METHOD FOR CONTROLLING THE MASS FILTER IN AN IMS / MS HYBRID SYSTEM

Abstract

A method for recording fragment mass spectra of substances in complex mixtures of substances using a mass spectrometric hybrid system comprising an ion source, an ion mobility separator, a mass filter, an ion fragmentation cell and a mass analyzer, comprising the following steps: - Temporal separation of the ions according to their mobility by the ion mobility separator, whereby the duration of the ion mobility signal differs for different types of ions; - Isolation of the temporally separated ion species of a predetermined set of ion species one after the other based on their mass using the mass filter; - Fragmentation of the isolated ion species in the ion fragmentation cell and recording of fragment mass spectra by the mass analyzer, wherein the residence time of the mass filter during isolation is not constant for at least some of the ion species of the predetermined set, but is adapted to the duration of the ion mobility signal of the respective ion species after separation in the ion mobility separator.

Description

Field of the invention

[0001] The invention relates to methods and devices for recording fragment mass spectra of selected precursor ions separated according to their ion mobility, whereby substances in complex mixtures, in particular digest peptides, are identified on the basis of the fragment mass spectra in a bottom-up proteomics process and the substances are quantified if necessary. Background of the invention

[0002] In protein research, there is growing interest in identifying as many peptides and proteins as possible in a single liquid chromatography / mass spectrometry (LC / MS) run of a proteolytic digest of proteins extracted from a biological sample. Hybrid mass spectrometry systems combining ion mobility spectrometry (IMS) and mass spectrometry (MS) have been shown to improve identification rates.

[0003] US Patent No. 6,960,761 B2 (“Instrument for separating ions in time as functions of preselected ion mobility and ion mass”; Clemmer) discloses a spectrometric IMS / MS hybrid system in Fig. and includes an ion source, a drift-type ion mobility separator, a quadrupole mass filter, an ion fragmentation cell, and an orthogonal ion injection (OTOF) time-of-flight mass analyzer. An ion trap is used to collect ions upstream of the drift-type mobility separator. The IMS / MS hybrid spectrometric system can optionally also include an upstream substance separator, such as a liquid chromatograph (LC). In the drift-type mobility separator, the ions are separated according to their ion mobility, and mass spectra of the separated ions are acquired by the downstream OTOF mass analyzer for a mass mobility map.

[0004] US Patent No. 7,838,826 B1 (“Apparatus and method for parallel flow ion mobility spectrometry combined with mass spectrometry”; Park) discloses a compact ion mobility separator called a “storage ion mobility spectrometer” (TIMS).

[0005] In a typical embodiment, a gas flow drives ions in the separation unit against an opposing DC electric field barrier, where the ions are axially stored during a collection phase and spatially separated according to their ion mobility. The ions are radially confined by a quadrupole radiofrequency field. During a scanning phase, the stored ions are subsequently eluted from the DC electric field barrier over time by reducing the magnitude of the axially directed DC electric field. The ions are released sequentially from low ion mobility to higher ion mobility.

[0006] The mobility resolution of a TIMS mobility separator increases with longer scan times or with a reduction in scan speed.

[0007] US Patent No. 9,683,964 B2 ("Trapping ion mobility spectrometer with parallel accumulation"; Park and Schubert) discloses a TIMS separator with a collection unit located upstream of the separation unit that stores ions during the scanning phase. TIMS with parallel collection offers the possibility of extending the ion collection time to detect additional detectable ion species without reducing the acquisition rate for the fragment mass spectra. The high acquisition rate for the fragment mass spectra allows for repeated measurement of rare ion species, thus improving the quality of the fragment mass spectra.

[0008] US Patent No. 9,891,194 B2 (“Acquisition of fragment mass spectra of ions separated by their mobility”; Mann et al.) discloses a multi-cycle method for acquiring fragment ion spectra of substances in complex mixtures using a mass spectrometric hybrid system, as described in Fig. The method has become commonly known as "PASEF" (Parallel Accumulation Sequential Fragmentation), particularly for a bottom-up proteomics workflow.

[0009] Fig. shows a flow diagram of the multi-cycle procedure for recording the fragment mass spectra of several ion species. Both of the Fig. The spectrometric IMS / MS hybrid systems shown can be used to perform the multicyclic procedure. Based on the fragment mass spectra, the corresponding substances, especially proteins of a proteome, can be identified and optionally quantified.

[0010] In a first measurement cycle of a first loop, the ions are separated temporally according to their mobility, and mass spectra of the separated ions are recorded by the mass analyzer for a mass mobility map. Separated ion species of interest with different mass and mobility are selected from the mass mobility map. In a second measurement cycle, new ions are separated temporally according to their mobility. The selected ion species of interest are sequentially isolated according to their mass by the mass filter and fragmented in the fragmentation cell. The fragment mass spectra are recorded with the mass analyzer. If other ion species of interest can be selected from the mass mobility map of the first measurement cycle, a new mass mobility map is not required, and fragment mass spectra of the remaining ion species of interest are recorded in one or more additional measurement cycles.Otherwise, a second loop begins with the acquisition of a new mass mobility map. Loops with new mass mobility maps can be repeated at time intervals of 0.5 to three seconds, depending, for example, on the temporal behavior of an upstream substance separation device.

[0011] The publication of patent application DE 10 2017 111 067 A1 discloses methods for analyzing ion mixtures from complex mixtures of organic substances in time-of-flight mass spectrometers equipped with a storage ion mobility spectrometer, a quadrupole mass selector, and a fragmentation cell. The ion signals from an initial mass-mobility survey, the fragment ion spectra, and the identifications of the corresponding substances are then analyzed to determine whether unresolved ion mixtures, for example, consisting of isomers or isobars, may be present. The ion signals of interest are subsequently measured using process parameters that allow the ion species to be measured separately with high mobility resolution.

[0012] Another method for analyzing ions is disclosed in publication DE 11 2015 002 566 T5. In the described method, an initial multidimensional survey scan is performed, separating ions according to a first physicochemical property and a second physicochemical property. Brief description of the invention

[0013] In a first aspect, the invention provides a method for recording fragment mass spectra of substances in complex substance mixtures using a mass spectrometric hybrid system comprising an ion source, an ion mobility separator, a mass filter, an ion fragmentation cell and a mass analyzer.The method comprises the following steps: temporal separation of the ions according to their mobility by the ion mobility separator, wherein a duration of the ion mobility signal is different for different ion species; isolation of the temporally separated ion species of a predetermined set of ion species one after the other based on mass by the mass filter; and fragmentation of the isolated ion species in the ion fragmentation cell and acquisition of fragment mass spectra by the mass analyzer, wherein the residence time of the mass filter during the isolation is not constant for at least some of the ion species of the predetermined set, but is adapted to the duration of the ion mobility signal of the respective ion species after separation in the ion mobility separator.

[0014] The duration of the ion signals can be estimated from the parameters of the ion mobility separator used in the separation or, preferably, it is determined experimentally.

[0015] In a second aspect, the invention provides a method for recording fragment mass spectra of substances in complex substance mixtures using a mass spectrometric hybrid system comprising an ion source, an ion mobility separator, a mass filter, an ion fragmentation cell, and a mass analyzer. In a first measurement cycle, ions are temporally separated according to their mobility. Furthermore, a mass-mobility map is measured by the mass analyzer, from which at least one set of time-separated virtual windows is selected. These windows comprise ion species with different ion masses and different ion mobility scan times, each with a different ion mobility signal duration. In a second measurement cycle, ions are again temporally separated according to their mobility.Each ion species from the at least one set is isolated sequentially by the mass filter, fragmented in the ion fragmentation cell, and measured as a fragment mass spectrum with the mass analyzer. This method is characterized by the fact that the length of the windows, and thus the dwell time of the mass filter, is not constant but is adapted to the duration of the respective ion mobility signal in the mass mobility map.

[0016] Preferably, at least two sets are selected, whereby the selected sets are clearly distinct from each other and do not contain the same ion species from the mass mobility map. However, weak ion signals from the mass mobility map can be present in multiple sets, whose fragment mass spectra are combined to improve their quality.

[0017] A second mass mobility map for a second measurement loop can be measured when a specified time interval has elapsed, when a specified number of fragment mass spectra have been acquired, or when all ion species of the existing mass mobility map have been fragmented and measured as fragment mass spectra. The acquisition parameters of the first and second mass mobility maps can differ, for example, with regard to the mobility range, scan duration, or collection time.

[0018] The duration of the ion signals in the mass mobility map can be estimated from the parameters of the ion mobility separator used in the separation or, preferably, it is determined experimentally from the mass mobility map of the first measurement cycle.

[0019] The methods according to the present invention preferably use a TIMS ion mobility separator, and most preferably a TIMS with parallel collection. Most preferably, the duration of the mobility scan essentially corresponds to the duration of the ion collection. The introduction and use of the TIMS ion mobility separator instead of a drift tube mobility spectrometer is not just a simple replacement: Different operating modes offer several unique advantages, some of which only become apparent upon in-depth study of the properties of the TIMS. For example, a TIMS ion mobility separator can be operated in a so-called temporal zoom mode with increased mobility resolution in at least one mobility range, as described in publication US 2012 / 0273670 A1.The increased mobility resolution is preferably achieved by reducing the rate at which the height of the DC electric field barrier is varied during a mobility scan. The mobility resolution in the limited region of interest can be higher than 100, in particular higher than 200, while the total scan time of the TIMS ion mobility separator is less than 200 ms, in particular less than 100 ms.

[0020] Optionally, the substance mixture is first separated by an upstream substance separator, e.g., a liquid chromatograph or an electrophoresis device, which is coupled to the ion source.

[0021] The mass filter is preferably an RF quadrupole mass filter. The RF quadrupole rod mass filter can be tuned to the mass of the next ion species to be measured so that the passage through the mass filter is initially closed, then tuned to the next mass, and then reopened at the correct time for the next ion species to be measured.

[0022] The fragmentation cell is preferably a flow cell, in which the ions are not stored. The ions are preferably fragmented by collision-induced dissociation (CID) in a gas-filled cell, but can also be fragmented by surface-induced dissociation (SID), electron impact (EI), electron capture dissociation (ECD), electron transfer dissociation (ETD), or photon-induced dissociation (using either infrared or ultraviolet light sources).

[0023] The mass analyzer is preferably a time-of-flight mass analyzer with orthogonal ion injection (OTOF). OTOF mass analyzers offer a high acquisition rate of mass spectra combined with high mass resolution. The acquisition rate is between 1 kHz and 20 kHz, typically around 10 kHz. High mass resolutions of more than 10,000, up to 100,000, or even more, typically around 30,000 to 50,000, can be achieved, particularly in the mass range of digest peptide ions generated with an electrospray source.

[0024] The mass filter residence time is preferably a multiple of the mass analyzer scan time and should be synchronized with the mass analyzer scans. If a time-of-flight mass spectrometer is used and operated at an acquisition rate of 10,000 mass spectra per second, the acquisition of a single (fragment) mass spectrum will take 100 microseconds. The minimum mass filter residence time should therefore be 100 microseconds, and longer residence times should each be a multiple of 100 microseconds. Furthermore, the residence times should be synchronized with the time-of-flight mass analyzer acquisition times, taking into account the delay due to the transport of ions from the ion mobility separator through the mass filter and fragmentation cell to the mass analyzer.

[0025] The temporal length of the windows and thus the residence time of the mass filter can essentially correspond to the duration of the ion mobility signal in the mass mobility map.

[0026] The time length of the windows, and thus the residence time of the mass filter, can be several times longer than the duration of the ion mobility signal in the mass mobility map, e.g., one to ten scan times, to avoid the difficult process of synchronization. This synchronization process can be difficult due to scattering during the transport of ions from the ion mobility separator to the mass analyzer. If the scan time of the mass analyzer is 100 microseconds, the selection process can add 100 microseconds (or even 200 microseconds) to the beginning and / or end of the ion signals. With 100 microseconds at the beginning and end, the minimum selection time, and thus the minimum residence time of the quadrupole mass filter, is 300 microseconds.With 200 microseconds at each end, the minimum residence time is 500 microseconds, which still allows for a high number of selectable ion species per cycle.

[0027] In a third aspect, the invention provides power supply electronics for the quadrupole mass filter, with which the residence time of the quadrupole mass filter can be controlled for each ion species and adapted to the duration of the ion mobility signal after the ion mobility separator, which is determined from a measured mass mobility map or estimated from parameters of the ion mobility separator. The power supply electronics of the quadrupole mass filter allow for rapid switching from one filter state to another as well as control of the residence time. Short description of the figures Fig. shows a schematic representation of a state-of-the-art IMS / MS hybrid system comprising an ion source, an ion trap, a drift-type mobility separator, a quadrupole mass filter, an ion fragmentation cell, and a downstream high-resolution orthogonal ion injection (OTOF) time-of-flight mass analyzer coupled to an upstream substance separator, e.g., a liquid chromatograph (LC). Fig. shows a schematic representation of a state-of-the-art IMS / MS system incorporating a parallel collection TIMS mobility separator as the ion mobility separator. Fig. shows a flow diagram of a multi-cycle procedure for the acquisition of fragment mass spectra of several ion species. Both of the Fig. The IMS / MS hybrid systems shown can be used to perform the multi-cycle procedure. Fig. shows a simulated mass mobility map, which is based on the Fig. IMS / MS hybrid system shown. Fig. shows a set of ion species (1) to (13) that are found in the Fig. shown mass mobility map, wherein the durations of the ion mobility signals of these ion species (1) to (13) are determined from the mass mobility map and used to control the residence times of the quadrupole mass filter in a subsequent measurement cycle. Fig. shows a simulated mass mobility map, which is based on the Fig. IMS / MS hybrid system shown. Fig. shows a set of ion species (1) to (13) that are found in the Fig. shown mass mobility map, wherein the durations of the ion mobility signals of these ion species (1) to (13) are determined from the mass mobility map and used to control the residence times of the quadrupole mass filter in a subsequent measurement cycle. Definitions

[0028] The term “residence time” refers to the quadrupole mass filter and is the time interval during which the mass filter is open so that ions of a selected mass range can pass through.

[0029] The term “selection time” or “selection interval” refers to virtual numbers that describe the time interval around a selected ion signal in the mass mobility map, which is used to control the residence time of the quadrupole mass filter.

[0030] The term "selection window" refers to the virtual numbers that specify the mass range and the selection interval around a signal in the mass mobility map. These numbers are used to control the aperture of the quadrupole mass filter in terms of mass range and residence time.

[0031] A “set of selection windows” is a temporal sequence of selected windows with the shortest possible time intervals between the windows, but without overlap.

[0032] The terms "separator" and "separation" are used here for devices and processes that separate different substances or different ion types over time. Chromatography, capillary electrophoresis, and ion mobility spectrometry are separation techniques. The term "filter" refers to devices and processes that allow only selected ion types from a larger range to pass through. An example of this is the RF quadrupole mass filter, which can be used to filter ions by mass.

[0033] The term “measurement loop” is used here in a special sense: A measurement loop begins with the measurement of a new mass mobility map and the selection of sets of non-overlapping selection windows, followed by several fragment mass spectrum measurement cycles.

[0034] A "fragment mass spectrum measurement cycle", called a "cycle" or "measurement cycle" for short, begins with the transport of the new ions from the collection unit to the ion mobility scanning unit and measures a set of fragment mass spectra of the ion species in a set of selection windows selected from the mass mobility map.

[0035] The “mobility scan time” or “mobility scan duration” is defined as the duration of an ion mobility scan over a range of ion mobilities of interest, usually the range of mobilities of the stored ions. Detailed description of the invention

[0036] While the invention has been shown and described with reference to a number of different embodiments, it will be appreciated by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined in the claims.

[0037] According to the state of the art, the residence time of a mass filter that is part of an IMS / MS hybrid system is as described in the Fig. and is used to record fragment mass spectra of selected ion species, is the same for all isolated ion species, especially for the Fig. multicyclic procedures shown.

[0038] Fig. show simulated mass mobility maps that are compared with the Fig. IMS / MS hybrid systems shown. Each mass mobility map is recorded without mass filtering and fragmentation and shows three different subsets of ion species for different charge states. The mass mobility map in Fig. is recorded with a TIMS separator and differs from the mass mobility map in Fig. , which is recorded with a drift-type mobility separator. The ion species leave the TIMS mobility separator in the order of their mobilities (lowest mobility first), not in the order of their reversed mobilities (highest mobility first). A detailed analysis of mass mobility maps shows that the duration of the ion mobility signal varies between the ion species. This fact is documented in the Fig. (and the corresponding Fig. ) are indicated by line signals of varying lengths; the thickness does not indicate mass fluctuations, but rather indicates the intensity of the signals. In the m / z direction, the signals show the usual isotopic distributions (in the Fig. not visible).

[0039] The duration of the ion mobility signal of an ion species during a single mobility separation depends, for example, on the time at which the ion species leaves the ion mobility separator and, in particular, on the number of configurational isomers not resolved by the ion mobility separator. When using a temporal zoom during a TIMS separation, the duration of an ion mobility signal depends on the instantaneous scan speed of the TIMS mobility separator. The duration of the ion mobility signal of zoomed-in ion species is longer than the ion mobility signal of ion species not included in the temporal zoom. Therefore, the residence time of the mass filter must be adjusted to include all ions of the zoomed-in ion species without reducing the total number of ion species by assuming a constant (long) residence time for the zoomed-in ion species.Adjusting the residence time has the advantage over a constant residence time that the number of selectable ion species in a single mobility separation can be increased, in particular by a factor of about two, thereby doubling the number of fragment spectra per cycle.

[0040] Fig. shows the mass mobility map of Fig. with a selected set of non-overlapping ion species (1) to (13). According to the present invention, the residence time of the quadrupole mass filter is adapted to the duration of the selected ion mobility signals determined from the mass mobility map.

[0041] Fig. shows the mass mobility map of Fig.with a selected set of non-overlapping ion species (1) to (13). According to the present invention, the residence time of the quadrupole mass filter is adapted to the duration of the selected mobility signals determined from the mass-mobility map.

[0042] The proposed adjustment of the mass filter's residence time allows, depending on the variation in the duration of the ion mobility signals, to select sets of selection windows with a significantly increased number of ion species per measurement cycle compared to the previous state-of-the-art method. For typical compositions of complex digest peptide mixtures, an increase of approximately two or three times is possible, doubling or tripling the number of fragment spectra per cycle.

Claims

[1] A method for recording fragment mass spectra of substances in complex substance mixtures using a mass spectrometric hybrid system comprising an ion source, an ion mobility separator, a mass filter, an ion fragmentation cell and a mass analyzer, comprising the following steps: - Temporal separation of ions according to their mobility by the ion mobility separator, whereby the duration of the ion mobility signal is different for different ion types; - Isolating the temporally separated ion species of a predetermined set of ion species one after the other based on their mass by the mass filter; - fragmentation of the isolated ion species in the ion fragmentation cell and acquisition of fragment mass spectra by the mass analyzer, wherein the residence time of the mass filter during isolation is not constant for at least some of the ion species of the predetermined set, but is adapted to the duration of the ion mobility signal from the respective ion species after separation in the ion mobility separator. [2] A method for recording fragment mass spectra of substances in complex substance mixtures using a mass spectrometric hybrid system comprising an ion source, an ion mobility separator, a mass filter, an ion fragmentation cell and a mass analyzer, wherein, in a first measurement cycle, a mass mobility map of ion species with ion mobility scan times is measured against ion masses, from which at least one set of temporally separated virtual windows is selected, comprising ion species with different ion masses and different ion mobility scan times, each with a different duration of the ion mobility signal, and wherein in a second measurement cycle, the ion species from the at least one set are successively isolated by the mass filter, which is controlled with respect to mass and residence time, fragmented in the ion fragmentation cell and measured with the mass analyzer as fragment mass spectra, characterized in that the temporal length of the windows and thus the residence time of the mass filter is not constant, but is adapted to the duration of the respective ion mobility signal in the mass mobility map. [3] The method according to claim 2, wherein the temporal length of the windows and thus the residence time of the mass filter substantially corresponds to the duration of the ion mobility signal in the mass mobility map. [4] The method according to claim 3, wherein the temporal length of the windows and thus the residence time of the mass filter corresponds to at least one scan duration of the mass analyzer and the residence time is synchronized with the scans of the mass analyzer. [5] The method of claim 4, wherein a scan duration of the mass analyzer is 100 microseconds and the time length of the windows and thus the residence time of the mass filter are multiples of 100 microseconds. [6] The method according to claim 4, wherein the temporal length of the windows and thus the residence time of the mass filter is at least one to four scan times of the mass analyzer longer than the duration of the ion mobility signal in the mass mobility map. [7] The method according to claim 4, wherein a scan duration of the mass analyzer is 100 microseconds and the time length of the windows and thus the residence time of the mass filter is 200 microseconds at the beginning and 200 microseconds at the end of the ion signal in the mass mobility map longer than the duration of the ion mobility signal in the mass mobility map. [8] The method according to any one of claims 2 to 7, wherein at least two sets of windows are selected and the selected sets of windows are significantly different from each other and do not contain the same ion species of the mass mobility map. [9] The method of claim 8, wherein weak ion signals from the mass mobility map are present in multiple sets of windows, and wherein their fragment mass spectra are combined to increase their quality. [10] The method according to any one of claims 2 to 9, wherein a second mass mobility map is measured when a predetermined time interval has elapsed, when a predetermined number of fragment mass spectra have been acquired, or when all ion species of the present mass mobility map have been fragmented and measured as fragment mass spectra. [11] The method of claim 10, wherein the mobility area of ​​the second mass mobility card is different from the mobility area of ​​a first mass mobility card. [12] The method according to any one of claims 1 to 11, wherein a storage ion mobility spectrometer (TIMS) with parallel ion collection is used as the ion mobility separator. [13] The method of claim 12, wherein the ion collection time of the second mass mobility map is different from the ion collection time of the first mass mobility map. [14] The method of claim 12 or claim 13, wherein the ion mobility scan durations substantially correspond to the ion collection durations. [15] The method according to any one of claims 1 to 14, wherein an orthogonal ion injection (OTOF) time-of-flight mass analyzer is used as the mass analyzer. [16] The method according to any one of claims 1 to 15, which comprises working with mixtures of substances provided by an upstream substance separator, such as a liquid chromatograph or a capillary electrophoresis unit.

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