mass spectrometer <NUM>. Ion source <NUM> ionizes a material under analysis (i.e., peptide <NUM>) by removing or adding charge-carrying entities (e.g., hydrogen nuclei or electrons) to or from the material to provide the material with a positive or negative charge. This results in ions <NUM> forming from the ionization of peptide <NUM>. Ion source <NUM> is usually of the ESI type, but may instead utilize any other suitable ionization technique, including atmospheric-pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI). As described above, ionization of the peptide-containing sample will typically produce both singly charged ions (which will include the +<NUM> state of the peptide as well as interfering species such as solvent clusters) and multiply charged ions.

In the block diagram of <FIG>, the ions produced by the ion source are then introduced into an ion mobility separation device (<NUM>) and the multiply charged ions are preferentially transmitted relative to the singly charged. For example, in <FIG>, ion mobility separation device <NUM> separates ions based on their mobility properties in the presence of a buffer gas and exposure to an electric field. That is, rather than separating ions based on a mass-to-charge ratio, ion mobility separation device <NUM> separates ions by their mobility properties (e.g., their mobilities in a fixed field, or the ratio of their high field to low field mobilities. In <FIG>, this is implemented using a high-field asymmetric waveform ion mobility spectrometry (FAIMS) device that is used as a filter.

A FAIMS device is depicted in a simplified example in <FIG> as having two parallel plates with electrode <NUM> and electrode <NUM>, but some implementations include different geometries such as electrodes <NUM> and <NUM> as cylindrical electrodes with one disposed or positioned within the other electrode. Electrode <NUM> can be grounded (e.g., at <NUM> V) while a high-voltage asymmetric radio frequency (RF) signal is applied to electrode <NUM>, or vice versa. The signal applied to electrode <NUM> is composed of two sine waves with different phases (e.g., one ninety degrees out-of-phase from the other) and different amplitudes such that they define a first portion that has a higher positive amplitude than a negative amplitude of a second portion (e.g., the first portion might range from <NUM> volts (V) to X V whereas the second portion might range from <NUM> V to -<NUM>. 5X V), but the first portion is asserted for a shorter time period than the second portion (e.g., the first portion might be asserted for t microseconds (µs) and the second portion might be asserted for 2t µs). This results in the ions introduced into and transmitting within ion mobility separation device <NUM> to be subjected to an electric field that is higher-strength in one direction for a shorter period of time, but then switched to an electric field that is lower-strength in a second another direction for a longer period of time. Based on the differential mobilities of the ions in the different higher-strength and lower-strength electric fields, ions will generally drift towards one of the electrodes as they pass through ion mobility separation device <NUM>. In other types of IMS, mobility separates ions (due to the electric field not changing), whereas in FAIMS, the ions separate due to differences in mobility caused by the changing electric field. For example, during the lower-strength field, ions can drift similar to other types of IMS, but in the higher-strength electric field, ions drift due to a differential mobility that adds up via the periodicity of the RF signal. Thus, in IMS devices (including FAIMS), the mobility properties or characteristics causes ions to be separated or filtered.

To account for the drift and allow selected ions to be able to transmit through without hitting one of the electrodes, a DC compensation voltage (CV) is applied to electrode <NUM>. The application of the CV counteracts the ion drift arising from the oscillatory field such that ions generally track path <NUM> and exit the ion separation device <NUM>. If an appropriate CV is applied to electrode <NUM>, then one type of ion might drift to and from path <NUM> but be able to transmit through ion mobility separation device <NUM>. By contrast, if the CV applied does not correct for enough of the drift of another ion, then that ion might drift to and from path <NUM>, but overall drift closer to one of the electrodes and eventually impact an electrode, thus resulting in that ion not transmitting through ion mobility separation device <NUM>. By scanning through multiple CV values (i.e., applying CVs within a range of CVs), ions can be filtered through ion mobility separation device <NUM> in accordance with their relative mobilities. If the CV range does not include a CV for an ion with a particular relative mobility to transmit through, then ion mobility separation device <NUM> effectively acts as a filter.

As previously discussed, ion source <NUM> might implement ESI which forms singly charged ions and multiply charged ions. The singly charged ions are of less analytical interest in comparison to the multiply charged ions. Filtering out the singly charged ions using ion mobility separation device <NUM> can allow for more multiply charged ions to be mass analyzed. Thus, in <FIG>, to enable transmission of the multiply charged ions with reduced transmission of the singly charged ions can be performed by using a CV range that results in fewer, or even none, of the singly charged ions to transmit through while the multiply charged ions transmit through using the CV range. This results in more of the multiply charged ions to transmit through than the amount of singly charged ions that transmit through.

In some implementations, a conventional ion mobility separation device (e.g., ones using drift tubes) can employ gating mechanisms to separate, and even filter out singly charged ions from the multiply charged ions.

<FIG> illustrates an example of compensation voltages (CVs) used for ion mobility separation of singly charged ions and multiply charged ions. In <FIG>, peak <NUM> represents the transmission of singly charged ions and peaks <NUM> represent the transmission of multiply charged ions. CV range <NUM>, for example, can range from -<NUM> V to -<NUM> V while CV range <NUM> can range from -<NUM> V to -<NUM> V. As depicted in <FIG>, peaks <NUM> are clustered and overlapping in ion transmission at CV voltages within CV range <NUM>. However, the ion transmission for peak <NUM> is not within CV range <NUM> and, therefore, singly charged ions would not transmit through ion mobility separation device <NUM> if CV voltages within CV range <NUM> are applied to electrode <NUM> and CV voltages within CV range <NUM> are not applied to electrode <NUM>. In one implementation, the CV voltages within CV range <NUM> might step from -<NUM> V to -<NUM> V to -<NUM> V. Alternating or incrementing the CV voltages among these three voltages, and repeating the sequence of CV voltages, allows for multiply charged ions of peptides to transmit while the singly charged ions do not transmit. However, <FIG> is an idealized example, and in other scenarios some singly charged ions can transmit, and the peaks of <FIG> can overlap including peak <NUM> overlapping with one or more peaks representing the multiply-charged ions.

As a result, all or a substantial portion of the singly charged ions (represented by the larger circles in ions <NUM> in <FIG>) are not transmitted through ion mobility separation device <NUM> whereas all or a substantial portion of multiply charged ions (represented by the smaller circles in ions <NUM>) are transmitted through. That is, more multiply-charged ions are transmitted through in comparison with singly charged ions. The transmitted multiply charged ions are therefore collected into an ion trap for storage (<NUM> in <FIG>). This is depicted in <FIG> as ions being stored in ion trap <NUM>, which can be implemented with storage trap <NUM>. In the implementation of <FIG>, ions are introduced into the storage trap by passing through split lens <NUM>, which governs the introduction of transmitted ions into storage trap <NUM> based on a voltage applied. That is, split lens <NUM> provides ion gating to allow or disallow the transmission of ions into storage trap <NUM>.

Storage trap <NUM> may be a curved linear ion trap that stores a population of ions corresponding to a maximum aggregate number of charges. When storage trap <NUM> is filled with the appropriate number of charges (e.g., as determined by the rate of ions transmitting from ion mobility separation device <NUM>), the operation of split lens <NUM> can be adjusted (e.g., by changing a voltage applied to it) such that ions are now no longer allowed to be transmitted into storage trap <NUM>.

Next, in <FIG>, the ions stored in the ion trap are transmitted to a mass analyzer for mass analysis (<NUM>). For example, in <FIG>, the multiply charged ions in storage trap <NUM> are transmitted from storage trap <NUM> to orbital electrostatic trap <NUM>, which is an orbital electrostatic trap, as previously discussed. Storage trap <NUM> may be defined by a curved central axis between electrodes (of which RF signals are applied) with a slot in the electrode closest to orbital electrostatic trap <NUM>. This type of storage trap can also be referred to as a C-trap. When the multiply charged ions are accumulated within storage trap <NUM>, the ions are "cooled" down via collisional cooling of ions with a gas such as nitrogen. Upon sufficient collisional cooling, the RF signal is quickly ramped down, resulting in the multiply charged ions being no longer confined by the RF field of storage trap <NUM>. With the application of appropriate DC potentials applied to the storage trap electrodes and lenses arranged between storage trap <NUM> and orbital electrostatic trap <NUM>, the multiply charged ions are then quickly moved towards the slot in the electrode closest to orbital electrostatic trap <NUM> and enter an aperture on an outer electrode of orbital electrostatic trap <NUM>. Thus, the multiply charged ions are injected into orbital electrostatic trap <NUM> in a relatively quick time.

In <FIG>, the ions injected into the mass analyzer are then analyzed to acquire a mass spectrum (<NUM>). In <FIG>, orbital electrostatic trap <NUM> includes an internal central electrode with a spindle-like shape and outer electrodes enclosing the internal central electrode. Ions stored in storage trap <NUM> are injected into orbital electrostatic trap140 such that the ions are in orbit around the central electrode and oscillate back-and-forth along the central electrode. The frequency of the longitudinal oscillatory motion of an ion species is a function of its mass-to-charge ratio and, therefore, an image current can be detected using the outer electrodes to determine the mass-to-charge ratios of the ions within orbital electrostatic trap <NUM> (e.g., using digital signal processing techniques such as Fourier transforms), resulting in controller <NUM> generating data that can be used to generate mass spectrum <NUM>, which depicts the mass-to-charge ratios and abundances of ions at those ratios.

Because all or a substantial portion of the singly charged ions are filtered out by ion mobility separation device <NUM>, relatively more multiply charged ions are stored within storage trap <NUM> and injected into orbital electrostatic trap <NUM>, resulting in mass spectrum <NUM> including information related primarily to multiply charged ions rather than singly charged ions.

In the aforementioned example, storage trap <NUM> has a relatively small storage capacity (i.e., the number of ions that can be trapped therein without incurring space charge effects, which is a function of the trap dimensions and geometry) and can fill to a threshold level or capacity relatively quickly. For example, if both singly charged ions and multiply charged ions are allowed to enter storage trap <NUM> (i.e., ion mobility separation device <NUM> is not used between ion source <NUM> and ion trap <NUM>), it might quickly fill to a maximum set threshold within <NUM> millisecond (ms) due to the relatively high number of singly charged ions. However, mass analysis using orbital electrostatic trap <NUM> might be accomplished within <NUM>. Thus, for <NUM>, during an analysis cycle, ions are generated by ion source <NUM>, but the ions might not be allowed to transmit into storage trap <NUM> by split lens <NUM>. Accordingly, a significant number of ions are wasted and not used for mass analysis. In this example, a duty cycle of mass spectrometer <NUM> is <NUM> / <NUM>. By filtering out the singly charged ions using ion mobility separation device <NUM> implementing FAIMS, storage trap <NUM> might fill in <NUM> due to the lower number of multiply charged ions generated by ion source <NUM>. This increases the duty cycle of mass spectrometer <NUM> to <NUM> / <NUM>, which is a significant improvement.

Further improving the duty cycle can be accomplished by storing the multiply charged ions transmitted through ion mobility separation device <NUM> within an accumulator positioned upstream in the ion path of the storage trap. <FIG> and <FIG> illustrate an example of a block diagram for operating a mass spectrometer using an accumulator in the form of an ion funnel to increase a duty cycle for mass analysis. <FIG> illustrates an example of a mass spectrometer using an accumulator in the form of an ion funnel to increase a duty cycle for mass analysis.

In <FIG>, a peptide is separated from a mixture (<NUM>) and received by an ion source of a mass spectrometer (<NUM>) to be ionized to form ions (<NUM>). This is depicted in <FIG>, in which LC system <NUM> separates various peptides of a mixture such that they are separated in space, or position, along a flow path (e.g., within a chromatographic column) such that peptide <NUM> is introduced into ion source <NUM> at a different time than other peptides.

Returning to <FIG>, the ions formed by the ion source are then introduced into an ion mobility separation device (<NUM>). For example, in <FIG>, ion mobility separation device <NUM> can implement FAIMS to filter out the singly charged ions but allow transmission of multiply charged ions by using CVs within an appropriate CV range. In <FIG>, the multiply charged ions are then stored in an ion funnel (<NUM>). For example, in <FIG>, ion funnel <NUM> is disposed, or positioned, following ion mobility separation device <NUM> but before ion trap <NUM> (which can include storage trap <NUM> in <FIG>). Ion funnel <NUM> is a device used to focus the ions produced from ion source <NUM> and that are transmitted through ion mobility separation device <NUM> into a beam using a series of ring electrodes having progressively smaller (in the direction of axial ion motion) apertures and application of RF signals to radially confine ions for efficient transfer to other components within mass spectrometer <NUM>.

In <FIG>, the multiply charged ions are then stored in the ion funnel (<NUM>). In <FIG>, by adjusting a DC voltage applied at an electrode at the endpoint of ion funnel <NUM> (e.g., an electrode near or at the outlet of ion funnel <NUM>), ion funnel can act as a confining device to maintain the ions within a potential well and not be allowed to transmit to ion trap <NUM>. Though an ion funnel described in this example, other types of ion storage devices can be used as the ion accumulator. For example, the accumulator may take the form of a ring trap having cylindrical ring electrodes without the decreasing diameter as used in an ion funnel, a quadrupole or multipole ion guide having elongated rod electrodes, or other types of devices that can accumulate and selectively release ions. In some implementations, the accumulator may be used together with an ion funnel, which provides focusing of ions released by the accumulator.

Next, in <FIG>, the controller of the mass spectrometer determines that the mass analyzer is available for mass analysis (<NUM>). In <FIG>, controller <NUM> determines that orbital electrostatic trap <NUM> is available for mass analysis (e.g., by determining that an elapsed amount of time has passed since the beginning of mass analysis, determining that no new data is being provided via mass analysis, etc.). This results in the controller to provide signals to the various components such that ions are released from from the ion funnel (<NUM>) and that the released ions are directed into the ion trap (<NUM>). For example, the operational state of the ion funnel can be adjusted from accumulating (without releasing) to releasing (while accumulating) ions by changing a voltage at the electrode of the ion funnel. As a result, ions stored in ion funnel <NUM> are no longer maintained in an axial potential well, and can exit the device. Meanwhile, controller <NUM> also adjusts the voltage on split lens <NUM> such that the ions can transmit into storage trap <NUM> for storage. That is, the operational state of split lens <NUM> can be changed from preventing transmission to allowing transmission of ions to storage trap <NUM>. In some implementations, the ion trap can be filled with ions while the orbital electrostatic trap is performing mass analysis.

Next, in <FIG>, controller <NUM> can determine that a threshold number of ions is stored in the ion trap (<NUM>). For example, in <FIG>, ion funnel <NUM> stores a significantly larger number of charges than storage trap <NUM>, for example, 20e6 elementary charges rather than a relatively lower 2e5 elementary charges that storage trap <NUM> can store. The ions stored in ion funnel <NUM> can empty out (i.e., be released towards storage trap <NUM>) relatively fast such that ion funnel <NUM> would empty in a few hundred microseconds. Thus, by adjusting the voltage of the electrode of ion funnel <NUM> for a few microseconds (i.e., a short time period in comparison to the overall time needed to empty the entirety of ion funnel <NUM>), a fraction of the stored ions in ion funnel <NUM> are released, but this small fraction is enough to fill storage trap <NUM>. After this short time period, the voltage on the electrode of ion funnel <NUM> can be adjusted such that the ions are confined within ion funnel <NUM> again (<NUM> in <FIG>), the voltage applied to split lens <NUM> is adjusted so that new ions cannot be introduced into storage trap <NUM> (<NUM> in <FIG>), and then the ions stored in storage trap <NUM> are injected into orbital electrostatic trap <NUM> for mass analysis (<NUM> in <FIG>). Thus, a mass spectrum of the ions in the mass analyzer is acquired (<NUM>) using a portion of the ions that were stored in the ion funnel. Meanwhile, any new ions formed by ion source <NUM> are stored within ion funnel <NUM>.

Controller <NUM> then determines that mass analysis is complete and, therefore, orbital electrostatic trap <NUM> is available for mass analysis again. This results in ion funnel being adjusted to allow for ions to transmit through during a short time period again, split lens <NUM> de-gating to allow ions to enter storage trap <NUM>, and storage trap <NUM> filling to capacity. Again, the ion funnel is adjusted to store ions without allowing transmission, the split lens <NUM> prevents ions from entering storage trap <NUM>, and the ions stored within storage trap <NUM> are injected into orbital electrostatic trap <NUM> for mass analysis. However, in other implementations, storage trap <NUM> can be filled with ions while orbital electrostatic trap <NUM> is performing mass analysis. That is, both storage trap <NUM> can begin storing ions while orbital electrostatic trap <NUM> is performing a mass analysis with ions that were previously stored in storage trap <NUM>. Accordingly, the synchronization of operational states of ion funnel <NUM> (e.g., to switch between storing-only or storing-and-transmitting) and storage trap <NUM> (e.g., from accepting ions for storage to no longer accepting ions for storage and providing the ions for mass analysis) allows more of the ions being used for mass analysis and, therefore, significantly increasing the duty cycle from <NUM>/<NUM> from the scenario if singly charged ions are not filtered out using ion mobility separation device <NUM>, and also the increasing the duty cycle from <NUM>/<NUM> from the implementation discussed with respect to <FIG>.

In some implementations, information related to how a peptide is introduced into the mass spectrometer can be used with any of the examples. This information can be used to modify the range of CVs applied to an electrode of a FAIMS, further improving the transmission of multiply charged ions. For example, due to how a LC system separates peptides within a column, usually (but not always) smaller molecules elute out before larger molecules. Additionally, smaller molecules often have a higher mobility than larger molecules. Thus, the CVs applied to smaller molecules are often different than the CVs applied to larger molecules. For example, a CV of -<NUM> V might be used for a molecule with a higher mobility and a CV of -<NUM> V might be used for a molecule with a lower mobility. Accordingly, peptides that elute from a column and introduced to an ion source earlier in time during the LC process might benefit from increasing the transmission of multiply charged ions from FAIMS by applying a different CV range than peptides that elute from the column at later times.

<FIG> illustrates an example of a block diagram for adjusting CVs based on separation characteristics of a peptide. In <FIG>, information regarding separation characteristics of a peptide is received by a controller of a mass spectrometer (<NUM>). For example, in <FIG>, LC system <NUM> separates peptides in a mixture such that they are introduced into mass spectrometer <NUM> at different times, as previously discussed. Controller <NUM> can receive data from LC system <NUM> regarding the separation process, for example, the retention time of peptide <NUM> (i.e., the time at from a setpoint in which peptide <NUM> emerges from column <NUM>), which is different than the retention time of the next peptide to elute from column <NUM>. Alternatively, controller <NUM> can determine the retention time based on the peptide being introduced to ion source <NUM>.

Returning to <FIG>, the peptide can be ionized (<NUM>) and the ions introduced into a FAIMS device (<NUM>). Moreover, the compensation voltages applied to an electrode of the FAIMS device can be selected or adjusted based on the separation characteristics (<NUM>). For example, controller <NUM> can select a CV range based on the retention time of the peptide currently being ionized by ion source <NUM>. Thus, peptide <NUM> might be ionized, the ions provided to the FAIMS device implemented by ion mobility separation device <NUM>, and the CV voltages applied to electrode <NUM> (in <FIG>) might alternate range from -<NUM> V to -<NUM> V. If the retention time was higher (e.g., above a threshold value), then another range might be selected, for example, -<NUM> V to - <NUM> V because the ions formed from the ionization of a peptide eluting at a later retention time would have a higher likelihood of having lower mobilities. Returning back to the block diagram of <FIG>, the ions are then filtered in accordance with the compensation values (<NUM>).

Other separation characteristics that can be considered to adjust compensation voltages can include hydrophobicity of the peptide. In some implementations, multiple characteristics can be considered, for example, both hydrophobicity and retention time.

FAIMS can be implemented in connection with another IMS device in a a mass spectrometer. <FIG> illustrates an example of a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device used with an ion mobility spectrometer (IMS). In <FIG>, FAIMS device <NUM> is disposed before ion funnel <NUM>, similar to <FIG>. Ion funnel trap <NUM> is an additional component that can accumulate and selectively release ions received from ion funnel <NUM> to IMS device <NUM>. IMS device <NUM> is employs a different mobility property-based separation technique than FAIMS in that IMS <NUM> can be a drift-time ion mobility spectrometer (DTIMS) in which smaller ions travel faster through the drift tube than larger ions due to differences in ion mobilities. The ions are separated based on their drift time through the drift tube. Thus, by using a FAIMS device followed by a (drift-tube type) IMS device, additional separation of the ions can be achieved for better mass analysis. In some implementations, IMS <NUM> can be implemented with a Structures for Lossless Ion Manipulations (SLIM) device, as described in <NPL>) and <NPL>). By using a SLIM device, the voltages used by FAIMS <NUM> do not have to be floated upon the voltages used by IMS <NUM>. Rather, the voltages used by FAIMS <NUM> can be offset by the maximum voltage used by IMS <NUM>, providing an easy implementation of the power supplies.

As depicted in <FIG>, rear ion funnel <NUM> receives the ions transmitting through IMS <NUM> before the ions are then further transmitted to ion funnel <NUM>, which can be an ion funnel positioned at an inlet of a mass spectrometer, though ion funnel <NUM> need not be necessarily implemented. Thus, the ions are ionized, filtered using FAIMS, and then further separated using IMS device <NUM>, which can be a drift tube separating ions into groups of similar mobilities that are sequentially transmitted. Not only does this provide more separation of the ions, but performing FAIMS first can allow for solvent clusters to shed off the ions due to the movement of the ions and internal heating generated through FAIMS. By shedding off the solvent clusters using FAIMS, better separation through IMS <NUM> can be achieved. Thus, if performing top-down proteomics with proteins, better results are achieved.

Using FAIMS and IMS with a mass spectrometer with orbital electrostatic trap <NUM> involves synchronizing the various components. <FIG> illustrates an example of a block diagram for using the FAIMS device with an IMS device. In <FIG>, a mass analyzer begins a mass analysis (<NUM>). For example, ions can be injected from a storage trap into an orbital electrostatic trap for mass analysis. This is caused by changing the voltages used by the storage trap to store ions and causing injection of the ions into the orbital electrostatic trap, as previously discussed. Thus, the changing of one or more voltages at this point is indicative of the beginning of a mass analysis to be performed by the orbital electrostatic trap.

Another component (e.g., controller <NUM>, or FAIMS <NUM> or IMS <NUM>) can determine that the mass analysis has started. Thus, FAIMS <NUM> might be instructed to change the CV (<NUM>) such that different ions are stored in ion funnel <NUM>. IMS <NUM> might also be instructed to begin separating ions in accordance with their ion mobility (<NUM>). Eventually, the ions are filtered through the IMS <NUM> (<NUM>) and stored into the storage trap via the split lens. Thus, while the orbital electrostatic trap is performing mass analysis of ions that were transmitted through FAIMS <NUM> at one CV, another set of ions that were transmitted through FAIMS <NUM> at another CV are being stored in the storage trap. When the first mass analysis is complete, the ions in the storage trap can then be injected into the orbital electrostatic trap for a new mass spectrum to be acquired (<NUM>). When the new mass analysis is begun, the CV of FAIMS can be adjusted again (<NUM>).

Many of the examples describe implementations with liquid chromatography (LC) for separating peptides. However, other types of mixture separation can be used including gas chromatography (GC) or capillary electrophoresis (CE).

The examples describe techniques for peptides, however, other biomolecules can be used with the techniques described herein. For example, in addition to proteins and their peptides, other types of biomolecules that can be used with the techniques include lipids, nucleic acids, metabolites, oligosaccharides, polysaccharides, and the like. Moreover, other large molecules other than biomolecules can be used, in addition to small molecules.

The examples described herein include using an orbital electrostatic trap mass analyzer, but other mass analyzers can also be used with the techniques. For example, quadrupole or time-of-flight (TOF) analyzers might be used. In another example, a tandem mass spectrometer might be used.

<FIG> illustrates an example of an electronic device which may be used to implement some of the implementations. In some implementations, the electronic device of <FIG> can store or use a computer program product including one or more non-transitory computer-readable media having computer programs instructed stored therein, the computer program instructions being configured such that, when executed by one or more computing devices, the computer program instructions cause the one or more computing devices to perform he techniques described herein.

In <FIG>, computer system <NUM> can implement any of the methods or techniques described herein. For example, computer system <NUM> can implement controller <NUM> in <FIG>. Thus, the operation of components of the associated mass spectrometer may be adjusted in accordance with calculations or determinations made by computer system <NUM>. In various embodiments, computer system <NUM> can include a bus <NUM> or other communication mechanism for communicating information, and a processor <NUM> coupled with bus <NUM> for processing information. In various embodiments, computer system <NUM> can also include a memory <NUM>, which can be a random-access memory (RAM) or other dynamic storage device, coupled to bus <NUM>, and instructions to be executed by processor <NUM>. Memory <NUM> also can be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor <NUM>. In various embodiments, computer system <NUM> can further include a read only memory (ROM) <NUM> or other static storage device coupled to bus <NUM> for storing static information and instructions for processor <NUM>. A storage device <NUM>, such as a magnetic disk or optical disk, can be provided and coupled to bus <NUM> for storing information and instructions.

In various embodiments, computer system <NUM> can be coupled via bus <NUM> to a display <NUM>, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device <NUM>, including alphanumeric and other keys, can be coupled to bus <NUM> for communicating information and command selections to processor <NUM>. Another type of user input device is a cursor control <NUM>, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor <NUM> and for controlling cursor movement on display <NUM>. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

A computer system <NUM> can perform the techniques described herein. Consistent with certain implementations, results can be provided by computer system <NUM> in response to processor <NUM> executing one or more sequences of one or more instructions contained in memory <NUM>. Such instructions can be read into memory <NUM> from another computer-readable medium, such as storage device <NUM>. Execution of the sequences of instructions contained in memory <NUM> can cause processor <NUM> to perform the processes described herein. In various embodiments, instructions in the memory can sequence the use of various combinations of logic gates available within the processor to perform the processes describe herein. Alternatively hard-wired circuitry can be used in place of or in combination with software instructions to implement the present teachings. In various embodiments, the hard-wired circuitry can include the necessary logic gates, operated in the necessary sequence to perform the processes described herein. Thus implementations described herein are not limited to any specific combination of hardware circuitry and software.

The term "computer-readable medium" as used herein refers to any media that participates in providing instructions to processor <NUM> for execution. Such a medium can take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Examples of non-volatile media can include, but are not limited to, optical or magnetic disks, such as storage device <NUM>. Examples of volatile media can include, but are not limited to, dynamic memory, such as memory <NUM>. Examples of transmission media can include, but are not limited to, coaxial cables, copper wire, and fiber optics, including the wires that comprise bus <NUM>.

Common forms of non-transitory computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

In various embodiments, the methods of the present teachings may be implemented in a software program and applications written in conventional programming languages such as C, C++, etc..

The embodiments described herein, can be practiced with other computer system configurations including hand-held devices, microprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers and the like. The embodiments can also be practiced in distributing computing environments where tasks are performed by remote processing devices that are linked through a network.

It should also be understood that the embodiments described herein can employ various computer-implemented operations involving data stored in computer systems. These operations are those requiring physical manipulation of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. Further, the manipulations performed are often referred to in terms, such as producing, identifying, determining, or comparing.

Any of the operations that form part of the embodiments described herein are useful machine operations. The embodiments, described herein, also relate to a device or an apparatus for performing these operations. The systems and methods described herein can be specially constructed for the required purposes or it may be a general-purpose computer selectively activated or configured by a computer program stored in the computer. In particular, various general-purpose machines may be used with computer programs written in accordance with the teachings herein, or it may be more convenient to construct a more specialized apparatus to perform the required operations.

Claim 1:
A mass spectrometer (<NUM>), comprising:
an ion source (<NUM>) configured to receive a sample and generate singly-charged ions and multiply-charged ions from the sample;
a field asymmetric-waveform ion-mobility spectrometer (FAIMS) (<NUM>) configured to receive the singly-charged ions and the multiply-charged ions, and configured to allow transmission, in response to differences of ion mobility between the multiply-charged ions and the singly-charged ions in a changing electric field, of more multiply-charged ions through an output of the FAIMS (<NUM>) than transmission of the singly-charged ions through the output of the FAIMS (<NUM>);
an ion accumulator configured to store the multiply-charged ions that drift through the output of the FAIMS (<NUM>);
a storage trap (<NUM>) configured to receive a portion of the multiply-charged ions stored by the ion accumulator;
a mass analyzer configured to receive the portion of multiply-charged ions stored in the storage trap (<NUM>) for mass analysis; and
a controller circuit configured to determine an operational state of the mass analyzer and adjust operation of the ion accumulator to allow the portion of the multiply-charged ions to transmit from the ion storage to the storage trap (<NUM>).