Time-of-flight mass spectrometry of labeled analyte molecules

Abstract

To provide methods of analyzing labeled analyte molecules and / or ions using time-of-flight (ToF) mass analyzers.SOLUTION: An analytical instrument comprises an ion source 10, a mass filter 20, a fragmentation device 30, and a time-of-flight (ToF) mass analyzer 40. A method of analyzing labeled analyte ions comprises fragmenting labeled analyte ions to produce analyte fragment ions and reporter ions or complementary ions, analyzing the analyte fragment ions using a time-of-flight mass analyzer operating in a first mode of operation, and analyzing the reporter ions or the complementary ions using the time-of-flight mass analyzer operating in a second mode of operation. In the first mode of operation, ions are caused to travel along a flight path having a first length; and in the second mode of operation, ions are caused to travel along a flight path having a second length, where the second length is greater than the first length.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for analyzing ions, and in particular to a method for analyzing labeled analyte molecules and / or ions using a Time-of-Flight (ToF) mass analyzer. [Background technology]

[0002] Chemical tagging is a long-standing tool for quantification of analytes and, in its simplest form, involves comparing peak intensities with those of labeled standards of known concentration. When multiple different chemical tags with different masses, such as multiple isotopomers, are available, it becomes possible to mix multiple samples together and analyze them in a single high-throughput multiplexed workflow.

[0003] An important application within this group is the Tandem Mass Tag (TMT) method (Thompson et al, Anal. Chem., 2003, 75, 1895-1904), commercially available as TMT or Isobaric Tagging for Relative Quantitation (iTRAQ). This is a tandem method in which multiple tagged peptides have the same m / z and therefore co-elute from liquid chromatography and are co-separated by a quadrupole mass filter. However, upon fragmentation, characteristic 1, 2 or 6 reporter ions with different m / z are generated and detected for quantification. Simultaneous detection of peptide fragments allows peptide identification in the same step.

[0004] It is believed that there remains room for improvement in methods for analyzing labeled analyte molecules. Summary of the Invention

[0005] A first aspect provides a method of analysing a labelled analyte ion, the method comprising: fragmenting the labeled analyte ions to produce analyte fragment ions and reporter or complementary ions; analyzing the analyte fragment ions using a time-of-flight mass analyzer operating in a first mode of operation that moves ions along a flight path having a first length; Analysing the reporter ions or complementary ions using a time-of-flight mass analyser operating in a second mode of operation that moves ions along a flight path having a second length, the second length being longer than the first length.

[0006] The embodiments provide a method for analyzing labeled analyte molecules, e.g., biomolecules labeled with isobaric tags (e.g., "Tandem Mass Tags" (TMT) or "Isobaric Tags for Relative and Absolute Quantitation" (iTRAQ)). Each tag may include a reporter region and a balancer region, such that each labeled analyte molecule may include a reporter region, a balancer region, and an analyte molecule. To analyze the labeled analyte molecules, they may first be ionized to generate labeled analyte ions, which are then fragmented. When the labeled analyte ions are fragmented, reporter ions may be generated together with the analyte molecule fragment ions (the reporter ions are ions of the reporter region). Additionally or alternatively, complementary ions may be generated (the complementary ions are ions of the balancer region and analyte molecule combined).

[0007] In this method, the analyte fragment ions and reporter or complementary ions are analyzed using a mass analyzer, i.e. to determine their mass to charge ratio (m / z) and / or intensity. The m / z and / or intensity information obtained for the analyte fragment ions can be used to identify the analyte molecules, and the m / z and / or intensity information for the reporter or complementary ions can be used to quantify the analyte molecules.

[0008] In conventional mass spectrometric (MS) methods for analyzing labeled analyte molecules, analyte fragment ions and reporter ions are analyzed together in the same mass analyzer scan. This may require a very high-resolution mass analyzer, especially when using highly multiplexed isobaric tag sets, especially at the lower end of the m / z spectrum where reporter ions appear. Therefore, conventional methods for analyzing labeled analyte molecules are generally performed using high-resolution electrostatic ion trap mass analyzers, such as electrostatic orbital traps, more specifically Orbitrap™ FT mass analyzers manufactured by Thermo Fisher Scientific. Although these analyzers are particularly well suited for analyzing labeled analyte molecules, they generally operate at a relatively slow repetition rate, especially when compared to time-of-flight (ToF) mass analyzers. This results in relatively slow experimental cycles and relatively low throughput.

[0009] Some conventional time-of-flight (ToF) mass analyzers can provide sufficiently high resolution to resolve both the analyte fragment ions and the reporter ions (or complementary ions), but such analyzers must be relatively complex and / or have long ion flight paths to be able to resolve with reasonable resilience to space charge effects.

[0010] In the methods of various embodiments, the analyte fragment ions and reporter ions (or complementary ions) are analyzed using a time-of-flight (ToF) mass analyzer capable of operating in (at least) two different modes of operation: a first mode of operation in which ions are moved along a flight path having a first length, and a second mode of operation in which ions are moved along a flight path having a second, longer length. Increasing the length of the ion flight path in the second mode of operation has the effect of increasing the resolving power of the analyzer, but reducing the m / z range of ions that can be analyzed. If the increase in path length is achieved by increasing the number of passes of ions through a cyclic segment of the ion path (as explained further below), this is because ions of lower m / z will overtake (i.e. wrap) ions of higher m / z, and therefore it becomes uncertain how many passes some ions (with m / z outside of some apparent m / z range) have made through the cyclic segment, and therefore the m / z of some peaks may become uncertain. Additionally, or alternatively, when the analyzer is operated at a constant repetition rate, for longer ion flight path lengths, the high m / z ions in a particular repetition do not have enough time to reach the detector before the next repetition begins. Thus, the second mode of operation may be referred to as a "zoom" mode of operation (as the analyzer actually "zooms in" on a narrower m / z region of the m / z spectrum).

[0011] While this loss of m / z range is problematic in conventional methods of analyzing labeled analyte molecules (because the analyte fragment ions of interest may have a relatively high m / z and the reporter ions may have a relatively low m / z), the inventors have recognized that by analyzing the analyte fragment ions using a first mode of operation and the reporter ions (or complementary ions) using a second mode of operation, this problem is circumvented. This is because the first mode of operation with its wide m / z range is particularly suitable for the analysis of analyte fragment ions (which typically appear across a relatively wide m / z range) and correspondingly the second ("zoom") mode of operation with its narrow m / z range but higher resolution is particularly suitable for the analysis of reporter ions (which typically appear within a relatively narrow m / z range at a relatively low m / z) or complementary ions. Furthermore, this has the effect of relaxing the resolution required in the first ("normal") mode of operation, primarily due to the fact that the reporter ions (and complementary ions) are located very close together in m / z.

[0012] Thus, the inventors have recognized that a time-of-flight (ToF) mass analyzer having a variable path length is particularly well suited for analyzing labeled analyte molecules. Using a ToF analyzer to analyze labeled analyte ions (e.g., instead of an electrostatic ion trap analyzer) in turn facilitates increased instrument repetition rates and experimental throughput. Thus, it will be appreciated that the embodiments provide improved methods of analyzing labeled analyte molecules.

[0013] A further aspect provides a method of analyzing a labeled analyte molecule, comprising ionizing the labeled analyte molecule to produce labeled analyte ions, and analyzing the labeled analyte ions using a method described herein. These aspects and embodiments may, and in embodiments do, include any one or more or each of the optional features described herein.

[0014] The analyte molecule analyzed in various aspects and embodiments can be any molecule suitable for analysis, such as an organic molecule, a biomolecule, DNA, RNA, a protein, a peptide, a nucleic acid, etc. In an embodiment, the analyte molecule is a peptide.

[0015] Analyte molecules can be labeled with a set of chemical labels, such as a set of isobaric tags. A set of isobaric tags is a set of tag molecules that have (approximately) the same mass, but produce characteristic reporter ions of different masses upon fragmentation of the labeled analyte ions. Suitable isobaric tags include tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ). Each tag can include (at least) a reporter region and a balancer region, for example, such that each labeled analyte molecule includes (at least) a reporter region, a balancer region, and an analyte molecule (e.g., a peptide). The embodiments are particularly suitable for the analysis of analyte molecules labeled with highly multiplexed sets of isobaric tags, such as TMT10, 16, or 18, where the minimum spacing between reporter ion channels can be on the order of a few MDa, for example about 6 MDa. The embodiments can also be used for lower multiplexed isobaric tag sets, such as TMT6 and 8, which have reporter ion channel spacing on the order of about 1 Da.

[0016] The method may be carried out using an analytical instrument such as a mass spectrometer. The analytical instrument may comprise (at least) an ion source, a mass filter, a fragmentation device, and a time-of-flight mass analyzer (described in more detail below). The analytical instrument may comprise a separation device coupled to the ion source.

[0017] The labeled analyte molecules can be provided in a solution, the solution can be separated using a separation device, and the separated solution can be provided to an ion source for ionization. The ion source can ionize the labeled analyte molecules to produce labeled analyte ions.

[0018] The labeled analyte ions can be first analyzed by an instrument operating in an MS1 ​​mode of operation to provide MS1 data. The MS1 data can include one or more ion peaks, each ion peak corresponding to a labeled analyte ion having a particular m / z (i.e., a particular precursor).

[0019] The labeled analyte ions may then be analyzed by an instrument operating in an MS2 mode of operation to provide MS2 data. Each precursor of interest identified from the MS1 data may be used to define an m / z window for a mass filter. The mass filter may then step through each m / z window in sequence to sequentially select each precursor of interest. The fragmentation device may be operated in a fragmentation mode of operation such that the mass filtered labeled analyte ions are fragmented. When the labeled analyte ions are fragmented, reporter ions may be generated along with the analyte molecule fragment ions (the reporter ions are ions of the reporter region). Additionally or alternatively, complementary ions may be generated (the complementary ions are ions of the combined balancer region and analyte molecule).

[0020] The resulting analyte fragment ions and reporter or complementary ions are then analyzed using a time-of-flight analyzer. The mass-to-charge ratio (m / z) and / or intensity information obtained for the analyte fragment ions can be used to identify the analyte molecules, and the mass-to-charge ratio (m / z) and / or intensity information for the reporter or complementary ions can be used to quantify the analyte molecules.

[0021] The analyte fragment ions are analyzed using a first mode of operation that causes the ions to travel along a flight path having a first length, and the reporter or complementary ions are analyzed using a second mode of operation that causes the ions to travel along a flight path having a second, longer length.

[0022] A time-of-flight mass analyzer may comprise one or more ion reflectors. In a first mode of operation, ions may be reflected n times in the one or more ion reflectors, where n is an integer > 0. In a second mode of operation, ions may be reflected m times in the one or more ion reflectors, where m is an integer > n. In general, an "ion reflector" may be, for example, an ion mirror, a reflectron, an ion deflector, a lens or the like.

[0023] The time-of-flight mass analyzer may be a multi-reflection time-of-flight (MR-ToF) mass analyzer, wherein the MR-ToF comprises: two ion mirrors spaced apart and facing each other in a first direction X, each mirror elongated generally along a drift direction Y between a first end and a second end, the drift direction Y being orthogonal to the first direction X; an ion injector for injecting ions into a space between the ion mirrors, the ion injector being located near a first end of the ion mirror; A detector for detecting ions after the ions have completed multiple reflections between the ion mirrors, the detector being located near a first end of the ion mirror.

[0024] Analyzing the analyte fragment ions using an analyzer operating in a first mode of operation includes: The method may include injecting analyte fragment ions from an ion injector into a space between the ion mirrors, where the ions (a) drift along a drift direction Y towards a second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) follow a zigzag ion path having multiple reflections in direction X between the ion mirrors while drifting along drift direction Y back to the first end of the ion mirror, and then moving the ions to a detector for detection.

[0025] Analyzing the reporter ion or the complementary ion using the analyzer operating in a second mode of operation includes: (i) injecting reporter ions or complementary ions from an ion injector into a space between the ion mirrors, where the ions complete a first cycle in which they (a) drift along a drift direction Y towards a second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) drift back along the drift direction Y towards the first end of the ion mirror while following a zigzag ion path having multiple reflections in a direction X between the ion mirrors; (ii) reversing the drift directional velocity of the ion near the first end of the ion mirror such that the ion completes a further cycle in which the ion (a) drifts along drift direction Y towards the second end of the ion mirror, (b) reverses the drift directional velocity near the second end of the ion mirror, and (c) follows a zigzag ion path having multiple reflections in direction X between the ion mirrors while drifting back along drift direction Y towards the first end of the ion mirror; (iii) optionally repeating step (ii) one or more times; and then (iv) transferring the ions to a detector for detection.

[0026] The multi-reflecting time-of-flight (MR-ToF) mass analyzer may further comprise a deflector or lens located near the first end of the ion mirror. The deflector or lens may be located approximately equidistant (in the X-direction) between the first and second ion mirrors. The deflector or lens may be positioned along the ion path after the first ion mirror reflection (at the first ion mirror) that the ion beam undergoes after being injected from the injector, but before the second ion mirror reflection (at the second ion mirror). Correspondingly, the deflector or lens may be positioned along the ion path before the last ion mirror reflection (at the second ion mirror) that the ion beam undergoes before reaching the detector, but after the penultimate ion mirror reflection (at the first ion mirror).

[0027] Analyzing the analyte fragment ions using an analyzer operating in a first mode of operation includes: The method may include injecting analyte fragment ions from an ion injector into a space between the ion mirrors, where the ions (a) drift along a drift direction Y from the deflector or lens towards a second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) follow a zigzag ion path having multiple reflections in direction X between the ion mirrors while drifting along the drift direction Y back to the deflector or lens, and then moving the ions from the deflector or lens to a detector for detection.

[0028] Analyzing the reporter ion or the complementary ion using the analyzer operating in a second mode of operation includes: (i) injecting reporter ions or complementary ions from an ion injector into a space between the ion mirrors, where the ions complete a first cycle in which they (a) drift along a drift direction Y from the deflector or lens towards a second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) drift back along the drift direction Y towards the deflector or lens while following a zigzag ion path having multiple reflections in direction X between the ion mirrors; (ii) using a deflector or lens to reverse the drift direction velocity of the ions such that the ions (a) drift along drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse the drift direction velocity near the second end of the ion mirror, and (c) complete a further cycle following a zigzag ion path having multiple reflections in direction X between the ion mirrors while drifting along drift direction Y back towards the deflector or lens; (iii) optionally repeating step (ii) one or more times; and then (iv) transferring the ions from the deflector or lens to a detector for detection.

[0029] The multi-reflection time-of-flight (MR-ToF) mass analyzer may include any suitable type of MR-ToF. For example, the analyzer may include an MR-ToF having a set of periodic lenses configured to keep the ion beam focused along its flight path, as described, for example, in A. Verenchikov et al., Journal of Applied Solution Chemistry and Modelling, 2017, 6, 1-22.

[0030] However, in certain embodiments, the analyzer is a tilted mirror type multi-reflecting time-of-flight mass analyzer, for example of the type described in U.S. Pat. No. 9,136,101, the contents of which are incorporated herein by reference. Thus, the ion mirrors may be at a non-constant distance from each other in the X direction along at least part, most, or all of their length in the drift direction Y. The drift direction velocity of the ions towards the second end of the ion mirror may be opposed by an electric field resulting from the non-constant distance of the two mirrors from each other. This electric field may cause the ions to reverse their drift direction velocity near the second end of the ion mirror and drift back along the drift direction towards the deflector.

[0031] Alternatively, the analyzer may be a single focusing lens type multi-reflection time-of-flight mass analyzer, for example of the type described in GB Patent No. 2,580,089, the contents of which are incorporated herein by reference. Thus, the deflector may be a first deflector and the analyzer may comprise a second deflector located near the second end of the ion mirror. The second deflector may be configured to cause the ions to reverse their drift direction velocity near the second end of the ion mirror and drift back along the drift direction towards the first deflector. To do this, a suitable voltage may be applied to the second deflector, for example in the manner described in GB Patent No. 2,580,089.

[0032] Analyzing the analyte fragment ions may include analyzing one or more first ion packets, for example, by injecting each first ion packet into a ToF analyzer. Analyzing the reporter ions or complementary ions may include analyzing one or more second, different ion packets, for example, by injecting each second ion packet into a ToF analyzer. Thus, for each precursor of interest, the analyzer may perform two (or more) scans, i.e., the analyte fragment ions may be analyzed using a different analyzer scan than the reporter ions or complementary ions.

[0033] In these embodiments, each first ion packet may contain only analyte fragment ions, or may contain analyte fragment ions together with reporter ions and / or complementary ions. If the first ion packet contains reporter ions and / or complementary ions, these ions may be optionally deflected by a deflector so as not to reach the detector (while almost all of the analyte fragment ions reach the detector). Each second ion packet may contain only reporter ions and / or complementary ions, or may contain reporter ions and / or complementary ions together with analyte fragment ions. If the second ion packet contains analyte fragment ions, these ions may be deflected by a deflector so as not to reach the detector (while almost all of the reporter ions or complementary ions reach the detector).

[0034] The method may include generating and / or processing and / or analyzing each first ion packet using a first set of one or more instrument parameters, and generating and / or processing and / or analyzing each second ion packet using a second, different set of one or more instrument parameters, the first set of one or more instrument parameters may be configured to provide improved sensitivity and / or resolution when generating and / or processing and / or analyzing analyte fragment ions (relative to reporter ions or complementary ions), and the second set of one or more instrument parameters may be configured to provide improved sensitivity and / or resolution when generating and / or processing and / or analyzing reporter ions or complementary ions (relative to analyte fragment ions).

[0035] The set of one or more instrument parameters may include, for example, one or more voltages (RF or DC) applied to any one or more of the instrument's components, such as the ion source, the ion inlet, any one or more ion transport devices, the mass filter, the fragmentation device, and / or the analyzer (e.g., its ion injector, ion reflector, and / or detector). In certain embodiments, the set of one or more instrument parameters includes (i) one or more fragmentation parameters, such as the collision energy of a fragmentation device, and / or (ii) the width of the transmission window of a mass filter.

[0036] Thus, for example, the method may include generating each first ion packet using a first mass filter transmission window width (i.e., a mass filter is used to select a particular precursor of interest to be fragmented and the resulting fragment ions are used to form each first ion packet), and, for example, if the first mass filter transmission window width is wider than the second mass filter transmission window width, generating each second ion packet using a second, different mass filter transmission window width (i.e., a mass filter is used to select a particular precursor of interest to be fragmented and the resulting fragment ions are used to form each second ion packet). Using a wider mass filter transmission window width advantageously increases sensitivity for the analysis of analyte fragment ions, while using a narrower mass filter transmission window width advantageously reduces interference for the analysis of reporter ions or complementary ions. Alternatively, it is also possible for the first mass filter transmission window width to be narrower than the second mass filter transmission window width.

[0037] The method may include generating each first ion packet using one or more first fragmentation parameters, such as a first collision energy (i.e., a particular target precursor is fragmented using one or more first fragmentation parameters, such as a first collision energy, and the resulting fragment ions are used to form each first ion packet), and generating each second ion packet using one or more second fragmentation parameters, such as a second, different collision energy (i.e., a particular target precursor is fragmented using one or more second fragmentation parameters, such as a second collision energy, and the resulting fragment ions are used to form each second ion packet). The one or more first fragmentation parameters, such as the first collision energy, may be configured to efficiently generate analyte fragment ions (relative to the reporter ions or complementary ions), and the one or more second fragmentation parameters, such as the second collision energy, may be configured to efficiently generate reporter ions or complementary ions (relative to the analyte fragment ions). Higher collision energies are typically beneficial for generating low m / z fragment ions such as reporter ions, while lower collision energies are typically beneficial for generating mid-range or high m / z fragment ions such as analyte fragment ions. Thus, the first collision energy may be lower than the second collision energy, for example, when reporter ions are generated and analyzed. Meanwhile, a relatively low collision energy is typically used to generate complementary ions, compared to the collision energy used to generate analyte fragment ions. Thus, the first collision energy may be higher than the second collision energy, for example, when complementary ions are generated and analyzed.

[0038] In alternative embodiments, analyzing the analyte fragment ions and analyzing the reporter or complementary ions may include analyzing one or more single ion packets, for example, by injecting each packet of ions into a ToF analyzer. Thus, for each precursor of interest, the analyzer may perform a single scan, i.e., the analyte fragment ions are analyzed using the same scan as the reporter or complementary ions. In these embodiments, each packet of ions may include the analyte fragment ions along with the reporter ions and / or complementary ions.

[0039] The analyzer may include an ion path including a circulation segment, an ion injector for injecting ions into the ion path, at least one ion reflector positioned along the ion path, and a detector positioned at an end of the ion path.

[0040] The method is: (i) injecting an ion packet comprising analyte fragment ions and reporter or complementary ions from an ion injector into an ion path such that both the analyte fragment ions and the reporter or complementary ions travel along the ion path to an ion reflector; (ii) transferring (only) the analyte fragment ions from the ion reflector to a detector for detection; (iii) using an ion reflector to cause the reporter ion or complementary ion (only) to complete one or more cycles along a circular segment of the ion path; and then (iv) transferring the reporter ions or complementary ions from the ion reflector to a detector for detection.

[0041] The method is: (i) injecting ion packets comprising analyte fragment ions and reporter or complementary ions from an ion injector into a space between the ion mirrors, wherein the ions complete a first cycle in which they (a) drift along a drift direction Y from the deflector or lens towards a second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) drift back along the drift direction Y towards the deflector or lens while following a zigzag ion path having multiple reflections in direction X between the ion mirrors; (ii) transferring (only) the analyte fragment ions through a deflector or lens to a detector for detection; (iii) using a deflector or lens to reverse the drift direction velocity of the reporter ions or complementary ions (only) to cause the ions to complete a further cycle in which they (a) drift along drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) follow a zigzag ion path having multiple reflections in direction X between the ion mirrors while drifting along drift direction Y back towards the deflector or lens; (iv) optionally repeating step (iii) one or more times; and then (v) transferring the reporter ion or complementary ion through a deflector or lens to a detector for detection.

[0042] According to another aspect, there is provided a method of operating a Time-of-Flight (ToF) mass analyzer, the ToF mass analyzer comprising: an ion pathway including a cyclic segment; an ion injector for injecting ions into the ion path; at least one ion reflector disposed along the ion path; a detector disposed at an end of the ion path; The method is: (i) injecting an ion packet including first ions and second ions from an ion injector into an ion path such that the first ions and the second ions travel along the ion path to an ion reflector; (ii) transferring (only) the first ions from the ion reflector to a detector for detection; (iii) using an ion reflector to cause (only) the second ions to complete one or more (e.g., further) cycles along a circular segment of the ion path; and then (iv) transferring the second ions from the ion reflector to a detector for detection.

[0043] These aspects and embodiments may, and in embodiments do, include any one or more, or each, of the optional features described herein.

[0044] In these aspects and embodiments, the ToF analyzer may be a cyclic analyzer. The ion path includes a cyclic segment, through which ions may pass zero or multiple times (repeatedly) as they travel along the ion path from the ion injector to the detector. For example, the ion path may comprise a first non-cyclic segment, a cyclic segment disposed downstream of the first non-cyclic segment, and a second non-cyclic segment disposed downstream of the cyclic segment. The ToF analyzer comprises at least one ion reflector, such as a deflector, disposed along the ion path. The ion reflector may be used to cause ions to complete one or more (further) cycles along the cyclic segment of the ion path, for example by suitable application of a (e.g., pulsed) voltage to the ion reflector.

[0045] In the method, an ion packet including first and second ions is injected into an ion path such that the ions travel along the ion path to an ion reflector. Before reaching the ion reflector, the first and second ions may follow the same ion path and may pass through a circular segment of the ion path zero or more times. The first ions (only) are then moved from the ion reflector to a detector for detection, while the ion reflector is used to allow the second ions (only) to complete one or more (e.g., further) cycles along the circular segment of the ion path before moving from the ion reflector to the detector for detection. This can be done by suitably pulsing the magnitude of the voltage applied to the ion reflector with suitable timing, such that only the second ions are allowed to complete one or more (e.g., further) cycles, and not the first ions.

[0046] The first ions can be ions having m / z within a first relatively broad range. The second ions can be ions having m / z within a second, different, relatively narrow range. The second range can overlap with or be encompassed by the first range.

[0047] According to another aspect, there is provided a method of operating a multi-reflecting time-of-flight (MR-ToF) mass analyzer, the MR-ToF mass analyzer comprising: two ion mirrors spaced apart and facing each other in a first direction X, each mirror elongated generally along a drift direction Y between a first end and a second end, the drift direction Y being orthogonal to the first direction X; an ion injector for injecting ions into a space between the ion mirrors, the ion injector being located near a first end of the ion mirror; a detector for detecting ions after the ions have completed multiple reflections between the ion mirrors, the detector being located near a first end of the ion mirror; a deflector or lens located near the first end of the ion mirror; The method is: (i) injecting an ion packet comprising first and second ions from an ion injector into a space between the ion mirrors, wherein the ions complete a first cycle in which they (a) drift along a drift direction Y from a deflector or lens towards a second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) follow a zigzag ion path having multiple reflections in a direction X between the ion mirrors while drifting back along the drift direction Y towards the deflector or lens; (ii) transferring (only) the first ions from the deflector or lens to a detector for detection; (iii) using the deflector or lens to reverse the drift direction velocity of the second ion (only) such that the second ion (a) drifts along drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverses the drift direction velocity near the second end of the ion mirror, and (c) causes the second ion to complete a further cycle following a zigzag ion path having multiple reflections in direction X between the ion mirrors while drifting along drift direction Y back towards the deflector or lens; (iv) optionally repeating step (iii) one or more times; and then (v) transferring the second ions from the deflector or lens to a detector for detection.

[0048] These aspects and embodiments may, and in embodiments do, include any one or more, or each, of the optional features described herein.

[0049] A further aspect provides a non-transitory computer readable storage medium storing computer software code which, when executed on a processor, performs the above-mentioned methods.

[0050] A further aspect provides a control system for an analytical instrument, such as a mass spectrometer, the control system configured to cause the analytical instrument to carry out the method described above.

[0051] A further aspect provides an analytical instrument, such as a mass spectrometer, comprising a control system as described above.

[0052] A further aspect provides an analytical instrument, such as a mass spectrometer, comprising: A fragmentation device; a time-of-flight (ToF) mass analyzer operable in a first mode of operation to move ions along a flight path having a first length and a second mode of operation to move ions along a flight path having a second length, the second length being greater than the first length; and a control system, the control system comprising: causing the fragmentation device to fragment the labeled analyte ions to generate analyte fragment ions and reporter or complementary ions; causing a time-of-flight mass analyzer to analyze the analyte fragment ions using a first mode of operation; and causing the time-of-flight mass analyzer to analyze the reporter ion or the complementary ion using a second mode of operation.

[0053] A further aspect provides a time-of-flight (ToF) mass analyzer, comprising: an ion pathway including a cyclic segment; an ion injector for injecting ions into the ion path; at least one ion reflector disposed along the ion path; a detector disposed at an end of the ion path; The analyzer is (i) injecting an ion packet including first ions and second ions from an ion injector into an ion path such that the first ions and the second ions travel along the ion path to an ion reflector; (ii) transferring (only) the first ions from the ion reflector to a detector for detection; (iii) using an ion reflector to cause (only) the second ions to complete one or more (e.g., further) cycles along a circular segment of the ion path; and then (iv) analyzing the ions by transferring the second ions from the ion reflector to a detector for detection.

[0054] A further aspect provides a multi-reflecting time-of-flight (MR-ToF) mass analyzer, the MR-ToF mass analyzer comprising: two ion mirrors spaced apart and facing each other in a first direction X, each mirror elongated generally along a drift direction Y between a first end and a second end, the drift direction Y being orthogonal to the first direction X; an ion injector for injecting ions into a space between the ion mirrors, the ion injector being located near a first end of the ion mirror; a detector for detecting ions after the ions have completed multiple reflections between the ion mirrors, the detector being located near a first end of the ion mirror; a deflector or lens located near the first end of the ion mirror, the analyzer comprising: (i) injecting an ion packet comprising first and second ions from an ion injector into a space between the ion mirrors, wherein the ions complete a first cycle in which they (a) drift along a drift direction Y from a deflector or lens towards a second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) follow a zigzag ion path having multiple reflections in a direction X between the ion mirrors while drifting back along the drift direction Y towards the deflector or lens; (ii) transferring (only) the first ions from the deflector or lens to a detector for detection; (iii) using the deflector or lens to reverse the drift direction velocity of the second ion (only) such that the second ion (a) drifts along drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverses the drift direction velocity near the second end of the ion mirror, and (c) causes the second ion to complete a further cycle following a zigzag ion path having multiple reflections in direction X between the ion mirrors while drifting along drift direction Y back towards the deflector or lens; (iv) optionally repeating step (iii) one or more times; and then (v) analyzing the ions by moving the second ions through the deflector or lens to a detector for detection.

[0055] A further aspect provides an analytical instrument, such as a mass spectrometer, comprising a Time-of-Flight (ToF) mass analyser or a Multi-Reflecting Time-of-Flight (MR-ToF) mass analyser as described above.

[0056] These aspects and embodiments may, and in embodiments do, include any one or more, or each, of the optional features described herein.

[0057] Various embodiments will now be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0058] [Figure 1] The base TMT C8H16N+ reporter ion with m / z 126 is shown diagrammatically. [Diagram 2] 1 illustrates a schematic diagram of an analytical instrument according to an embodiment; [Diagram 3] 1 illustrates a schematic diagram of an analytical instrument according to an embodiment; [Figure 4] 1 illustrates a schematic representation of a multi-reflecting time-of-flight mass analyzer according to an embodiment; [Diagram 5] 1 illustrates a schematic representation of a multi-reflecting time-of-flight mass analyzer according to an embodiment; [Figure 6]1 illustrates a schematic of a method of operating a multi-reflecting time-of-flight mass analyzer according to an embodiment; [Figure 7] 1 illustrates a DDA method according to an embodiment. [Figure 8] 13 illustrates a method using 5x zoom of TMT reporter ions and non-zoomed peptide fragment ions, according to an embodiment. [Figure 9] 1 illustrates a DDA method according to an embodiment. [Figure 10A] 1 shows a plot of resolution versus ion number for a typical single pass mode of operation of a multi-reflecting time-of-flight mass analyzer. [Figure 10B] 1 shows a plot of resolution versus ion number for a 3x zoom mode of operation of a multi-reflecting time-of-flight mass analyzer. [Figure 11] 1 shows a plot of resolution and signal versus number of passes through a multi-reflecting time-of-flight mass analyzer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Chemical tagging is a long-standing tool for quantification of analytes and, in its simplest form, involves comparing peak intensities with those of labeled standards of known concentration. When multiple different chemical tags with different masses, such as multiple isotopomers, are available, it becomes possible to mix multiple samples together and analyze them in a single high-throughput multiplexed workflow.

[0060] An important application within this group is the tandem mass tag (TMT) method (Thompson et al, Anal. Chem., 2003, 75, 1895-1904), commercially available as TMT or isobaric tags for relative and absolute quantification (iTRAQ). This is a tandem method in which multiple tagged peptides have the same m / z and therefore co-elute from liquid chromatography and are co-separated by a quadrupole mass filter. However, upon fragmentation, characteristic 1, 2, or 6 reporter ions with different m / z are generated and detected for quantification. Simultaneous detection of peptide fragments allows peptide identification in the same step. The great advantage of this method lies in the simplification of the complete mass spectrum and the sensitivity of the co-separation by a factor of the level of multiplexing.

[0061] Early commercial implementations of TMT6plex include: 13 Using isotopic mass separation generated by substitution with C, the reporter ions shown in Figure 1 were generated from m / z 126-131. More recent advances include 15 Adding nitrogen substitution by N, 13 A 10-plex approach provided a small 6.32 mDa mass defect for the C-only reporter, increasing the number of accessible substitution tags and splitting the reporter region into a series of isobaric doublets (McAlister et al, Anal. Chem., 2012, 84, 7469-7478). Even higher levels of multiplexing, such as 18-plex, have also been demonstrated.

[0062] However, this method has its own drawbacks. Interference between co-separated isobaric peptides can contaminate the reporter ion channel, preventing accurate quantification and preventing fast experimental cycles using shorter, less well-resolving LC gradients. An additional separation and fragmentation step (MS3) can be used to remove interferences, but this also impacts speed and sensitivity. High-resolution separation, such as that provided by specialized time-of-flight (ToF) instruments, is a more optimal solution, especially when applied in series with quadrupole separation.

[0063] A second problem is that the reporter ion doublets of 10-plex and above methods require a resolving power of approximately 50K at m / z 127 in order to be distinguished and measured. This is outside the scope of most time-of-flight (ToF) analyzer designs and imposes severe space-charge related limitations on the dynamic range for advanced ToF analyzers capable of providing such resolution.

[0064] To date, the TMT 10-plex method has been performed on Orbitrap™ FT instruments, which excel at providing high resolution at low m / z. However, the acquisition time required to achieve such resolution limits the acquisition speed and restricts the number of measurements. For Orbitrap™ analyzers, improved data processing methods such as Phi-SDM have been used to significantly improve the resolution at speed (Bekker-Jenson et al, Mol. Cell. Proteomics, 2020, 14, 716-729). Space charge effects also cause fusion / coalescence of reporter ion doublets in Orbitrap™ instruments, which can require high ion loading that is usually limited by automatic gain control (AGC) procedures (Werner et al, Anal. Chem., 2014, 86, 3594-3601).

[0065] An important modification to the TMT method is TMT complementary ion quantification (Johnson et al, J. Proteome Res., 2021, 20, 3043-3052). Here, rather than quantifying from the reporter ion, the tagged peptide minus the reporter ion is generated and measured at low fragmentation energy. After reporter separation, the tag still contains a "balancer" component, which normally serves to balance the mass of all tagged ions, but without the reporter component, this generates a mirror image distribution of complementary ion channels. These channels can have a similar relative intensity to the reporter ion distribution and can therefore be used for quantification. The advantage is that they are more robust to interference from co-isolated peptides, since they still have the original peptide bound. However, the tradeoff is that much higher resolution is required to resolve the channels, since the complementary ions are much heavier than the reporters, but the mass difference between the channels is the same. So far, this process has been limited to 8-channel TMT methods with 1 Da spacing, but the equivalent 16-channel spacing, which typically requires a resolution of 50K, may require 500K when applied to ions ten times heavier.

[0066] Few time-of-flight (ToF) analyzers provide sufficient resolution to be compatible with higher TMT multiplexing methods. Resolution in such analyzers is typically limited by the length of the ion flight path and the width of the peak resulting from both the arrival time spread of the ions at the detector and the time response of the detector itself. This latter fixed response results in a mass dependency in the analyzer resolution, preventing low m / z measurements such as TMT reporter regions (the opposite trend to Orbitrap™ analyzers).

[0067] Conventional ToF analyzers typically incorporate an ion implantation device, such as an orthogonal accelerator or extraction trap, a detector, and an ion mirror that serves to focus the ions onto the detector surface. They also generally include lenses or deflectors after the ion source that are configured to appropriately shape and direct the beam.

[0068] A more advanced ToF analyzer with very high resolution has been introduced by Wollnik (DE3025764C2), which incorporates a pair of ion mirrors that allow trapped ions to undergo multiple oscillations before being released to the detector. The drawback of this system was that lighter ions overtake / wrap around heavier ions, making mass analysis very complicated. Increasing the resolution of a target ion via increased dwell time, at the expense of limiting the mass range, can be referred to as "zooming in".

[0069] An extension of the ion mirror by Nazarenki (SU1725289) led to the development of an "open trap" or "multiple reflection time-of-flight (MR-ToF)" analyzer with a fixed zigzag flight path that eliminated the wrapping problem. It was improved and commercialized by Verentchikov (UK Patent No. 2,403,063) by incorporating periodic lenses between the mirrors that systematically corrected for ion dispersion orthogonal to the ion oscillations.

[0070] This latter method of controlling ion dispersion has been proposed by Sudakov (WO 2008 / 047891), Stewart (US Pat. No. 10,964,520), and most notably by Grinfeld in US Pat. No. 9,136,101. This latter method involves slightly tilting the ion mirrors so that the injected ions are allowed to spread out as they drift in the drift direction between the elongated mirrors. After each pass of the tilted mirror and supporting stripe electrodes, the ions are deflected back until they completely reverse their drift velocity and are focused onto a detector on the starting side of the mirror system. A schematic of this analyzer is shown in FIG. 4 (described in more detail below).

[0071] Such analyzers are capable of resolving TMT reporters, but must be relatively large (approximately 1 m) to do so with reasonable space charge restoring forces. 2 ) The detectable ratio of the two peaks in a doublet can still be limited because baseline resolution of the peaks is not easily achievable due to tailing or other effects.

[0072] A special mode of operation covering this wide family of analyzers, termed the "zoom mode", has been previously reported by Verenchikov et al. (Verenchikov et al., Journal of Applied Solution Chemistry and Modelling, 2017, 6, 1-22). A deflector placed early in the ion path can be switched to a trapping voltage, forcing the ions to make multiple passes up and down the mirror, each involving the same number of oscillations between the mirrors. This significant extension of the flight path demonstrated a greatly increased resolving power up to 500,000, but, as the name suggests, caused a severe loss of mass range.

[0073] The embodiments described herein address the difficulty of resolving and thereby accurately quantifying peaks within TMT reporter doublets or similar isobaric fragment tags, particularly as applied to multi-reflection time-of-flight (MR-ToF) analyzers (and other ToF analyzers).

[0074] As mentioned above, Orbitrap™ instruments are commonly used for TMT methods, but these instruments can suffer somewhat from the slow repetition rates required to operate at such high resolution. This also imposes slower experimental cycles and lower throughput, although it is not always clear whether this or contamination of precursor targets by co-eluting and co-isolated peptides predominates.

[0075] Most ToF analyzers have resolution that is too low to be entirely compatible with the more advanced multiplexed TMT methods, and even relatively large MR-ToF systems may struggle to maintain their resolution at higher ion loads, limiting the dynamic range and the ratio of doublet peaks that can be characterized. High repetition rate implantation devices such as orthogonal accelerators reduce the space charge load per shot, but these can come with a significant sensitivity cost to the extraction trap, and advanced operating methods involving deconvolution (such as via coded frequent pulsing) may be required to make such sources compatible with the long flight times of multi-reflecting ToF analyzers. Space charge tolerant analyzers such as the tilted mirror analyzer shown in FIG. 4 can be compatible with these methods, but improved performance is still desired.

[0076] Therefore, certain embodiments are directed to improving the resolution and space charge restoration, and therefore the dynamic range, of multi-reflecting time-of-flight (MR-ToF) analyzers for the TMT reporter ions used for quantification, while minimizing the impact on the peptide fragments used for identification.

[0077] According to the embodiments described herein, the zoom mode is used only for the TMT reporter (or complementary) ions, and the normal operation mode is used for peptide fragment identification, e.g., in data-dependent TMT methods. As described in more detail below, the reporter ions (or complementary ions) and fragment ions can be analyzed either in separate scans or in a single integrated scan. The zoom mode increases the achievable resolution of the TMT reporters, which also improves the space charge resilience and dynamic range of the analyzer.

[0078] 2 shows a schematic diagram of an analytical instrument, such as a mass spectrometer, that may be used to carry out methods according to embodiments. As shown in FIG. 2, the analytical instrument includes an ion source 10, a mass filter 20, a fragmentation device 30, and a time-of-flight (ToF) mass analyzer 40.

[0079] The ion source 10 is configured to generate ions from a sample. The ion source 10 may be coupled to a separation device, such as a liquid chromatography separation device or a capillary electrophoresis separation device (not shown), whereby the sample that is ionized in the ion source 10 comes from the separation device. The ion source 10 may be any suitable ion source, such as an ion source compatible with the separation device. In an embodiment, the ion source 10 is an electrospray ionisation (ESI) ion source, an atmospheric pressure ionisation (API) ion source, a chemical ionisation ion source, an electron impact (EI) ion source, or the like.

[0080] The mass filter 20 may be disposed downstream of the ion source 10 and configured to receive ions from the ion source 10. The mass filter 20 may be configured to filter the received ions according to their mass to charge ratio (m / z), for example such that only ions within an m / z window are transmitted onward by the mass filter 20 and ions outside the mass filter's m / z window are rejected by the mass filter 20 and not transmitted onward. The m / z width and centre m / z of the mass filter's transmission window may be controllable (variable), for example, by suitable control of RF and DC voltages applied to the mass filter 20. Thus, for example, the mass filter may be operable in a transmission mode of operation in which most or all ions within a relatively wide m / z window are transmitted onward by the mass filter 20, and a filtering mode of operation in which only ions within a relatively narrow m / z window (centreed about a desired m / z) are transmitted onward by the mass filter 20. The mass filter 20 may be any suitable type of mass filter, such as a quadrupole mass filter.

[0081] The fragmentation device 30 may be disposed downstream of the mass filter 20 and configured to receive most or all of the ions transmitted by the mass filter 20. The fragmentation device 30 may be configured to selectively fragment some or all of the received ions, i.e., generate fragment ions. The fragmentation device 30 may be operable in a fragmentation mode of operation in which most or all of the received ions are fragmented to generate fragment ions (which may then be transmitted onward from the fragmentation device 30), and a non-fragmentation mode of operation in which most or all of the received ions are transmitted onward without (intentionally) being fragmented. The non-fragmentation mode of operation may also be implemented by allowing ions to bypass the fragmentation device 30. The fragmentation device 30 may also be operable in one or more intermediate modes of operation, e.g., in which the degree of fragmentation is controllable (variable). The fragmentation device 30 may also be operable in higher order (MS) modes, e.g., in which the fragment ions are further fragmented one or more times by the fragmentation device 30. N ) fragmented mode of operation.

[0082] The fragmentation device 30 may be any suitable type of fragmentation device that can be used to fragment labeled analyte ions to produce analyte fragment ions and reporter or complementary ions, such as, for example, a collision induced dissociation (CID) fragmentation device, an electron induced dissociation (EID) fragmentation device, a photodissociation fragmentation device, etc. Many other types of fragmentation are possible.

[0083] In an embodiment, the fragmentation device 30 is a collision induced dissociation (CID) fragmentation device. The fragmentation device may thus include a collision cell, which may be filled with a collision gas maintained at a relatively high pressure, for example. Ions may be selectively fragmented in the collision cell by controlling (varying) the kinetic energy with which the ions are caused to enter the collision cell. In a fragmentation mode of operation, ions may be accelerated to enter the collision cell with a relatively high kinetic energy, which may fragment most or all of the accelerated ions. In a non-fragmentation mode of operation, ions may be caused to enter the collision cell with a relatively low kinetic energy, which may be insufficient to fragment most or all of the ions. In an intermediate mode, ions may be caused to enter the collision cell with an intermediate kinetic energy.

[0084] The ToF analyzer 40 may be positioned downstream of the fragmentation device 30 and may be configured to receive most or all of the ions transmitted onward from the fragmentation device 30. For example, if the instrument is configured such that ions bypass the fragmentation device 30 in a non-fragmenting mode of operation, it is also possible to configure the instrument such that the ToF analyzer 40 can receive ions from the mass filter 20. Thus, in general, the ToF analyzer 40 may be configured to receive ions from various upstream stages of the instrument, which may include unfragmented ("precursor" or "parent") ions, fragment ("product" or "daughter") ions, fragmented fragment ("grand-daughter") ions, etc.

[0085] The ToF analyzer 40 is configured to analyze the received ions to determine their mass to charge ratio (m / z). To do this, the ToF analyzer 40 is configured to analyze the ions received to determine their mass to charge ratio (m / z). To do this, the ToF analyzer 40 is configured to analyze the ions received in a drift region of the analyzer 40 (the drift region is a high vacuum (e.g., <1×10 -5 The system is configured to pass ions along an ion path within a pressure range of 100 psi (100 psi) at 100 psi (mbar) and measure the time it takes for the ions to pass along the ion path (drift time). The ions may be accelerated into the drift region by the electric field and detected by an ion detector located at the end of the ion path. The acceleration may cause ions with a relatively low m / z to achieve a relatively high velocity and reach the ion detector before ions with a relatively high m / z. Thus, the ions reach the ion detector after a time determined by their velocity and the length of the ion path, which allows the m / z of the ion to be determined. Each ion or group of ions arriving at the detector may be sampled by the detector and the signal from the detector may be digitized. The processor may then determine a value indicative of the time of flight and / or m / z of the ion or group of ions. Data for multiple ions may be collected and combined to generate a time-of-flight ("ToF") spectrum and / or a mass spectrum.

[0086] The ToF mass analyzer 40 may be any suitable ToF analyzer. In general, the ToF analyzer 40 may include an ion injector disposed at the beginning of an ion path and an ion detector disposed at the end of the ion path. The analyzer 40 may be configured to analyze the ions by determining the arrival time of the ions at the detector (i.e., the time it takes for the ions to travel from the injector, through the ion path, and arrive at the detector).

[0087] The ion implanter may be in any suitable form, such as, for example, one or more (e.g., orthogonal) accelerating electrodes. However, in certain embodiments, the ion implanter comprises an ion trap. The ion trap may be configured to receive ions (from the fragmentation device 30) and may be configured to accumulate packets of ions, for example, by accumulating the ions during an accumulation period. The ion trap may be configured to inject each accumulated packet ion into an ion path (e.g., by accelerating the ion packet along the ion path), which may then travel along the ion path to a detector.

[0088] The detector may be any suitable ion detector, such as one or more conversion dynodes, optionally followed by one or more electron multipliers, one or more scintillators, and / or one or more photon multipliers, etc. The detector may be configured to detect ions received at the detector and may be configured to produce a signal indicative of the intensity of the ions received at the detector as a function of (arrival) time. The m / z of the ions may then be determined from the measured arrival times.

[0089] The ion path may have any suitable form, such as being linear in the case of a linear ToF analyzer, or including one or more reflections in the case of a ToF analyzer comprising a reflectron or a multi-reflection time-of-flight (MR-ToF) analyzer. The ion path may include a circular segment.

[0090] In a particular embodiment, the analyzer 40 is a multi-reflection time-of-flight (MR-ToF) analyzer. The analyzer 40 may thus comprise two ion mirrors spaced apart and facing each other in a first direction X. Each mirror is elongated generally along a drift direction Y between a first end and a second end, the drift direction Y being orthogonal to the first direction X. The ion injector may be located near the first ends of the ion mirrors and may be configured to inject ions into the space between the ion mirrors. The detector may be located near the first ends of the ion mirrors and may be configured to detect ions after they have completed multiple reflections between the ion mirrors. The analyzer may be configured to analyze ions by injecting ions from an ion injector into the space between the ion mirrors, such that the ions may follow a zigzag ion path having multiple reflections in direction X between the ion mirrors while (a) drifting along drift direction Y towards the second end of the ion mirror, (b) reversing the drift direction velocity near the second end of the ion mirror, and (c) drifting back to the first end of the ion mirror along drift direction Y. The ions may then be moved to a detector for detection.

[0091] It should be noted that Figure 2 is merely schematic, and that the analytical instrument can, and in embodiments does, include any number of one or more additional components. For example, the instrument will typically include one or more ion transport stages disposed between the various illustrated components 10, 20, 30, 40 and configured to transport ions from one component to the next. The one or more ion transport stages may include any suitable arrangement of one or more ion guides, lenses, and / or other ion optical devices.

[0092] 2, the instrument may be under the control of a control unit 50, such as a suitably programmed computer, which may be configured to control the operation of various components of the instrument, including mass filter 20, fragmentation device 30, and analyzer 40, for example to cause the instrument to operate in a particular mode of operation and / or perform methods described herein. The control unit 50 may also receive and process data from the various components, including, for example, mass spectral data from analyzer 40 according to embodiments described herein.

[0093] The device may be capable of operating in various modes of operation, including an MS1 ​​mode of operation and an MS2 mode of operation.

[0094] In the MS1 (or "full mass scan") mode of operation, for example, the mass filter 20 is operated in its transmission mode of operation and the fragmentation device 30 is operated in its non-fragmentation mode of operation such that unfragmented ("precursor" or "parent") ions of a wide m / z range (e.g., full mass range) are analyzed by the analyzer 40.

[0095] In the MS2 mode of operation, the mass filter 20 operates in its filtering mode of operation and the fragmentation device 30 operates in its fragmentation mode, e.g., such that precursor ions in a selected narrow m / z range are fragmented and the resulting fragment ("product" or "daughter") ions are analyzed by the analyzer 40. In the MS2 mode of operation, the center of the (narrow) m / z window of the mass filter can be changed continuously between each of a plurality of different m / z values, e.g., to successively select (and fragment) each of a plurality of different precursor ions with respective different m / z. In a data dependent acquisition (DDA) MS2 mode of operation, the plurality of different m / z values ​​can correspond to a plurality of different precursor ions identified from the corresponding MS1 data (i.e., the full mass scan). In a data independent acquisition (DIA) MS2 mode of operation, the plurality of different m / z values ​​can be acquired from a predetermined (fixed) list, i.e., without reference to the MS1 data.

[0096] The instrument may also be configured to perform one or more higher order fragmentation (MS) such as, for example, an MS3 mode of operation. N ) mode of operation whereby precursor ions are fragmented, at least some of the resulting fragment ions are themselves fragmented, and second generation fragment ions (“granddaughter ions”) are analyzed by the analyzer 40.

[0097] The ToF analyzer 40 is operable in at least two modes of operation: a first mode of operation in which ions are moved along a flight path (between the ion implanter and the ion detector) having a first length, and a second mode of operation in which ions are moved along a flight path (between the ion implanter and the ion detector) having a second, longer length. Increasing the length of the ion flight path in the second mode of operation has the effect of increasing the resolution of the analyzer, but reducing the m / z range of ions that can be analyzed. The second mode of operation may therefore be referred to as a "zoom" mode of operation (as the analyzer actually "zooms in" on a narrower m / z region of the m / z spectrum). The ToF analyzer 40 may optionally be operable in one or more further modes of operation in which ions are moved along flight paths (between the ion implanter and the ion detector) having one or more different lengths.

[0098] A time-of-flight (ToF) mass analyzer can be configured to have a variable path length in any suitable manner. In general, the ToF analyzer 40 can have one or more ion reflectors (such as one or more ion mirrors and / or one or more reflectrons) configured to reflect ions. The ion path may be lengthened by increasing the number of reflections in one or more ion reflectors that an ion takes before being detected.

[0099] Thus, for example, in a first mode of operation, ions may not be reflected by an ion reflector before being detected, and in a second mode of operation, ions may be reflected one or more times by one or more ion reflectors before being detected. Alternatively, in the first mode of operation, ions may be reflected one or more times by one or more ion reflectors before being detected, and in the second mode of operation, ions may be reflected more times by one or more ion reflectors before being detected (than in the first mode). For example, the ToF analyzer may be switchable between a first ("V") mode of operation in which ions are reflected once at an ion reflector (reflectron) before being detected, and a second ("W") mode of operation in which ions are reflected three times (in two ion reflectors) before being detected.

[0100] The ion path may also, or alternatively, be lengthened by increasing the number of passes through a circular segment of the ion path that an ion takes before being detected.

[0101] In a particular embodiment, analyzer 40 is a multi-reflecting time-of-flight (MR-ToF) mass analyzer capable of operating in a single-pass "normal" mode of operation and a multi-pass "zoom" mode of operation (described in more detail below).

[0102] Figure 3 shows in more detail a schematic of a mass spectrometer suitable for carrying out the methods of the various embodiments. The instrument is a hybrid instrument incorporating an MR-ToF analyzer 40 (of the type described in US Patent No. 9,136,101), a quadrupole mass filter 20, and an Orbitrap™ analyzer 60. The instrument also includes an electrospray source 10, a collision cell 30, and various ion guides for a complete mass analyzer, etc. It will be understood that the instrument shown in Figure 3 is a non-limiting example and that many variations are possible.

[0103] In the embodiment shown in Figure 3, the instrument's ion source 10 is an electrospray ionization (ESI) ion source. The instrument includes a transfer tube 21, an ion funnel 22, a quadrupole pre-filter ion guide 23, and a vacuum interface including a so-called "bent flat pole" ion guide 24. The ion guide 24 may be of the design described in US Patent No. 9,536,722.

[0104] The instrument also includes a mass filter in the form of a quadrupole mass filter 20, an ion trap 31 in the form of a curved linear ion trap ("C-trap"), and a collision cell 30 in the form of an ion routing multipole collision cell ("IRM"). Ions from the ion source 10 may be accumulated in the C-trap 31 and / or the collision cell 30 by opening and closing gate electrodes located in a charge detector assembly 26 that is disposed between the C-trap 31 and the mass filter 20.

[0105] The instrument includes a time-of-flight (ToF) mass analyzer 40 in the form of a multi-reflecting time-of-flight (ToF) mass analyzer. In the instrument shown in Figure 3, the analyzer is of the tilting mirror type described in U.S. Patent No. 9,136,101, although it will be understood that any type of ToF analyzer may be used.

[0106] As shown in Figure 3, the instrument includes a multipole ion guide 32 that allows ions to be transferred from the collision cell 30 to a time-of-flight mass analyzer 40. The time-of-flight mass analyzer 40 includes an extraction trap 41 whereby ions are delivered from the collision cell 30 through the multipole ion guide 32 to the extraction trap 41. The ions are accumulated and cooled in the extraction trap 41.

[0107] The extraction trap 41 may incorporate two trapping regions, one at relatively high pressure for rapid ion cooling and a second low pressure region for ion extraction. Ions are cooled in the high pressure region and then transferred to the low pressure region where they are pulsed into the ToF analyzer via a pair of deflectors 42. The ions oscillate between a pair of mirrors 43, which are tilted relative to each other such that the ion path is slowly deflected back to the detector 44. Compensation stripe electrodes 45 counter loss of ion focusing otherwise caused by mirror non-parallelism.

[0108] As also shown in Figure 3, the instrument may optionally include a second mass analyzer in the form of an electrostatic mass analyzer 60, such as an orbital ion trap mass analyzer, more specifically an Orbitrap™ FT mass analyzer manufactured by Thermo Fisher Scientific. This hybridized instrument is described in more detail in U.S. Patent No. 10,699,888, the contents of which are incorporated herein by reference.

[0109] Ions may be collected in the ion trap 31 and then ejected orthogonally into the Orbitrap™ analyzer 60 for analysis without entering the collision or reaction cell 30, or the ions may be transferred axially into the collision or reaction cell 30. Ions sent to the collision or reaction cell 30 may either be fragmented by collisions with collision gases and / or reagents in the collision cell 30, or simply cooled by collisions with lower energy gases that cause the ions to fragment. Once accumulated in the collision cell 30, the ions may either be ejected (via the multipole ion guide 32) into the mass analyzer 40 for analysis, or ejected (via the C-trap 31) into the Orbitrap™ analyzer 60 for analysis.

[0110] 4 and 5 show schematic details of exemplary embodiments of a variable path-length analyzer 40. In these embodiments, the analyzer 40 is a multi-reflecting time-of-flight (MR-ToF) mass analyzer capable of operating in a single-pass "normal" mode of operation and in a multi-pass "zoom" mode of operation.

[0111] 4 and 5, the multi-reflecting time-of-flight analyzer 40 includes a pair of ion mirrors 43a, 43b spaced apart and facing each other in a first direction X. The ion mirrors 43a, 43b are elongated along an orthogonal drift direction Y between first and second ends.

[0112] An ion source (injector) 41, which may be in the form of an ion trap, is located at one end (first end) of the analyzer. The ion source 41 may be positioned and configured to receive ions from the fragmentation device 30. Ions may be accumulated in the ion source 41 before being injected into the space between the ion mirrors 43a, 43b. As shown in Figures 4 and 5, ions may be injected from the ion source 41 at a relatively small injection angle or drift directional velocity, producing zigzag ion trajectories, whereby different oscillations between the mirrors 43a, 43b are spatially separated.

[0113] One or more lenses and / or deflectors may be positioned along the ion path between the ion source 41 and the ion mirror 43b that the ions first encounter. For example, as shown in Figures 4 and 5, a first out-of-plane lens 46, an injection deflector 42a, and a second out-of-plane lens 47 may be positioned along the ion path between the ion source 41 and the ion mirror 43b that the ions first encounter. Other configurations may be possible. In general, the one or more lenses and / or deflectors may be configured to suitably condition, focus, and / or deflect the ion beam, i.e., to cause the ion beam to take a desired trajectory through the analyzer.

[0114] The analyzer 40 also includes another deflector 42b disposed along the ion path between the ion mirrors 43a, 43b. As shown in Figures 4 and 5, the deflector 42b may be disposed approximately equidistant along the ion path between the ion mirrors 43a, 43b after its first ion mirror reflection (at ion mirror 43b) and before its second ion mirror reflection (at the other ion mirror 43a).

[0115] The analyzer also includes a detector 44. The detector 44 may be any suitable ion detector configured to detect ions and, for example, record the intensity and arrival time associated with the arrival of the ions at the detector. Suitable detectors include, for example, one or more conversion dynodes, optionally followed by one or more electron multipliers, or the like.

[0116] In its "normal" mode of operation, ions are injected from the ion source 41 into the space between the ion mirrors 43a, 43b such that ions follow a zigzag ion path that reflects multiple times in the X direction between the ion mirrors 43a, 43b while (a) drifting along drift direction Y from the deflector 42b towards the opposite (second) ends of the ion mirrors 43a, 43b, (b) reversing the drift direction velocity near the second ends of the ion mirrors 43a, 43b, and (c) drifting back along drift direction Y towards the deflector 42b. The ions may then be transferred from the deflector 42b to the detector 44 for detection.

[0117] In the analyzer of Fig. 4, both ion mirrors 43a, 43b are tilted with respect to the X direction and / or the drift Y direction. Alternatively, only one of the ion mirrors 43a, 43b can be tilted, for example with the other of the ion mirrors 43a, 43b arranged parallel to the drift Y direction. Typically, the ion mirrors are at a non-constant distance from each other in the X direction along most or all of their length in the drift direction Y. The drift direction velocity of the ions towards the second end of the ion mirror is opposed by an electric field resulting from the non-constant distance of the two mirrors from each other, which causes the ions to reverse their drift direction velocity near the second end of the ion mirror and drift back along the drift direction towards the deflector.

[0118] The analyzer shown in Figure 4 further comprises a pair of correction stripe electrodes 45. Ions moving down the drift length are slightly deflected each time they pass a mirror 43a, 43b, and the additional stripe electrodes 45 are used to correct time-of-flight errors caused by changes in the distance between the mirrors. For example, the stripe electrodes 45 can be electrically energized such that the period of ion oscillation between the mirrors is substantially constant along the entire drift length (despite the fact that the distance between the two mirrors is not constant). The ions are eventually reflected back down the drift space and focused onto the detector 44.

[0119] Further details of the tilted mirror multi-reflecting time-of-flight mass analyzer of FIG. 4 are described in US Pat. No. 9,136,101, the contents of which are incorporated herein by reference.

[0120] In the analyzer of Figure 5, the ion mirrors 43a, 43b are parallel to one another. In this embodiment, the analyzer includes a second deflector 48 at the second end of the ion mirrors 43a, 43b to cause the ions to reverse their drift direction velocity near the second end of the ion mirror and drift back along the drift direction towards the deflector.

[0121] Also, as shown in FIG. 5, in this embodiment, a lens may be included in the implant deflector 42a and / or the deflector 42b. The ion beam may then expand its short path into the analyzer before impinging on the long focal lens, which has the effect of focusing the ion beam along its length. The lens may be an elliptical drift focusing lens mounted in the deflector 42b. The second deflector 48 may also include a lens, but is used to reverse the beam direction while maintaining control of the focal properties.

[0122] Further details of the single lens multi-reflecting time-of-flight mass analyser of Figure 5 are described in UK Patent No. 2,580,089, the contents of which are incorporated herein by reference.

[0123] In the analyzers shown in Figures 4 and 5, the ion beam is allowed to spread relatively widely (in the drift direction Y) over most of its flight path. This is in contrast to multi-reflection time-of-flight (MR-ToF) mass analyzers that use a set of periodic lenses to focus the ion beam along its entire flight path, as described, for example, in A. Verenchikov et al., Journal of Applied Solution Chemistry and Modelling, 2017, 6, 1-22. An important advantage of allowing the ion beam to spread widely over most of its flight path is that space charge effects are reduced, which can be a significant issue for time-of-flight analyzers, especially when analyzing labeled analyte ions. Nevertheless, the embodiments described herein are also applicable to other MR-ToF analyzer designs, such as Verenchikov-type MR-ToF analyzers.

[0124] 4 and 5, the fact that the ion beam is relatively wide in the drift dimension Y means that the deflector 42b should be able to accommodate such a wide beam without introducing clipping or non-uniform deflection. A preferred deflector design is a trapezoidal or prismatic deflector.

[0125] Thus, the deflector 42b may comprise a trapezoidal or prismatic electrode located above the ion beam and another trapezoidal or prismatic electrode located below the ion beam. The electrodes may be located out of the plane of deflection, thereby allowing the electrodes to be easily made wide enough to accommodate a wide ion beam (at least compared to more conventional deflection plates that would be located on either side of the beam). The electrodes may be angled with respect to the ion beam, such that when a suitable (DC) voltage is applied to the electrodes, the resulting electric field induces a deflection of the ion beam. Ions may experience a relatively strong electric field at the edges of the angled electrodes, inducing a deflection. A suitable deflection voltage is on the order of ± a few volts, ± tens of volts, or ± hundreds of volts. The deflector should be (and in embodiments is) configured to be able to deflect the ion beam by a desired (selected) angle. The angle at which the ion beam is deflected by the deflector may be adjustable, for example, by adjusting the magnitude of the (DC) voltage applied to the deflector.

[0126] In an embodiment, the multi-reflecting time-of-flight (MR-ToF) mass analyzer is operated in a multi-pass "zoom" mode of operation. In this mode of operation, ions are cycled multiple times in the drift direction Y within the analyzer. Increasing the number of cycles N increases the length of the ion path that the ions take within the analyzer (between the injector 41 and the detector 44), thereby increasing the resolution of the analyzer. In a Verenchikov analyzer, this can be done by controlling the voltage to the entrance lens. In the case of the analyzer shown in Figures 4 and 5, the deflector 42b before the analyzer, which is usually used to reduce the injection angle and / or optimize the number of oscillations in one drift pass, can (also) be used to reverse the drift direction velocity of the ions to make them complete another cycle through the analyzer.

[0127] Thus, in a multi-pass "zoom" mode of operation, ions are made to complete multiple cycles within the analyzer 40, in which, in each cycle, the ions drift in drift direction Y from the deflector 42b (or entrance lens) towards the opposite (second) ends of the ion mirrors 43a, 43b and then return to the deflector 42b (or entrance lens). In each cycle, the ions also complete multiple reflections in the X direction between the ion mirrors. Thus, in each cycle, the ions follow a zig-zag ion path through the space between the ion mirrors 43a, 43b.

[0128] In the analyzer shown in Figures 4 and 5, an initial cycle may begin by injecting ions from the injector 41 into the space between the ion mirrors 43a, 43b. The ions may be reflected off one of the ion mirrors 43b and then travel to the deflector 42b. An appropriate (e.g., relatively small) voltage may be applied to the deflector 42b such that the ions exit the deflector 42b in a direction toward the second end of the ion mirror. In the presence of the deflector 42b, the ions follow a zigzag ion path with multiple reflections in direction X between the ion mirrors 43a, 43b while (a) drifting along drift direction Y from the deflector 42b toward the second end of the ion mirror, (b) reversing the drift direction velocity near the second end of the ion mirror, and (c) drifting back along drift direction Y toward the deflector 42b.

[0129] After the ions have completed this initial cycle, each further cycle is initiated by using the deflector 42b to reverse the drift direction velocity of the ions (near the first end of the ion mirror). To do this, a suitable voltage may be applied to the deflector 42b that causes the ions to exit the deflector 42b with a drift direction velocity opposite to the drift direction velocity with which the ions initially entered the deflector 42b. This voltage may be applied during the period in which the ions are expected to return to the deflector 42b. A suitable deflection voltage to reverse the drift direction of the ions is on the order of several hundred volts.

[0130] The deflector may be used to reverse the drift directional velocity of the ions one or more times. Thus, the method may include causing the ions to complete multiple (N) cycles within the analyzer, where a first cycle is initiated by injecting the ions into a space between the ion mirrors, and after the ions have completed the first cycle, each further cycle may be initiated by reversing the drift directional velocity of the ions using the deflector.

[0131] After the ions have completed a desired number (N) of cycles within the analyzer, the ions are allowed to travel from the deflector 42b to the detector 44 for detection. To do this, an appropriate voltage may be applied to the deflector 42b, and the ions are ejected from the deflector 42b in a direction towards the detector 44. The ions may be reflected off the (other) one of the ion mirrors 43a before traveling to (and being detected by) the detector 44.

[0132] FIG. 6 illustrates this zoom mode of operation diagrammatically. As shown in FIG. 6, ions are injected from the ion trap source 41 through a deflector 42a between the mirrors at a relatively high angle. After a first half oscillation, the ions pass through a second prism-shaped deflector 42b that reduces the injection angle by approximately half. The oscillating ions then drift up the length of the elongated mirror and are returned by a set tilt of the mirror, for example in the case of the analyzer of FIG. 4. By the time the ions return to this second deflector 42b, the voltage may be switched from an injection / extraction potential of about −150V to a trapping potential of about +350V, which reflects the ion beam back into the analyzer body for the second pass. After the desired number of passes (N) have been traversed by the ions, the deflector 42b is switched back to the injection / extraction potential and the ions escape to the detector 44.

[0133] The embodiments are directed to a method of analyzing labeled analyte molecules, such as peptides labeled with isobaric tags. The analyte molecules may be labeled with a set of isobaric tags. Isobaric tags and their use have been described in the literature (see, for example, Thompson et al, Anal. Chem., 2003, 75, 1895-1904). A set of isobaric tags is a set of tag molecules that have (approximately) the same mass, but that produce characteristic reporter ions of different masses upon fragmentation of the labeled analyte ions. Suitable isobaric tags include tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ). Each tag may, for example, include (at least) a reporter region and a balancer region, such that each labeled analyte molecule includes (at least) a reporter region, a balancer region, and an analyte molecule (e.g., a peptide).

[0134] The labeled analyte molecules may be provided in a solution, the solution may be separated using a separation device, and the separated solution from the separation device may be provided to the ion source 10 for ionization. The ion source 10 may ionize the labeled analyte molecules to produce labeled analyte ions.

[0135] The labeled analyte ions may optionally be first analyzed in an MS1 ​​mode of operation to provide MS1 data. The MS1 data may include one or more ion peaks, each ion peak corresponding to a labeled analyte ion having a particular m / z (i.e., a particular precursor).

[0136] The labeled analyte ions may then be analyzed in an MS2 mode of operation to provide MS2 data. Each precursor of interest identified from the MS1 data may be used to define an m / z window for the mass filter 20. The mass filter 20 may then sequentially step through each m / z window corresponding to each precursor of interest. In this MS2 mode of operation, the fragmentation device 30 is operating in a fragmentation mode of operation. Thus, the mass filtered labeled analyte ions are sequentially fragmented in the fragmentation device 30. When the labeled analyte ions are fragmented, reporter ions may be generated along with the analyte molecule fragment ions (the reporter ions are ions of the reporter region).

[0137] Additionally or alternatively, complementary ions may be generated (complementary ions are ions of the combined balancer region and analyte molecules). The generation of complementary ions requires careful control of the collision energy, e.g., to a relatively low value. This low collision energy may also generate some peptide fragments (but less efficiently than a relatively high collision energy). Alternatively, two different collision energies may be used, i.e., a low collision energy may be used to generate complementary ions and a high collision energy may be used to generate peptide fragments. Thus, for each target precursor, two injections into the fragmentation device 30 may be provided with different collision energies. The resulting fragment ions may be injected (and analyzed) into the analyzer 40 either in a single injection (as a single ion packet) or in two injections (as two ion packets).

[0138] The analyte fragment ions and reporter or complementary ions are analyzed using an analyzer 40 .

[0139] With reference to the instrument shown in FIG. 3, in an embodiment, multiplexed and tagged samples are delivered from a liquid chromatography (LC) separation device to an electrospray ion source 10, ionized, and passed through a vacuum to a quadrupole 20.

[0140] To perform a full mass scan (MS1), the quadrupole 20 is configured to minimize its level of separation and transmits ions to the C-Trap 31 and Orbitrap™ analyzer 60 or to the MR-ToF analyzer 40 for analysis.

[0141] To perform a fragment scan (MS2), suitable precursors, either identified from the MS1 scan (Data Dependent Acquisition (DDA)) or run off from a fixed list (Data Independent Acquisition (DIA)), are sent to the collision cell 30 at increased energy and fragmented. The fragments are then delivered to the MR-ToF analyzer 40 for analysis, where TMT reporter ion peaks are measured for quantification and peptide fragments are detected for precursor identification.

[0142] This DDA process is illustrated in Figure 7. As shown in Figure 7, an MS1 ​​scan is first performed (step 60) using either the Orbitrap™ analyzer 60 or the ToF analyzer 40. In the instrument design of Figure 3, the Orbitrap™ analyzer 60 may be advantageous for performing MS1 precursor identification, although it is possible to use only the ToF analyzer 40 and the Orbitrap™ analyzer 60 is not a required part of the instrument.

[0143] A precursor ion is then selected from the MS1 scan (step 62) and an MS2 scan is performed on that precursor ion using the ToF analyzer 40 (step 64). As shown by step 66, this process is then repeated until the list of precursor ions identified from the MS1 scan is exhausted. Once the list of precursor ions is exhausted, a new MS1 scan is performed and the process repeats itself.

[0144] This DDA process is advantageous for the TMT method described herein (and may not be preferred for DIA processes), because the quadrupole separation width tends to be too wide to blindly cover the ~300-1100 mass range (as is typically done in DIA methods) fast enough to match the time scale of the chromatographic peaks from the separation device, while a very narrow separation window is preferred to minimize interference between co-separated peptides. However, complementary ion methods have fewer difficulties with such interferences and are much more compatible with DIA.

[0145] In an embodiment, the zoom mode described above may or may not be applied in the MS1 scan, however, according to an embodiment, the zoom mode is applied to the MS2 scan.

[0146] In this regard, it should be noted that the reporter ions appear very early in the mass spectrum, and in fact are lower than the instrument can normally be set to detect most of the precursor fragment ions. They are also closely spaced over several m / z units. The consequence of this is that the zoom mode can be advantageously applied to only the reporter ions, allowing the remaining fragment ions to pass through the instrument uninterrupted. It is also possible to apply the zoom mode only to the complementary ions, which are also closely spaced over several m / z units.

[0147] Thus, in an embodiment, analyte fragment ions are analyzed using a time-of-flight mass analyzer 40 operating in a first mode of operation which causes ions to travel along a flight path having a first length. Reporter ions or complementary ions are analyzed using a time-of-flight mass analyzer 40 operating in a second mode of operation which causes ions to travel along a flight path having a second, longer length. It is recognized that the wide m / z range normal mode of operation of the analyzer is particularly suitable for analysis of peptide fragment ions (which typically appear over a relatively wide m / z range) and the narrow m / z range, but higher resolution zoom mode of operation is particularly suitable for analysis of reporter ions (which typically appear within a relatively narrow m / z range at a relatively low m / z) or complementary ions.

[0148] The m / z and / or intensity information obtained for the analyte fragment ions can be used to identify the analyte molecules, and the m / z and / or intensity information for the reporter ions or complementary ions can be used to quantify the analyte molecules.

[0149] In the MR-ToF instrument of Figures 3 and 4, with a flight path of 20-25 m, all injected ions pass through the second deflector 42b relatively quickly, within about 100 ps. The TMT reporter ions with m / z of about 128 return to the deflector 42b after about 250 ps, ​​thereby leaving a large overhead time for the deflector 42b to be switched to its internal trap zoom mode. The reporter ions can then be reflected back into the instrument within a few microseconds, and the deflector 42b can be switched back to its transmission mode, thereby allowing the peptide fragment ions to leave the system along with the TMT reporter ions returning from their second pass. This has the effect of shifting the reporter region to a location beyond 500 ps (570 ps in the actual experiment, but this varies substantially with analyzer tuning), but this should be understood by the data analysis software.

[0150] In some embodiments, multiple short deflector voltage pulses can be aligned with the return of the TMT reporter ions, causing these ions to pass through the analyzer multiple times for even higher levels of performance.

[0151] Figure 8 shows a scheme in which reporter ions are sent for five passes through the analyzer 40. As shown in Figure 8, the voltage applied to deflector 42b is pulsed from its injection / extraction potential (approximately -150V) to its trapping potential (approximately +350V) in time with the return of the TMT reporter ions, so that only the reporter ions are sent for five passes through the analyzer 40, while the peptide fragment ions make one normal pass through the analyzer 40.

[0152] Thus, in an embodiment, the method includes injecting an ion packet containing analyte fragment ions and reporter or complementary ions from an ion injector 41 into the space between the ion mirrors 43a, 43b. The ions complete a first cycle in which they (a) drift along a drift direction Y from the deflector 42b or lens towards the second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) drift back along the drift direction Y towards the deflector 42b or lens while following a zigzag ion path having multiple reflections in direction X between the ion mirrors 43a, 43b. Only the analyte fragment ion region is then allowed to move from the deflector 42b or lens to the detector 44 for detection. The deflector 42b or lens is used to reverse the drift direction velocity of the reporter ions or complementary ions (only) to cause them to complete a further cycle of (a) drifting along drift direction Y from the deflector 42b or lens towards the second end of the ion mirror, (b) reversing the drift direction velocity near the second end of the ion mirror, and (c) following a zigzag ion path with multiple reflections in direction X between the ion mirrors 43a, 43b while drifting back along drift direction Y towards the deflector 42b or lens. This step may be optionally repeated, for example, to allow the reporter ions or complementary ions to complete a desired number N of passes within the analyzer 40. Finally, the reporter ions or complementary ions are transferred from the deflector 42b or lens to the detector 44 for detection.

[0153] This mode of operation has a greater advantage in terms of TMT resolution, but signal loss has been noted experimentally for multiple passes (see FIG. 11), and notches in the deflector voltage become notches in the mass spectrum. Resolution increases with the number of passes, but so does signal loss, and the possibility that the notches will trap peptide fragment ions and generate false positive peaks increases. Relatively fast deflector switching is required for this operation, as is a minimum dwell time within the deflector 42b itself, which is typically around 1 ps, so the speed of power supply switching seems more critical. Prism voltage switch timing can be calibrated and optimized for each instrument.

[0154] The focal plane position of ions arriving at the detector should be aligned to the detector surface for best resolution. However, the focal plane position may be shifted for zoom mode relative to non-zoomed normal mode. Usually, focal plane adjustment can be performed by small perturbations of mirror voltages, but this is generally not feasible when ions with mixed trajectories are flying together through the analyzer.

[0155] In this regard, it should be noted that an advantage of the method is that only the zoomed reporter ions require the highest resolution, whereas the peptide fragment ions require much less demanding resolution. Thus, in some embodiments, the focal plane may be optimized only for the zoomed ions. Additionally or alternatively, it is possible to take advantage of the fact that the reporter ions are the lowest m / z ions in the spectrum, emerge earliest from the trap 41, and therefore can be subjected to different controlled voltages in the trap 41 (e.g., a slow rising extraction pulse, etc.), in the injection optics 46, 42a, 47, 42b, in the fringes of the mirrors 43a, 43b, and / or at the detector 44 to adjust the focal plane without affecting other ions. Additionally or alternatively, it is possible to employ m / z-dependent ion energy correction methods, such as those described in U.S. Pat. No. 10,727,039, the contents of which are incorporated herein by reference. Additionally or alternatively, a small oscillating voltage of controlled frequency and phase can be applied to mirrors 43a, 43b that are in resonance with the oscillations of the low m / z reporter ions to provide a focal plane adjustment that affects only the reporter ions.

[0156] FIG. 9 shows a workflow according to a further embodiment in which reporter ion measurements are not made in the same scan as the peptide fragments, but instead are measured in a separate scan.

[0157] As shown in Figure 9, an MS1 ​​full mass scan is first performed (step 70) and precursor ions are selected from the MS1 scan (step 72). A first MS2 scan is then performed using zoom mode to detect TMT reporter ions (step 74), and then a second MS2 scan is performed using normal operation mode to detect peptide fragment ions (step 76). The order of steps 74 and 76 can be reversed. As shown by step 78, this process is then repeated until the list of precursor ions identified from the MS1 scan is exhausted. Once the list of precursor ions is exhausted, a new MS1 scan is performed and the process repeats itself.

[0158] Thus, in an embodiment, the analyte fragment ions are analyzed as one or more first ion packets injected into the analyzer 40, and the reporter ions or complementary ions are analyzed as one or more second, different ion packets injected into the analyzer 40.

[0159] This method has the advantage that instrument settings can be optimized for each of the two MS2 scans, e.g. by adjusting mirrors 43a, 43b for resolution and / or by optimizing RF and DC voltages for different target mass ranges of reporter ions and peptide fragment ions, e.g. to optimize the collision energy for each of the two scans, to optimize the injection voltage applied to the ion trap 41 for each of the two scans and / or to optimize the isolation window of the mass filter for each of the two scans.

[0160] In these embodiments, a notch method similar to that shown by FIG. 8 may still be optionally used for TMT scans, except that deflector 42b is set to an extreme voltage to remove all peptide fragment ions from the spectrum, rather than being set to transmit ions between the notches.

[0161] These embodiments have additional costs in terms of raw sensitivity and acquisition speed, but the maximum acquisition speed of the MR-ToF analyzer 40 is many times higher than an Orbitrap™ analyzer operating at 50 Hz, and some sensitivity can be recovered by separate optimization of collision energy (higher energies are better for low m / z ions such as reporter ions, but less favorable for mid / high m / z fragment ions), trap RF, etc. Advantageously, the quadrupole 20 isolation window may be narrowed for TMT scans to eliminate interferences and widened for peptide scans to maximize sensitivity. Generally, about 10,000 detected ions are preferred for peptide identification, while far fewer ions are required for reporter ion quantification, especially given the single ion level detection offered by ToF analyzers.

[0162] Figure 10 shows experimental results of space charge induced resolution loss for the analyzer of Figure 4 for single pass and zoom modes using MRFA ions generated from a Flexmix™ sample. It can be seen that in zoom mode not only is the top end resolution nearly doubled, but the resilience to space charge is also nearly doubled (i.e., the number of ions in the peak at which the 50K resolution requirement is achieved is nearly doubled).

[0163] Figure 11 shows plots of experiments where the level of zoom mode (i.e. the number of passes that ions drift up and down the analyzer) is varied. Three passes are found to be optimal for achieving high resolution and minimal signal loss with this analyzer design. However, other MR-ToF analyzer designs may behave differently, for example 500K resolution has previously been observed on analyzers incorporating periodic lenses (e.g. as described in GB Patent No. 2,403,063).

[0164] From the above, it will be appreciated that embodiments provide a way to enhance detection of a TMT reporter region (or complementary ion region) using a zoom mode. This may be done as a scan in its own right, with optimized isolation windows, collision energies, etc., or may be integrated into a wider scan, for example via notched switching of the deflector 42b voltage.

[0165] According to embodiments, TMT reporter ions can be identified via zoom mode using MR-ToF analyzers that would otherwise not meet the requirement of >50K resolution at the desired dynamic range. Although reporter regions often contain thousands of ions, MR-ToF analyzers may be capable of resolving 100:1 TMT doublets with only a few thousand ions total. This results in a significant limit to the dynamic range over which smaller peaks are observable. Figures 10 and 11 show the inherent advantages of using zoom mode to circumvent these limitations.

[0166] As mentioned above, integrating the zoom mode TMT scan into the standard main scan means that there is no loss in repetition rate or sensitivity. However, this is optional, and since ToF analyzers are already much faster than required, it may be advantageous to perform these scans separately. Relatively long / slow throughput experimental cycles may be implemented due to the need for high quality chromatographic separation. This may be improved by having a separate narrow separation window reporter ion scan with lower interference. Sensitivity becomes a more significant issue, but this can be remedied by more optimal instrument settings.

[0167] The zoom mode can also increase the resolution enough to enable 16-plex or 18-plex complementary ion TMT methods, which are not currently possible in any ToF system. The improved resolution is also valuable for lower multiplexed complementary ions, for example to resolve chemical noise and co-separated peptides, allowing for larger separation widths and even higher throughput.

[0168] While various specific embodiments have been described above, various alternative embodiments are possible.

[0169] For example, the methods described herein can be performed on any instrument incorporating a ToF analyzer capable of varying ion flight path length, for example, by changing the number of reflections taken by ions passing through the analyzer. For example, this can be from zero (linear ToF) reflections to a single reflection. This can also include switching between a low-sensitivity W-shaped ion flight path and a high-sensitivity low-resolution V-shaped ion flight path. In general, all multi-reflection and multi-turn ToFs can be used to perform the methods described herein.

[0170] With reference to the embodiment described in connection with Figure 9, instead of the alternating sequence of reporter ion scans and peptide fragment ion scans shown in Figure 9, a sequence of TMT reporter ion scans can be made, followed by a sequence of peptide fragment ion scans. This can be beneficial when there is a relatively slow voltage shift that needs to occur when switching from normal to zoom mode. For example, a regulated power supply can take several milliseconds to regulate.

[0171] A more efficient method can be implemented in which tagged analytes are identified by one MS2 scan and then a second MS2 scan is performed to quantify the tag.

[0172] Similar levels of interference can be detected from MS1 scans. Chemical interference in reporter ions comes from fragmentation of co-isolated tagged peptides. In principle, it may be possible to see these interfering peptide peaks in MS1 ​​scans within the same mass isolation window as the target peptides. Based on the MS1 scan (or analyte MS2 scans, which usually still show substantial amounts of unfragmented precursor ions), the isolation window may be dynamically set to balance transmission and minimize interference.

[0173] The MS2 TMT scan (or combined TMT / precursor scan) may be replaced by an MS3 scan, which is less sensitive but better for low interference quantification. Such methods may be performed by instruments containing devices for further separation and fragmentation, such as a linear ion trap, or by passing the ions back through the quadrupole 20. Again, there is an advantage to using a more sensitive method for fragment ion detection and a cleaner method for improved reporter ion quantification.

[0174] The notched switching method described above may be more broadly applicable for extracting target analytes requiring high resolution in a range of other applications.

[0175] For example, notched switching mode may be used to pick out targets from an unknown background. This may be useful for protein DIA / DDA, where precursors may be analyzed in zoom mode to optimally resolve the isotopic envelope (requiring high resolution), while lower m / z fragment ions (with lower resolution requirements) may be successfully detected. Even standard peptide and / or regular m / z ion DDA / DIA may benefit, since better mass accuracy and separation of different precursors are obtained in the MS2 spectrum. Targeted applications such as SIM scans may also be beneficial, where it is desired that the target ions are detected with maximum performance, but the masses of any fragments are also desired.

[0176] In general, these methods may be applied to any application where one or more target ions (known or determined by previous scans) require high resolution / dynamic range against a background of unknown or low abundance ions, preferably without suffering from the m / z measurement uncertainty or sensitivity loss induced by zoom mode.

[0177] Although the invention has been described with reference to various embodiments, it will be understood that various modifications can be made without departing from the scope of the invention as set forth in the appended claims.

Claims

1. 1. A method for analyzing labeled analyte ions, the method comprising: fragmenting the labeled analyte ions to produce analyte fragment ions and reporter or complementary ions; analyzing the analyte fragment ions using a time-of-flight mass analyzer operating in a first mode of operation that moves ions along a flight path having a first length; analyzing the reporter ions or the complementary ions using the time-of-flight mass analyzer operating in a second mode of operation that moves ions along a flight path having a second length, the second length being longer than the first length.

2. the time-of-flight mass analyzer comprises one or more ion reflectors; in the first mode of operation, ions are caused to make n reflections within the one or more ion reflectors, n being an integer >= 0; 2. The method of claim 1, wherein in the second mode of operation ions are caused to make m reflections in the one or more ion reflectors, where m is an integer > n.

3. The time-of-flight mass analyzer is a multi-reflecting time-of-flight (MR-ToF) mass analyzer, the MR-ToF mass analyzer comprising: two ion mirrors facing each other and spaced apart in a first direction X, each mirror being elongated along a drift direction Y between a first end and a second end, the drift direction Y being orthogonal to the first direction X; an ion injector for injecting ions into a space between the ion mirrors, the ion injector being located proximate the first end of the ion mirror; a detector for detecting ions after the ions have completed multiple reflections between the ion mirrors, the detector being located near the first end of the ion mirror; analyzing analyte fragment ions using the analyzer operating in the first mode of operation; injecting analyte fragment ions from the ion injector into the space between the ion mirrors, wherein the ions (a) drift along the drift direction Y towards the second end of the ion mirror, (b) reverse drift directional velocity near the second end of the ion mirror, and (c) follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back to the first end of the ion mirror; and then and transferring the ions to the detector for detection.

4. analyzing reporter ions or complementary ions using the analyzer operating in the second mode of operation; (i) injecting reporter ions or complementary ions from the ion injector into the space between the ion mirrors, wherein the ions (a) drift along the drift direction Y towards the second end of the ion mirror, (b) reverse their drift directional velocity near the second end of the ion mirror, and (c) complete a first cycle in which the ions follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting back along the drift direction Y towards the first end of the ion mirror; (ii) reversing the drift directional velocity of the ions near the first end of the ion mirror such that the ions (a) drift along the drift direction Y towards the second end of the ion mirror, (b) reversing the drift directional velocity near the second end of the ion mirror, and (c) completing a further cycle in which the ions follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting back along the drift direction Y towards the first end of the ion mirror; The method of claim 3 , comprising: (iii) transferring the ions to the detector for detection.

5. the multi-reflecting time-of-flight (MR-ToF) mass analyzer further comprising a deflector or lens located near the first end of the ion mirror; analyzing reporter ions or complementary ions using the analyzer operating in the second mode of operation; (i) injecting reporter or complementary ions from the ion injector into the space between the ion mirrors, wherein the ions (a) drift along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) complete a first cycle in which the ions follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; (ii) using the deflector or lens to reverse the drift direction velocity of the ions such that the ions (a) drift along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse the drift direction velocity near the second end of the ion mirror, and (c) complete a further cycle in which the ions follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; 4. The method of claim 3, comprising: (iii) moving the ions from the deflector or lens to the detector for detection.

6. 3. The method of claim 1 or 2, wherein analyzing the analyte fragment ions comprises analyzing one or more first ion packets and analyzing the reporter ions or the complementary ions comprises analyzing one or more second, different ion packets.

7. 7. The method of claim 6, further comprising generating and / or processing and / or analyzing each first ion packet using a first set of one or more instrument parameters, and generating and / or processing and / or analyzing each second ion packet using a second, different set of one or more instrument parameters.

8. 7. The method of claim 6, further comprising generating each first ion packet using a first mass filter transmission window width, and generating each second ion packet using a second, different mass filter transmission window width.

9. 7. The method of claim 6, further comprising generating each first packet of ions using a first collision energy and generating each second packet of ions using a second, different collision energy.

10. 3. The method of claim 1 or 2, wherein analyzing the analyte fragment ions and analyzing the reporter ions or the complementary ions comprises analyzing one or more single ion packets.

11. The analyzer comprises: an ion pathway including a cyclic segment; an ion injector for injecting ions into the ion path; at least one ion reflector disposed along the ion path; a detector disposed at an end of the ion path; The method further comprising: (i) injecting ion packets comprising analyte fragment ions and reporter or complementary ions from the ion injector into the ion path such that the analyte fragment ions and the reporter or complementary ions travel along the ion path to the ion reflector; (ii) transferring the analyte fragment ions from the ion reflector to a detector for detection; (iii) causing the reporter ion or the complementary ion to complete one or more cycles along the circular segment of the ion path using the ion reflector; and then 11. The method of claim 10, comprising: (iv) transferring the reporter ion or the complementary ion from the ion reflector to the detector for detection.

12. The time-of-flight mass analyzer is a multi-reflecting time-of-flight (MR-ToF) mass analyzer, the MR-ToF mass analyzer comprising: two ion mirrors facing each other and spaced apart in a first direction X, each mirror being elongated along a drift direction Y between a first end and a second end, the drift direction Y being orthogonal to the first direction X; an ion injector for injecting ions into a space between the ion mirrors, the ion injector being located proximate the first end of the ion mirror; a deflector or lens located near the first end of the ion mirror; a detector for detecting ions after the ions have completed multiple reflections between the ion mirrors, the detector being located near the first end of the ion mirror; The method further comprising: (i) injecting ion packets comprising analyte fragment ions and reporter or complementary ions from the ion injector into the space between the ion mirrors, wherein the ions (a) drift along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) complete a first cycle in which the ions follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; (ii) transferring the analyte fragment ions from the deflector or lens to the detector for detection; (iii) using the deflector or lens to reverse the drift direction velocity of the reporter ion or the complementary ion to cause the ions to complete a further cycle in which they (a) drift along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; 11. The method of claim 10, further comprising: (iv) transferring the reporter ion or the complementary ion from the deflector or lens to the detector for detection.

13. The method further comprising: analyzing the labeled analyte ions in an MS1 ​​mode of operation to generate MS1 data and identifying one or more precursors of interest in the MS1 data; 3. The method of claim 1 or 2, wherein fragmenting the labeled analyte ions comprises sequentially selecting and fragmenting each identified precursor of interest.

14. 1. A method for analyzing a labeled analyte molecule, the method comprising: ionizing the labeled analyte molecules to produce labeled analyte ions; and analyzing the labeled analyte ions using the method of claim 1 or 2.

15. identifying the analyte molecules using mass to charge ratio (m / z) and / or intensity information from the analysis of the analyte fragment ions; 15. The method of claim 14, further comprising quantifying the analyte molecules using mass-to-charge ratio (m / z) and / or intensity information from the analysis of the reporter ions or the complementary ions.

16. 3. The method of claim 1 or 2, wherein the labeled analyte ion is an ion of a peptide labeled with an isobaric tag.

17. 1. A method of operating a Time-of-Flight (ToF) mass analyzer, comprising: an ion pathway including a cyclic segment; an ion injector for injecting ions into the ion path; at least one ion reflector disposed along the ion path; a detector disposed at an end of the ion path; The method further comprising: (i) injecting an ion packet including the first ions and the second ions from the ion injector into the ion path such that the first ions and the second ions travel along the ion path to the ion reflector; (ii) transferring the first ions from the ion reflector to the detector for detection; (iii) causing the second ions to complete one or more cycles along the circular segment of the ion path using the ion reflector; and then (iv) transferring the second ions from the ion reflector to the detector for detection.

18. The time-of-flight (ToF) mass analyzer is a multi-reflecting time-of-flight (MR-ToF) mass analyzer, the MR-ToF mass analyzer comprising: two ion mirrors facing each other and spaced apart in a first direction X, each mirror being elongated along a drift direction Y between a first end and a second end, the drift direction Y being perpendicular to the first direction X; the ion injector is configured to inject ions into a space between the ion mirrors, the ion injector being located proximate the first end of the ion mirror; the detector is configured to detect ions after the ions have completed multiple reflections between the ion mirrors, the detector being located proximate the first end of the ion mirror; the ion reflector comprises a deflector or lens located near the first end of the ion mirror; The method further comprising: (i) injecting an ion packet comprising first and second ions from the ion injector into the space between the ion mirrors, wherein the ions (a) drift along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) complete a first cycle in which the ions follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; (ii) moving the first ions from the deflector or lens to the detector for detection; (iii) using the deflector or lens to reverse the drift direction velocity of the second ion such that the second ion (a) drifts along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverses the drift direction velocity near the second end of the ion mirror, and (c) completes a further cycle following a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; 20. The method of claim 17, comprising: (iv) moving the second ions from the deflector or lens to the detector for detection.

19. 20. A non-transitory computer readable storage medium storing computer software code which, when executed on a processor, causes an analytical instrument to perform the method of claim 1, 2, 17 or 18.

20. 20. A control system for an analytical instrument, said control system being configured to cause said analytical instrument to perform the method of claim 1, 2, 17 or 18.

21. 21. An analytical instrument comprising the control system of claim 20.

22. An analytical instrument comprising: A fragmentation device; a time-of-flight (ToF) mass analyzer operable in a first mode of operation to move ions along a flight path having a first length and a second mode of operation to move ions along a flight path having a second length, the second length being greater than the first length; and a control system, wherein when the instrument is being used to analyze labeled analyte ions, the control system causing the fragmentation device to fragment the labeled analyte ions to generate analyte fragment ions and reporter or complementary ions; causing the time-of-flight mass analyzer to analyze the analyte fragment ions using the first mode of operation; causing the time-of-flight mass analyzer to analyze the reporter ion or the complementary ion using the second mode of operation.

23. 1. A time-of-flight (ToF) mass analyzer comprising: an ion pathway including a cyclic segment; an ion injector for injecting ions into the ion path; at least one ion reflector disposed along the ion path; a detector disposed at an end of the ion path; The analyzer comprises: (i) injecting an ion packet including the first ions and the second ions from the ion injector into the ion path such that the first ions and the second ions travel along the ion path to the ion reflector; (ii) transferring the first ions from the ion reflector to the detector for detection; (iii) causing the second ions to complete one or more cycles along the circular segment of the ion path using the ion reflector; and then (iv) an analyzer configured to analyze ions by transferring the second ions from the ion reflector to the detector for detection.

24. the time-of-flight (ToF) mass analyzer is a multi-reflecting time-of-flight (MR-ToF) mass analyzer, the MR-ToF mass analyzer comprising two ion mirrors spaced apart and facing each other in a first direction X, each mirror being elongated along a drift direction Y between a first end and a second end, the drift direction Y being orthogonal to the first direction X; an ion injector configured to inject ions into a space between the ion mirrors, the ion injector positioned proximate the first end of the ion mirrors; the detector is configured to detect ions after the ions have completed multiple reflections between the ion mirrors, the detector being located proximate the first end of the ion mirror; an ion reflector being a deflector or a lens located near the first end of the ion mirror; The analyzer comprises: (i) injecting an ion packet comprising first and second ions from the ion injector into the space between the ion mirrors, wherein the ions (a) drift along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverse their drift direction velocity near the second end of the ion mirror, and (c) complete a first cycle in which the ions follow a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; (ii) moving the first ions from the deflector or lens to the detector for detection; (iii) using the deflector or lens to reverse the drift direction velocity of the second ion such that the second ion (a) drifts along the drift direction Y from the deflector or lens towards the second end of the ion mirror, (b) reverses the drift direction velocity near the second end of the ion mirror, and (c) completes a further cycle following a zigzag ion path having multiple reflections in the direction X between the ion mirrors while drifting along the drift direction Y back towards the deflector or lens; 24. The analyzer of claim 23, configured to analyze ions by: (iv) moving the second ions from the deflector or lens to the detector for detection.

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