Systems and methods for processing IONS in mass spectrometry

Abstract

In one aspect, provided are methods for processing ions in mass spectrometry. The methods, in various embodiments, apply an RF field to the ion optical element upstream of a collision cell at a first amplitude to deliver a first population of transmitted sample ions into the collision cell and apply the RF field to the ion optical element at a second amplitude to deliver a second population of transmitted sample ions into the collision cell, and detect a first population of fragment ions associated with the first population of transmitted sample ions and detecting a second population of fragment ions associated with the second population of transmitted sample ions, to generate a mass spectrum based on the detected first and second populations of fragment ions.

Description

4277-0417WO01 (2024-23860-P-US)SYSTEMS AND METHODS FOR PROCESSING IONS IN MASS SPECTROMETRYRelated Applications

[0001] This application claims priority to and the benefit of U.S. Provisional Application No.63 / 745,999 filed on January 16, 2025, which is incorporated herein by reference in its entirety.Technical Field

[0002] The present disclosure relates generally to methods of processing ions and mass spectrometers incorporating an ion containment device, including the processing of ions within such mass spectrometers.Background

[0003] The present disclosure relates to systems and methods for performing mass spectrometry, and particularly systems and methods for declustering and / or fragmenting ions for mass analysis.

[0004] Mass spectrometry (MS) is an analytical technique for determining the structure of test chemical substances (the analytes) with both qualitative and quantitative applications. MS can be useful for identifying unknown compounds, determining the composition of atomic elements in a molecule, determining the structure of a compound by observing its fragmentation, and quantifying the amount of a particular chemical compound in a mixed sample. Mass spectrometers detect these chemical entities as ions such that a conversion of the analytes to charged ions must occur.

[0005] Analyte ions, often given their nature of preparation and / or introduction into the mass spectrometer, may be non-covalently bonded (e.g. clustered) with solvent ions, water vapor, etc. Often, accurate analysis requires these non-covalently bonded ions to be removed from the analyte ions (often referred to as declustering). In addition, many analysis methods rely upon fragmenting an analyte ion and measuring the fragments to produce a fragment ion spectrum. The analyte ion that is fragmented is often referred to as the “precursor ion” of the fragments, and the fragments of the precursor ion are often referred to as daughter ions of the precursor. One frequently used method for fragmenting ions is collision induced dissociation (CID) where4277-0417WO01 (2024-23860-P-US)the ions are collided with a collision gas, typically a neutral inert gas such as helium, argon, nitrogen, etc.

[0006] The fragment ion spectrum of a precursor ion is almost always dependent on the collision energy (CE) of the ion with the collision gas, and a full and reliable investigation of a sample often requires fragmentation mass spectra to be obtained at different collision energies. However, a mass spectrometer can often only be reliably operated at one nominal collision energy at a time given the complex train of ion optical elements that traditionally need to be adjusted to vary the CE. It is to be understood that although there is often a spread of collision energies for any nominal CE (for example, due to variations in ion position, thermodynamics, etc.), this spread is still about a central or average collision energy value and few ions experience collision energies that are more than two standard deviations away from this average. In addition, fragmentation that occurs only on the fringes of such CE distributions is often poorly reproducible. Thus, a full and reliable investigation of a sample often requires several separate independent mass spectra to be acquired, one for each average collision energy, over a large range of collision energies.

[0007] Such multiple independent mass spectra acquisition takes time, consumes sample, and always introduce the possibility for mistakes, systemic and random errors between each independent acquisition. In addition, for generic complex sample analysis, any a priori knowledge of the CE required for detection can be difficult. Thus, there is a need to be able to generate fragmentation ion mass spectra over a large range of collision energies in a fast, reliable manner, that eliminates or minimizes the differences and errors that can inherently arise when such mass spectra must be acquired separately.Summary

[0008] The following summary is intended to introduce the reader to the inventions and their embodiments. One or more inventions and / or embodiments may reside in a combination or subcombination of the apparatus elements or method steps described below or in other parts of this document. The inventors do not waive or disclaim their rights to any invention or inventions disclosed in this specification merely by not describing such other invention or inventions in the claims.4277-0417WO01 (2024-23860-P-US)

[0009] In various aspects and embodiments, provided are methods and systems for processing ions in mass spectrometry that apply an RF field to an ion optical element upstream of a collision cell and vary this RF field’s peak-to-peak amplitude (VPP) during fragment spectrum acquisition, instead of, for example, acquiring a first fragment ion spectrum at a first RF amplitude, then changing the RF amplitude to second RF amplitude to acquire a second fragment ion spectrum. It is to be understood that fragment ions can be, and often are, stored in an ion trap before transfer to an ion detector for detection and generation of a fragment spectrum. The systems and methods disclosed can be used and / or processed with an ion trap, but do not require an ion trap or ion trap processing, and can be used and / or processed with mass spectrometric systems comprising a time-of-flight (TOF) analyzer.

[0010] In various aspects, provided are methods of fragmenting ions to produce a mass spectrum; in various embodiments the methods comprise: a) receiving sample ions from an ion source; b) transmitting the sample ions through an ion optical element into a collision cell for collision with a collision gas to produce fragment ions; c) applying an RF field to the ion optical element at a first amplitude to deliver a first population of transmitted sample ions into the collision cell; d) applying the RF field to the ion optical element at a second amplitude to deliver a second population of transmitted sample ions into the collision cell; e) detecting a first population of fragment ions associated with the first population of transmitted sample ions and detecting a second population of fragment ions associated with the second population of transmitted sample ions; and f) generating a mass spectrum based on the detected first and second populations of fragment ions.

[0011] A single RF field has a characteristic peak-to-peak amplitude (VPP) and frequency. It is to be understood that the electric field strength generated by an RF field changes with time at the field frequency such that the instantaneous electric filed strength varies from +1 / 2 VPPto -1 / 2 VPPabout some reference electrical potential. This reference electrical potential can be ground but can also be a non-zero potential, such as for example a DC potential which is often referred to as an offset potential. It is to be understood that when reference is made herein to an amplitude of an RF field, for example, such as in “applying an RF field ... at a first amplitude,” “applying the RF field ... at a second amplitude,” etc., the term amplitude in this usage is referring to the VPPof the RF field.4277-0417WO01 (2024-23860-P-US)

[0012] A stream of ions passing through an RF field can experience a range of electrical potentials depending on where in its cycle and RF field is, for example, at +1 / 2 VPP, at +1 / 4(VPP, at -1 / 8 VPP, etc. This can lead to different ions in the ion stream acquiring different amounts of kinetic energy, that is there will be a range of kinetic energies, and a distribution in the number of ions with a given kinetic energy level. This distribution in the number of ions with a given kinetic energy level can be referred to as a kinetic energy profile. The peak-to-peak amplitude (VPP) and frequency of the RF field, at least in part, determine the kinetic energy profile.

[0013] Ions with different kinetic energies, can produce different populations of fragment ions when, for example, they collide with a collision gas because different kinetic energies can produce different collision energies (CE). In various embodiments, fragment ion populations are distinguished by the RF field amplitude (VPP) experienced by the precursor ions. For example, ions that traverse a RF field having a first amplitude produce a first population of fragment ions, ions that traverse a RF field having a second amplitude produce a second population of fragment ions, ions that traverse a RF field having a third amplitude produce a third population of fragment ions, etc. It is to be understood that such differing populations of fragment ions may contain ions of the same mass, that is, for example, there may be an overlap in the fragment ion population distributions between these fist, second, third, etc., fragment ion populations.

[0014] It is to be understood that although a given ion kinetic energy profile will produce a range of collision energies, such profiles can be distinguished by an average collision energy even though they produce a range of collision energies.

[0015] In various embodiments, the methods comprise: a) receiving sample ions from an ion source; b) transmitting the sample ions through an ion optical element into a collision cell for collision with a collision gas to produce fragment ions, wherein the sample ions have a kinetic energy and the kinetic energy of the sample ions determines at least in part a collision energy with the collision gas; c) varying the average collision energy over a range between about lOeV and about 150 eV by varying substantially only the amplitude of an RF field applied to the ion optical element from a first amplitude to a second amplitude, d) detecting a first population of fragment ions associated with sample ions transmitted through the ion optical element at the first RF amplitude, and detecting a second population of fragment ions associated with sample ions transmitted through the ion optical element at the second RF amplitude; and e) generating a mass spectrum based on the detected first and second populations of fragment ions.4277-0417WO01 (2024-23860-P-US)

[0016] In various embodiments, methods provided include applying the RF field at two or more amplitudes (VPP) during fragmentation spectrum acquisition. In various embodiments, the RF amplitude is changed: (i) in substantially discrete steps, for example, from a first amplitude (V1PP) to a second amplitude (V2PP), etc., (ii) in a substantially continuously manner, for example, where the amplitude (VPP) is scanned from a first value (VIPP) to a second (V2PP), or (iii) combinations thereof. The various embodiments of the systems and methods provided include scanning the RF field from a first amplitude (V1PP) to a second amplitude (V2PP) and substantially continually detecting fragment ion populations during the scan.

[0017] In various embodiments, the methods comprise applying an RF field to the ion optical element at a first amplitude to deliver a first population of transmitted sample ions into the collision cell and applying the RF field to the ion optical element at a second amplitude to deliver a second population of transmitted sample ions into the collision cell, wherein the difference between the first amplitude and the second amplitude is such that a difference in average collision energy for the first population of transmitted sample ions and the second population of transmitted sample ions with the collision gas (that is the collision energy spread) is in a range between about 5 eV and about 100 eV, between about 15 eV and about 100 eV, between about 20 eV and about 50 eV, and / or between about 10 eV and about 70 eV.

[0018] In various embodiments of the systems and methods, the RF field has a RF field frequency in the range between about 60 kHz and 1 MHz, between about 50 kHz and about 100 kHz, and / or between about 60 kHz to about 150 kHz.

[0019] In various embodiments of the systems and methods, detecting populations of fragment ions comprises transmitting precursor ions from an ion filtering field having a filter scanning rate defined in Daltons per second (Da / sec) to a collision cell wherein collisions with the collision gas generate populations of fragment ions, and wherein each RF field has an RF field frequency that is at least about: (i) 10 times the filter scanning rate, (ii) 25 times the filter scanning rate, (iii) at least about 50 times the filter scanning rate, (iii) at least about 100 times the filter scanning rate, and / or (iv) 200 times the filter scanning rate.

[0020] In various embodiments of the systems and methods, the RF amplitude is change from the first amplitude to the second amplitude in less than: (i) about 20 ms, (ii) about 10 ms, (iii) about 5 ms, and / or (i) about 2 ms.4277-0417WO01 (2024-23860-P-US)

[0021] In various embodiments of the systems and methods, the second amplitude is: (i) about 200 VPPdifferent than the first amplitude, (ii) about 100 VPPdifferent than the first amplitude, (iii) about 75 VPPdifferent than the first amplitude, (iv) about 50 VPPdifferent than the first amplitude, (v) about 20 VPPdifferent than the first amplitude, (vi) about 10 VPPdifferent than the first amplitude.

[0022] In various embodiments of the systems and methods, the second amplitude differs from the first amplitude in the range between: (i) about 10 VPPto about 200 VPPdifference, (ii) about 15 Vpp to about 100 VPPdifference, (iii) about 20 VPPto about 75 VPPdifference, (iv) about 25 Vpp to about 90 VPPdifference, and / or (v) about 10 VPPto about 80 VPPdifference.

[0023] In various embodiments of the systems and methods, the collision gas is at a pressure between about 1 millitorr and about 15 millitorr in the collision cell. In various embodiments of the systems and methods, the collision gas is at a pressure between about 0.1 torr and about 1 torr, and the RF field has an RF field frequency is in the range between about 50 kHz and 1 MHz.

[0024] In various embodiments of the systems and methods, the first RF amplitude, second RF amplitude, or both are selected to decluster most of the analyte ions by breaking non-covalent bonds between the analyte ions and solvent ions without breaking covalent bonds within most of the analyte ions. In various embodiments, the first RF amplitude, second RF amplitude, or both, and the RF frequency are selected to cause declustering without substantially causing breaking covalent bonds within most of the analyte ions.

[0025] In various embodiments of the systems and methods, the ion optical element comprises an aperture lens. In various embodiments the ion optical element comprises one or more of: (i) an aperture lens, (ii) an interquad lens, (iii) an ion optical lens having a skimmertype lens geometry, (iv) a flat plate orifice, (v) a conical orifice, (vi) a wire grid (such as for example but not limited to a mesh), and (vii) a two-wire element mounted transverse to the ion flow. In various embodiments, the ion optical element comprises an element selected from the group consisting of: an interquad lens, a conical orifice, a skimmer plate, and a flat plate orifice.

[0026] In various embodiments of the systems and methods, a force is provided to at least a portion of ions upstream of the ion optical element, wherein the force is substantially directed towards the ion optical element. In various embodiments, the method further comprises4277-0417WO01 (2024-23860-P-US)providing a force to at least a portion of ions upstream of the ion optical element wherein the force is substantially directed away from the ion optical element.

[0027] In various embodiments of the systems and methods, a DC voltage is applied to the ion optical element. Thus, in various embodiments both a DC voltage and an RF field are applied to the ion optical element. In various embodiments, no DC voltage is applied to the ion optical element.

[0028] In a related aspect, provided are systems for fragmenting ions to produce a mass spectrum, the system comprising: a) an ion source; b) an ion detector; c) a collision cell disposed in an ion dissociation region between the ion source and ion detector, the collision cell having a collision gas; d) an ion filter between the ion source and collision cell; e) an ion optical element between the ion source and collision cell; f) a mass analyzer between the collision cell and the ion detector; g) a RF power supply connected to said ion optical element and configured to apply a RF field to the ion optical element; and h) a controller connected to the RF power supply, wherein the controller and RF power supply are configured to change the peak-to-peak amplitude (Vpp) of the RF field applied to the ion optical element from a first amplitude to a second amplitude during acquisition of a fragmentation ion spectra.

[0029] In various embodiments of the systems, the ion optical element is between the ion filter and ion dissociation region. In various embodiments of the systems, the ion dissociation region has an entrance to receive ions from the ion source and the ion optical element is disposed at the entrance to the ion dissociation region.

[0030] In various embodiments of the systems, the controller, RF power supply and connection of the RF power supply to the ion optical element are configured to change the peak-to-peak amplitude (Vpp) of the RF field applied to the ion optical element from a first amplitude to a second amplitude in less than: (i) about 20 ms, (ii) about 10 ms, (iii) about 5 ms, and / or (iv) about 2 ms.

[0031] Further understanding of various aspects of the present teachings can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.Brief Description of the Drawings

[0032] FIG. 1 is a schematic illustration of various conventional hybrid quadrupole mass spectrometers, it is to be understood that this figure is a schematic only, where various items and4277-0417WO01 (2024-23860-P-US)features are not to scale and may be in other configurational relationships (e.g. orthogonal) to each other;

[0033] FIGS.2A and 2B are schematic illustrations of how collision energy is conventionally varied;

[0034] FIG. 3 is a schematic illustration of how collision energy is varied in accordance with various embodiments of the systems and methods provided herein;

[0035] FIGS.4A to 4E are graphs illustrating collision energy as a function of mass spectrum accumulation time (FIG 4A is a graph illustrating conventional collision energy variation (none) over the accumulation time, and FIGS 4B to 4E are graphs illustrating various collision energy variations over the accumulation time in accordance with various embodiments of the systems and methods provided herein); and

[0036] FIG. 5 is a graph of the measured ratio of fragment ion (m / z =280) to precursor ion (Bosentan III, protonated m / z =528.17) signal as a function of mass spectra accumulation time for various collision energy spread workflows of Example 1, it is to be noted that in FIB 5 the y-axis is a logarithmic scale.Detailed Description

[0037] It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of the specifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

[0038] As used herein, the terms “about” and “substantially equal” refer to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the4277-0417WO01 (2024-23860-P-US)real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the terms “about” and “substantially” as used herein means 10% greater or less than the value or range of values stated or the complete condition or state. For instance, a concentration value of about 30% or substantially equal to 30% can mean a concentration between 27% and 33%. The terms also refer to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art.

[0039] As used herein the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0040] As used herein the terms "fragmentation spectrum," “fragment spectrum” and “ion fragment spectrum” as used herein interchangeably to refer to the spectrum of ions produced from fragmenting the analyte precursor ions; where it is to be understood that such spectra can include precursor ions and that fragmenting analyte precursor ions can include declustering.

[0041] The present disclosure is generally based on a discovery that applying and varying the RF peak-to-peak amplitude (VPP) to an ion optical element upstream of an collision cell during fragment spectrum acquisition can provide reliable fragmentation mass spectra over a large range of collision energies with significantly reduced accumulation times.

[0042] In various aspects and embodiments, provided are methods and systems for processing ions in mass spectrometry that apply an RF field to an ion optical element upstream of a collision cell and vary this RF field’s peak-to-peak amplitude (VPP) during fragment spectrum acquisition, instead of, for example, acquiring a first fragment ion spectrum at a first RF amplitude, then changing the RF amplitude to second RF amplitude to acquire a second fragment ion spectrum.

[0043] In various embodiments, in addition to the RF field, a DC offset voltage is also applied to the ion optical element. In certain instances it is desirable to add attractive or repulsive DC voltages to the ion optical element to better control the resulting average kinetic energy of the transmitted ion beam. In general, an attractive DC voltage will add energy to the ions transmitted through the ion optical element, while a repulsive DC voltage will reduce the average ion energy of the ions transmitted through the ion optical element and, in some embodiments, may cause some ions not to be transmitted at all.4277-0417WO01 (2024-23860-P-US)

[0044] As discussed in more detail below, in various embodiments methods and systems for performing mass spectrometry are disclosed that improve obtaining fragmentation ion mass spectra in mass spectrometry workflows and instrumentation, including but not limited to independent data acquisition (IDA), SWATH®analysis, scanning SWATH® analysis, and high resolution multiple reaction monitoring (MRM-HR) mass spectrometric workflows.

[0045] Referring first to FIG. 1, shown is a generalized conventional hybrid quadrupole mass spectrometer apparatus comprising a collision cell. Two general configurations of an ion dissociation region 140 and 140’ are shown, one comprising a collision cell 143, and the other 140’ comprising a collision cell 143 and an electron activated dissociation (EAD) cell 144’, upstream of the collision cell. In referring to directions within the mass spectrometer, “upstream” refers to a direction towards the ion source 101, while “downstream” refers to a direction towards the ion detector 160. The two embodiments are similar and will be described together except for the parts that differ between embodiments, which will be separately described. It is to be understood that the upstream regions and ion optics for the EAD + collision cell embodiment 140’ are substantially the same as for an ion dissociation region 140 without an EAD, but have been omitted for clarity. In addition, for the sake of clarity, typical features and structures well known to those in the art, such as for example, pumps, chambers, gas inlets, power supplies, etc. are not shown.

[0046] In general, such mass spectrometers comprise an ion source 101 that generates ions directed towards a curtain plate 103. The ion source can include any suitable ion source, including, for example, ion sources that provide ions through electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), ion bombardment, application of electrostatic fields (e.g., field ionization and field desorption), chemical ionization, etc.

[0047] Behind the curtain plate 103 there is an orifice plate 105. A curtain chamber 108 can be formed between the curtain plate 103 and the orifice plate 105, and a flow of curtain gas can be used reduce the flow of unwanted neutrals into the downstream sections of the mass spectrometer. Ions exit the curtain chamber 108 through the orifice plate 105 and are received by an intermediate chamber 110 between the orifice plate 105 and the ion lens IQ0 (112). The intermediate chamber can contain an ion guide 111 that collects ions and focuses the ions; in various instruments this region is referred to as the QJet region. The pressure in chamber 110 is typically of the order of 2 Torr. Ions pass through the ion lens 112 into an ion guide chamber of4277-0417WO01 (2024-23860-P-US)the mass spectrometer QO (120), A quadruple rod set 121 is provided in this chamber 120, for collecting and focusing ions. In various embodiments of instrument configuration and operation, chamber 120 can be used to extract solvent from the ion stream, and typically operates at a pressure of 8-10 milliTorr.

[0048] An ion lens IQ1 (122) focuses the ions exiting the Q0 ion guide into a mass filter chamber QI (130) The mass filter QI can include stubby lenses (e.g. a Brubaker lens) at entry 133a and / or exit 133b formed by a set of quadrupoles for focusing of the ions. The mass filter chamber QI further includes a set of quadrupole rods 131, to allow the selection of one or more precursor ions having m / z ratios within a target m / z range for transmission to a downstream ion dissociation device region Q2 (140, 140’) via an ion lens IQ2 (142). In configurations where the ion dissociation region comprises an EAD and a collision cell, the EAD 144’and collision cell 143 are typically separated by an ion lens IQ2b (142’). The ion dissociation region contains ion confinement optics 141, e.g. a quadrupole, 145’ to confine and transmit ions downstream towards an analyzer region 150.

[0049] Precursor ions selected by the mass filter QI pass through an ion lens IQ2 located in upstream of the collision cell 143 where they undergo collision with a collision gas, typically a neutral inert gas such as helium, argon, nitrogen, etc. to remove molecules non-covalently bound to the precursor ion (e.g. to decluster) and / or fragment the precursor ion. The pressure of the collision gas in the collision cell is typically on the order of about 1 millitorr to about 15 millitorr, but higher pressures are sometimes used up to the order of 1 Torr.

[0050] Fragment ions are then transmitted for analysis to the analyzer region 150, which contains a mass analyzer (not shown) which can sperate ions based on their mass to charge ration (m / z) and transmit them to an ion detector 160 to generate the mass spectrum. A variety of different mass analyzers can be used including, but not limited to, time-of-flight (TOF) mass analyzers. It is to be understood that the ion analyzer region 150 can comprise an ion trap, and often does, upstream of the mass analyzer. The ion trap (not shown) serves to trap ions, for example, to accumulate ions and / or cool them, prior to transmittal to the mass analyzer. A wide variety of ion traps can be used, including but not limited to, linear ion traps (LIT), ring traps, 3D traps, Zeno™ traps, etc. It is to be understood that the ion trap can reside primarily or completely within the analyzer region, or make use of ion optical and / or other elements4277-0417WO01 (2024-23860-P-US)associated with the ion dissociate region, such as, for example, in certain configurations of a Zeno™ trap.

[0051] The kinetic energy of the ions entering the collision cell, and hence their collision energy with the collision gas, in conventional approaches is determined primarily by the electrical potential difference between the collision cell and the electrical potential of upstream ion optical elements. In convectional approaches, the electrical potential on multiple ion optical components upstream of the collision cell to need to be change the average collision energy. Changing electrical potentials on multiple ion optical elements rapidly presents several problems. For example, such changes can interrupt ion transmission, change the focusing conditions, produce unwanted transient electrical fields, etc. all of which can decreasing signal, increasing data acquisition time, and / or reduce reproducibility.

[0052] FIGS.2A and 2B schematically illustrate the conventional process used to change to the average collision energy between two values, to subject, e.g., precursor ions to a collision energy spread (CES). FIG 2A illustrates in the lower portion the electrical potential on various ion optical elements of a mass spectrometer in accord with those schematically depicted in FIG 1. In FIGS 2A and 2B, illustration of system having an EAD + collision cell ion dissociation region has been omitted for clarity; however it is to be understood that the discussion with respect to the system illustrated in FIGS.2A and 2B applies as well to systems comprising an EAD + collision cell ion dissociation region.

[0053] FIG. 2A illustrates a set of electrical potentials for obtaining a 10 eV average collision energy of the precursor ions (e.g., those transmitted by the mass filter QI) with the collision gas using conventional methods. In this illustration, the collision cell is held at +31 V, while upstream elements have ever increasing voltages to provide the ions with energy as they move “down” the potential gradient. As illustrated in FIG.2A, ion guide chamber Q0 is held at + 41V to impart a potential difference of 10 V between the ion entering the mass filter QI and the collision cell of the ion dissociation region Q2. FIG.2B illustrates the changes needed to obtain a 40 eV average collision energy using conventional methods. As can be seen in FIG. 2B, the electrical potential on six ion optical elements needs to be changed QJet, IQ0, Q0, IQ1, STI, and RO1, to provide the 40 eV average collision energy. One of skill in the art understands that lowering the collision cell potential (or ion dissociation region in general), while adjusting multiple other optic elements associated with transmission and detection, requires changing4277-0417WO01 (2024-23860-P-US)multiple high voltages (>1000 V), can be technically very challenging to perform accurately to obtain reliable and reproducible results. FIGS.2A and 2B, illustrate obtaining data at just two collision energies. In general, data at multiple collision energies, or a spread of collision energies is desired.

[0054] In various embodiments of the systems and methods, it has been discovered that a range (or spread) of average collision energies can be obtained by applying a RF field to an ion optical element upstream of the collision cell, and changing this RF field’s peak-to-peak amplitude (VPP) during fragment spectrum acquisition, without, in various embodiments, substantially changing the electrical potential on any other ion optical element upstream of the collision cell. In addition, it was surprisingly discovered that in various embodiments this allows for more rapid accumulation of data, with shorter ion fragment accumulation times, and more reproducible data at these shorter times.

[0055] Referring to FIG. 3 and FIGS.4A to 4E, FIG.3 schematically illustrates providing a collision energy spread from 10 eV to 30 eV by changing the peak-to-peak amplitude (VPP) of an RF field applied to an ion optical element IQ2 upstream of the collision cell 143 from a first amplitude to a second amplitude. It is to be understood that FIG.3 is highly schematic, and meant to illustrate the concept because the electrical potential created by an RF field is not readily conveyed as a static potential non-time varying potential. FIGS.4A to 4E, schematically illustrate the time varying nature.

[0056] In FIGS.4A to 4E, the accumulation time refers to how long ions transmitted by the ion dissociation region to an analyzer region are measured to generate the mass spectrum. In various embodiments, accumulation time refers to how long ions transmitted by the ion dissociation region to a ion trap are accumulated by the trap prior to release and mass analysis to generate a mass spectrum. FIG.4A illustrates the conventional approach, where the average collision energy is not varied during fragment ion spectrum acquisition over the accumulation time. In FIG 4A, the average collision energy (CE) of 45 eV is not varied, collision energy spread (CES) = 0, over the accumulation time of 100 ms. FIGS.4B to 4E illustrate changing the average collision energy, in accord with various embodiments, to provide a range of average collision energies (a non-zero CES) over various accumulation times, by varying the RF peak-to-peak amplitude (VPP) from a first amplitude to a second amplitude. FIGS.4B to 4E illustrate a4277-0417WO01 (2024-23860-P-US)20 eV CES, that is ± 20 eV from a 45 eV average collision energy, over various accumulations times, 100ms, 35ms, 25ms, and 8ms, for FIGS.4B to 4E respectively.

[0057] In FIGS.4B to 4E, the change in RF amplitude (VPP) from a first amplitude to a second amplitude is illustrated as substantially linear and continuous to provide a substantially linear and continuous change in the average collision energy, but it is to be understood that the present teachings are not limited to linear and / or and continuous RF amplitude (VPP) changes but include, for example, discrete, and non-linear changes as well. In addition, it is to be understood that the eV y-axis refers to the resultant average collision energy and is not the RF amplitude (VPP) applied to ion optical element upstream of the collision cell. Further, FIGS.4B to 4E illustrate that in various embodiments the RF amplitude (VPP) is changed from a first amplitude to a range of amplitudes and thus provides fragmentation at a range of collision energies.

[0058] The present systems and methods thus produce a composite fragment ion mass spectrum, that is, a mass spectrum that is a composite of the fragmentation obtained at two or more average collision energies, and do so over a large range of CE-values (large CES) in a reproducible fashion even with, in various embodiments, accumulation times of 1 ms or lower.

[0059] Given the difficulties and time associated with conventional approaches to obtaining fragmentation ion mass spectra at different average collision energies, such experiments often rely on determining an optimal CE ahead of time so that experiments can start at and cover the most relevant collisions energies. In contrast, the present systems and methods allow for a large range of collision energies (large CES) to be used in single fragmentation ion spectrum acquisition, to generate a larger range of fragments, with short accumulation times.

[0060] For example, when using a wide CES value (>20 eV), to generate composite fragmentation ion spectra, current systems and methods require longer accumulation time (>30ms) to get representative and / or reproducible spectra. This requirement for longer accumulation times is observed, for example, on a Sciex ZenoTOF® 7600 system with MRM-HR as well as SWATH® analysis. Thus, using conventional approaches, a user will either resolve to use longer accumulation time, thus reducing duty cycle, or shorter accumulations times, thus impairing data reliability and reproducibility. Conventional approaches thus present a quandary for the user and certain experiments, such as for example when large panels of precursor ion are monitored by MRM-HR and short accumulation times may be required, cannot be readily conducted if at all.4277-0417WO01 (2024-23860-P-US)

[0061] The following example is provided for further elucidation of various aspects of the present teachings, and is not presented to provide necessarily an optimal way of practicing the present teachings and / or optimal results that may be obtained.

[0062] Example 1

[0063] A series of experiments were conducted to compare conventional approaches to acquiring composite fragment ion mass spectra (composites of mass spectra over a range of collision energies, that is, for various collision energy spreads (CES)) to various embodiments of the present teachings.

[0064] In contrast to conventional methods, it has been surprisingly discovered that when an RF voltage (VPP) is applied to the ion optical element in a Sciex ZenoTOF® 7600 system upstream of the collision cell, an ion optical element equivalent to that of IQ2 in FIG. 1, and varied from a first amplitude (V1PP) to a second amplitude (V2PP) a distribution of average collision energies is provided that generate substantially reproducible composite MSMS obtainable, in various embodiments, in less than 10ms and in various embodiments in less than 5ms.

[0065] The data in this example was obtained using a Sciex ZenoTOF® 7600 instrument (hereinafter the ZenoTOF) modified with a RF power supply and controller configured to change the RF amplitude (VPP) during the course of acquiring an ion fragment spectrum. The quadrupole QI of the ZenoTOF was used to select the m / z of the precursor ion. The selected precursor ions were passed through the aperture lens IQ2 located in front of a quadrupole collision cell and finally into the ZenoTOF ion trap TOF mass analyzer and data acquired for various accumulation times.

[0066] Referring to FIG. 5, all the traces in FIG. 5 are for a starting average collision energy of 45 eV and various collision energy spreads (CES) about this value. The traces plot the observed ratio of a selected fragment ion (m / z = 280) to that of its precursor ion (Bosentan III, protonate m / z =528.17) in the acquired composite fragment ion spectra as a function of accumulation time.

[0067] The traces labeled 501-507 are experiments conducted using conventional approaches, that is, the collision energy spread was generated by changing the DC voltage applied to multiple ion optical elements upstream of the collision cell, as schematically illustrated, for example, in FIGS.2A and 2B. Trace 501 used no CES, trace 502 used a CES of4277-0417WO01 (2024-23860-P-US)5 eV, trace 503 used a CES of 10 eV, trace 504 used a CES of 15 eV, trace 505 used a CES of 25 eV, trace 506 used a CES of 35 eV, and trace 507 used a CES of 50 eV.

[0068] Trace 510 shows the data for a composite fragment spectra using various embodiments of the systems and methods herein. Briefly, an RF field with a frequency of 100 kHZ was applied to IQ2 (entrance lens to EAD cell) of the ZenoTOF system and scanned from a first amplitude (V1PP= 0 V) to a second amplitude (V2PP= 80 V) to generate a CES of 30 eV and produce a composite MSMS spectra without significantly changing the voltages on other upstream ion optical elements. Using this approach, the energy spread no longer depended on ramping the voltages on multiple ion optic elements upstream to generate a CES, significantly increasing the speed needed to generate an equivalent CES composite spectrum.

[0069] In conventional approaches, the absolute value of the fragment and / or precursor ion ratio is expected to vary based on the CE range applied. In FIG. 5, the plateau region indicates where a reproducible ion ratio for a given CES value and accumulation time. As the data in FIG. 5 show, using conventional approaches, traces 501-507, for CES above roughly 5 eV or 10 eV, it is not possible to obtain reproducible spectra with accumulation times less than ~20ms, and this trend worsens as the CES spread increases to practical experimental values. For example, for a CES of 15 eV a user could only rely on data with an accumulation times of greater than about 30 ms, while for a CES of 35 eV this accumulation time increases to greater than about 40 ms. It should be noted that the ion ratio is a log scale, so the non-linearity (and indication of lack of reproducibility) of the non-plateau regions is more significant than it may appear.

[0070] In comparison, the data in FIG. 5 shows that for a composite fragmentation ion spectra generated according to various embodiments of the present teachings (dotted line trace 510), a constant ion ratio was observed down to the lowest accumulation time, 5 ms, studied in this example.

[0071] The ability to obtain, in various embodiments, composite fragment ion mass spectrum (i.e. combined spectra over multiple average CE) with short accumulation times makes it possible to generate such composite fragment ion mass spectra with mass spectrometric workflows that are not amenable to use of long accumulation times (e.g. > 30ms), such as, for example, Scanning-SWATH® analysis where the ion filter quadruple is scanned during data4277-0417WO01 (2024-23860-P-US)collection and other data independent acquisition (DIA) workflows where short accumulation times are required.

[0072] Scanning-SWATH® analysis is a data-independent acquisition (DIA) method in mass spectrometry, specifically used in proteomics, that allows for rapid and comprehensive quantification of proteins by continuously scanning across a range of precursor masses, enabling the identification and measurement of a large number of peptides within a short timeframe, often used for fast proteomic analysis in drug discovery or clinical studies; it essentially improves upon traditional SWATH® analysis by assigning precursor masses to MS / MS traces through its scanning feature, leading to increased identification accuracy compared to standard DIA methods.

[0073] While the above description provides example embodiments, it will be appreciated that the present invention is susceptible to modification and change without departing from the fair meaning and scope of the accompanying claims.

[0074] Accordingly, what has been described is merely illustrative of the application of aspects of embodiments of the invention and numerous modifications and variations of the present

[0075] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent description of a corresponding block or item or feature of a corresponding apparatus correspondingly configured to perform such steps. Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some embodiments, some one or more of the most important method steps may be executed by such an apparatus.

[0076] Depending on certain implementation requirements, embodiments of the invention can be implemented in hardware and / or in software. The implementation can be performed using a non-transitory storage medium such as a digital storage medium, for example a floppy disc, a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable.4277-0417WO01 (2024-23860-P-US)

[0077] While various embodiments have been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; embodiments of the present disclosure are not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing embodiments of the present disclosure, from a study of the drawings, the disclosure, and the appended claims.

[0078] In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single processor or other processing unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

Claims

4277-0417WO01 (2024-23860-P-US)What is claimed is:

1. A method of fragmenting ions to produce a mass spectrum, the method comprising:receiving sample ions from an ion source;transmitting the sample ions through an ion optical element into a collision cell for collision with a collision gas to produce fragment ions;applying an RF field to the ion optical element at a first amplitude to deliver a first population of transmitted sample ions into the collision cell;applying the RF field to the ion optical element at a second amplitude to deliver a second population of transmitted sample ions into the collision cell;detecting a first population of fragment ions associated with the first population of transmitted sample ions and detecting a second population of fragment ions associated with the second population of transmitted sample ions; andgenerating a mass spectrum based on the detected first and second populations of fragment ions.

2. The method of claim 1, wherein the RF field is applied at more than two amplitudes.

3. The method of claim 1, wherein the ion optical element comprises an ion lens defining an upstream end of the collision cell.

4. The method of any of claims 1 to 3, wherein the first population of transmitted sample ions comprise a first kinetic energy profile imparted by the RF field at the first amplitude, and wherein the second population of transmitted sample ions comprise a second kinetic energy profile imparted by the RF field at the second amplitude.

5. The method of any of claims 1 to 4, wherein the difference between the first amplitude and the second amplitude is such that a difference in average collision energy for the first population of transmitted sample ions and the second population of transmitted sample ions with the collision gas is in a range between about 15 eV and about 100 eV.4277-0417WO01 (2024-23860-P-US)6. The method of any of claims 1 to 5, wherein the collision energy spread is in the range between about 20 eV and about 60 eV.

7. The method of any of claims 1 to 6, wherein the RF field has a RF field frequency in the range between about 50 kHz and 1 MHz.

8. The method of claim 7, wherein the RF field has a RF field frequency in the range between about 60 kHz to about 150 kHz.

9. The method of claim 1 , wherein the step of detecting the populations of fragment ions comprises transmitting to the collision cell precursor ions from an ion filtering field having a filter scanning rate defined in Daltons per second (Da / sec), and wherein the RF field has an RF field frequency that is at least about 10 times the ion filter scanning rate.

10. The method of claim 9, wherein the RF field frequency is at least about 50 times the ion filter scanning rate.

11. The method of claim 9, wherein the RF field frequency is at least about 100 times the ion filter scanning rate.

12. The method of any of claims 1 to 11, wherein the RF amplitude is change from the first amplitude to the second amplitude in less than about 10 ms.

13. The method of any of claims 1 to 11, wherein the RF amplitude is change from the first amplitude to the second amplitude in less than about 5 ms.

14. The method of any of claims 1 to 11, wherein the RF amplitude is change from the first amplitude to the second amplitude in less than about 2 ms.

15. The method of claim 1, wherein the first amplitude is about 0 volts peak-to-peak (Vpp) and the second amplitude is about 100 Vpp.

16. The method of claim 1, wherein the second amplitude is 50 Vpp or more greater than the first amplitude Vpp.4277-0417WO01 (2024-23860-P-US)17. A method of fragmenting ions to produce a mass spectrum, the method comprising:receiving sample ions from an ion source;transmitting the sample ions through an ion optical element into a collision cell for collision with a collision gas to produce fragment ions, wherein the sample ions have a kinetic energy and the kinetic energy of the sample ions determines at least in part a collision energy with the collision gas;varying the average collision energy over a range between about 15 eV and about 100 eV by varying substantially only the amplitude of an RF field applied to the ion optical element from a first amplitude to a second amplitude,detecting a first population of fragment ions associated with sample ions transmitted through the ion optical element at the first RF amplitude, and detecting a second population of fragment ions associated with sample ions transmitted through the ion optical element at the second RF amplitude; andgenerating a mass spectrum based on the detected first and second populations of fragment ions.

18. The method of claim 17, wherein the RF field is applied at more than two amplitudes.

19. The method of any of claims 17 to 18, wherein the collision energy spread is in the range between about 20 eV and about 60 eV.

20. The method of any of claims 17 to 19, wherein the collision gas is at a pressure between about 1 millitorr and about 15 millitorr.

21. The method of any of claims 17 to 19, wherein the collision gas is at a pressure between about 0.1 torr and about 1 torr, and the RF field has an RF field frequency is in the range between about 50 kHz and 1 MHz.

22. The method of any of claims 17 to 19, wherein the varying RF field has an RF field frequency is in the range between about 60 kHz to about 150 kHz.

23. The method of claim 17, wherein the step of detecting each group of product ions comprises transmitting to the collision cell precursor ions from an ion filtering field having a filter scanning rate defined in Daltons per second (Da / sec), and wherein the4277-0417WO01 (2024-23860-P-US)varying RF field has an RF field frequency that is at least about 10 times the ion filter scanning rate.

24. The method of claim 23, wherein the RF field frequency is at least about 50 times the ion filter scanning rate.

25. The method of claim 23, wherein the RF field frequency is at least about 100 times the ion filter scanning rate.

26. The method of any of claims 17 to 23, wherein the RF amplitude is change from the first amplitude to the second amplitude in less than about 10 ms.

27. The method of any of claims 17 to 23, wherein the RF amplitude is change from the first amplitude to the second amplitude in less than about 5 ms.

28. The method of any of claims 17 to 23, wherein the RF amplitude is change from the first amplitude to the second amplitude in less than about 2 ms.

29. A system for fragmenting ions to produce a mass spectrum, the system comprising:an ion source;an ion detector;a collision cell disposed in an ion dissociation region between the ion source and ion detector, the collision cell having a collision gas;an ion filter between the ion source and collision cell;an ion optical element between the ion source and collision cell;a mass analyzer between the collision cell and the ion detector;a RF power supply connected to said ion optical element and configured to apply a RF field to the ion optical element; anda controller connected to the RF power supply, wherein the controller and RF power supply are configured to change the peak-to-peak amplitude (Vpp) of the RF field applied to the ion optical element from a first amplitude to a second amplitude during acquisition of a fragmentation ion spectra.4277-0417WO01 (2024-23860-P-US)30. The system of claim 29, wherein the ion optical element comprises anaperture ion lens.

31. The system any of claims 29 to 30, wherein the ion optical element is between the ion filter and ion dissociation region.

32. The system of any of claims 29 to 31, wherein the ion dissociation region has an entrance to receive ions from the ion source and the ion optical element is disposed at the entrance to the ion dissociation region.

33. The system of any of claims 29 to 32, wherein the controller, RF power supply and connection of the RF power supply to the ion optical element are configured to change the peak-to-peak amplitude (Vpp) of the RF field applied to the ion optical element from a first amplitude to a second amplitude in less than about 10 ms.

34. The system of claim 33, wherein the controller, RF power supply and connection of the RF power supply to the ion optical element are configured to change the peak-to-peak amplitude (Vpp) of the RF field applied to the ion optical element from a first amplitude to a second amplitude in less than about 2 ms.

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