Mass spectrometer and mass spectrometry analysis methods

Abstract

A mass spectrometry method for analyzing a sample within a target mass range is disclosed, comprising the following steps: ionizing the sample to generate a plurality of precursor ions; performing an MS1 ​​scan on the precursor ions, including mass analysis of the precursor ions within the target mass range to obtain an MS1 ​​mass spectrum of the precursor ions; determining ion intensity values ​​in the MS1 mass spectrum; selecting precursor mass segments within the target mass range, each precursor mass segment having a mass range, and for each precursor mass segment: fragmenting the precursor ions within the precursor mass segment; and performing an MS2 scan of the fragmented ions by controlling the amount of fragmented ions in the precursor mass segment based on the intensity values ​​of the precursor mass segment derived from the MS1 spectrum; and performing mass analysis on the amount of fragmented ions. A mass spectrometer for performing mass spectrometry analysis on a sample within a target mass range is also disclosed, the mass spectrometer having a controller configured to perform such a method.

Description

Technical Field

[0001] This invention relates to mass spectrometry analysis of organic samples, particularly biological samples such as proteins, peptides, metabolites, and lipids. Specifically, this invention relates to a high-resolution identification and quantification technique applicable to proteomics, metabolomics, and lipidomics. Background Technology

[0002] Mass spectrometry is a long-established technique used to identify and quantify complex mixtures, often large organic molecules. In recent years, techniques have been developed that allow the analysis of a wide range of biological and non-biological materials and have applications in law enforcement (e.g., the identification of drugs and explosive materials), the environment, scientific research, and biology (e.g., in proteomics, studying simple and complex mixtures of proteins, and applications in drug discovery, disease identification, etc.).

[0003] Proteins, containing a large number of amino acids, typically have considerable molecular weights. Therefore, accurate identification and quantification of proteins via direct mass spectrometry is challenging. Consequently, fragmentation of precursor sample materials is a well-known method. Various fragmentation techniques are known, each leading to the generation of different fragment ions from the precursor ions. Furthermore, the applied fragmentation energy can influence the fragmentation mechanism.

[0004] To determine the molecular structure of a sample molecule, mass spectrometry is first used to analyze the mass of all sample ions (precursor ions) within a selected mass-to-charge ratio (m / z) window. This type of scan is typically referred to as an MS1 ​​scan. Next, the selected sample ions are fragmented, and the resulting fragments are subsequently analyzed for mass within a selected m / z range. Scans of the fragmented ions are typically referred to as MS2 scans.

[0005] Sample analysis can be broadly categorized into data-independent analysis / acquisition (DIA) and data-dependent analysis / acquisition (DDA) techniques. DIA attempts to determine what is present in a sample with potentially unknown properties.

[0006] DDA (Differentiated Analytical Catalysis) attempts to confirm the presence of one or more substances in a given sample. The DDA method identifies a fixed number of precursor ion species and selects and analyzes these species using mass spectrometry analysis with MS2 scans. In DDA, determining which precursor ion species are target species can be based on intensity ranking (e.g., the top ten most abundant species observed through peaks in the MS1 spectrum) or by defining a "containment list" of precursor mass spectrum peaks (e.g., selected by the user), from which MS2 spectra are always acquired regardless of the intensity ranking of peaks in the MS1 mass spectrum. Additionally, an "exclusion list" of peaks in MS1 ​​can be defined, for example, by the user based on prior knowledge about the expected sample abundance.

[0007] DIA avoids the decision-making required in DDA by simply segmenting the target mass range (usually user-defined) and obtaining the MS2 spectrum for each segment. For conventional DIA, the acquisition of MS1 precursor spectra is more or less optional because the parameters of the sample ion selection window carry information about the possible range of sample ions within that window.

[0008] In MS1 ​​scans, i.e. precursor mass, the ion intensity can vary significantly (e.g., by several orders of magnitude) across the entire spectrum, thus requiring a high dynamic range to detect both high and very low ion intensities.

[0009] One objective of this invention is to solve the dynamic range problem in mass spectrometry analysis, particularly the dynamic range problem in data-independent mass spectrometry analysis. Summary of the Invention

[0010] This invention provides a mass spectrometry analysis method, a mass spectrometer, and a computer program product.

[0011] According to the present invention, a mass spectrometry analysis method for analyzing samples within a target mass range can be provided. The method includes the following steps:

[0012] - Ionize the sample to generate multiple precursor ions;

[0013] - Perform MS1 scans on the precursor ions, including mass analysis of the precursor ions within the target mass range, to obtain the MS1 mass spectra of the precursor ions;

[0014] - Determine the ion intensity values ​​within the MS1 mass spectrum;

[0015] - Select the precursor mass segment within the target mass range. Each precursor mass segment has a mass range, and for each precursor mass segment:

[0016] Fragmentation of precursor ions within the precursor mass segment; and

[0017] MS2 scans of fragmented ions are performed using the following method:

[0018] - The amount of fragmented ions in each precursor mass segment is controlled based on the intensity value derived from the MS1 spectrum; and

[0019] - Perform mass analysis on the amount of fragmented ions.

[0020] The amount of fragmented ions can be controlled by controlling the injection time of fragmented ions to accumulate an ion set from fragmented ions in the extraction trap, and performing an MS2 scan includes ejecting the ion set from the extraction trap into a mass analyzer for performing an MS2 scan.

[0021] In this scenario, the injection time can be determined such that the total charge or the number of fragmented ions is substantially equal for each precursor mass segment of the ion pack.

[0022] The amount of ions can be controlled by controlling the number of pulses generated by fragmented ions from the precursor mass segment by an ion implanter, preferably an orthogonal accelerator implanter, which are injected into a time-of-flight mass analyzer to generate an MS2 scan.

[0023] Multiple intensity levels can be defined for the intensity value, and each precursor mass segment is assigned to one of the multiple intensity levels, and the amount of fragmented ions is controlled based on the assigned intensity level.

[0024] In this scenario, the order in which precursor mass segments are processed can be defined based on strength grades, preferably such that precursor mass segments assigned to strength grades associated with higher strength values ​​are processed before precursor mass segments assigned to strength grades associated with lower strength values.

[0025] Multiple mass sub-ranges are defined within the target mass range. Each mass sub-range includes multiple adjacent precursor mass segments, such that all precursor mass segments within the mass sub-range are assigned the same intensity value.

[0026] Intensity peaks can be identified within the target quality range, and the intensity values ​​can include the peak intensity values ​​of the identified intensity peaks.

[0027] Each of the plurality of precursor mass segments may have substantially the same mass range, and preferably wherein the mass range is 20 Da (Dalton) or less, 15 Da (Dalton) or less, 10 Da (Dalton) or less, 8 Da (Dalton) or less, 6 Da (Dalton) or less, 4 Da (Dalton) or less, 3 Da (Dalton) or less, or 2 Da (Dalton) or less.

[0028] Precursor mass segments may include adjacent but non-overlapping precursor mass segments.

[0029] The mass ranges of the precursor mass segments may or may not overlap.

[0030] The same quality analyzer can be used to perform both the MS1 scan and the MS2 scan, and preferably, the quality analyzer is a time-of-flight quality analyzer.

[0031] MS1 scan can be performed using a first quality analyzer, and MS2 scan can be performed using a second quality analyzer, wherein preferably the first quality analyzer is a Fourier transform quality analyzer, more preferably an Orbitrap quality analyzer, and wherein preferably the second quality analyzer is a time-of-flight quality analyzer, more preferably a multi-reflection time-of-flight quality analyzer.

[0032] Based on this teaching, a mass spectrometer for performing mass spectrometric analysis of samples within a target mass range can be provided. A mass spectrometer comprising:

[0033] An ionization source, said ionization source being used to generate multiple precursor ions from a sample provided by a chromatographic system;

[0034] A mass selector for selecting a mass range of precursor ions;

[0035] A mass analyzer for performing an MS1 ​​scan on precursor ions in the MS1 domain;

[0036] Fragmented devices;

[0037] A mass analyzer for performing an MS2 scan of fragmented ions in the MS2 domain; an ion group or ion pulse forming device;

[0038] and a controller, the controller being configured to:

[0039] (i) Enables the mass selector to select precursor ions within the target mass range;

[0040] (ii) The mass analyzer used to perform the MS1 scan performs an MS1 ​​scan on the precursor ions in the target mass range to obtain the MS1 spectrum of the precursor ions in the MS1 domain;

[0041] (iii) Determine the ion intensity values ​​in the MS1 spectrum;

[0042] (iv) Enables the quality selector to select multiple precursor quality segments within the target quality range, each precursor quality segment having a quality range;

[0043] (v) The amount of fragmented ions in each precursor mass segment is controlled based on the intensity value of the precursor mass segment derived from the MS1 spectrum;

[0044] (vi) enables the fragmentation device to fragment precursor ions within each precursor mass segment;

[0045] (vii) The ion group or ion pulse forming device forms the amount of fragmented ions into one or more ion groups or ion pulses, and injects the ion groups or ion pulses of fragmented ions into the mass analyzer to perform an MS2 scan.

[0046] (viii) enables the mass analyzer used to perform MS2 scans to perform mass analysis on the amount of fragmented ions in one or more ion groups or ion pulses.

[0047] In a mass spectrometer used to perform mass spectrometry analysis, the controller may be susceptible to modifications made using any of the methods described above.

[0048] According to this teaching, a computer program product may be provided, the computer program product including program instructions that, when loaded into the memory of a controller, cause the controller of a mass spectrometer to perform any of the methods described above. Attached Figure Description

[0049] Figure 1 A schematic diagram of a mass spectrometer suitable for carrying out the present invention is shown;

[0050] Figure 2 An exemplary flowchart of a method according to an embodiment of the present invention is shown;

[0051] Figure 3 An example diagram of the MS1 spectrum for analysis and the mass subrange within the MS1 spectrum is shown according to an embodiment of the present invention;

[0052] Figure 4 A schematic diagram of an alternative mass spectrometer suitable for performing the method according to an embodiment of the invention is shown;

[0053] Figure 5 A schematic diagram of another alternative mass spectrometer suitable for performing the method according to an embodiment of the invention is shown;

[0054] Figure 6 Examples of methods according to embodiments of the present disclosure, superimposed on the characteristics of chromatographic peaks, are shown. Detailed Implementation

[0055] This invention can be embodied in many ways, and specific embodiments will now be described by way of example only and with reference to the accompanying drawings. It should be understood that the invention is not limited to the described embodiments and some embodiments may not include all the features described below. However, it will be apparent that various modifications and changes can be made herein without departing from the broader scope of the invention as set forth in the claims.

[0056] In this paper, the term mass may be used to refer to the mass-to-charge ratio m / z. Unless otherwise stated, the resolution of the mass analyzer is understood to refer to the resolution of the mass analyzer determined at a mass-to-charge ratio of 200. Intensity values ​​in a mass spectrum may be expressed differently as ion abundance or peak height, etc.

[0057] Figure 1A schematic arrangement of a mass spectrometer 10 suitable for performing the method according to an embodiment of the invention is shown.

[0058] exist Figure 1 In this process, the sample to be analyzed (e.g., from an autosampler) is supplied to a chromatographic device such as a liquid chromatography (LC) column. Figure 1 (Not shown in the image). One such example of an LC column is the ProSwift monolithic column from Thermo Fisher Scientific, Inc., which provides high-performance liquid chromatography (HPLC) by forcing a sample transported in the mobile phase through a stationary phase composed of irregularly or spherically shaped particles under high pressure. In an HPLC column, sample molecules elute at different rates depending on their degree of interaction with the stationary phase.

[0059] The sample may contain molecules selected from one or more of the following: biopolymers, proteins, peptides, polypeptides, amino acids, carbohydrates, sugars, fatty acids, lipids, vitamins, hormones, polysaccharides, phosphorylated peptides, phosphorylated proteins, glycopeptides, glycoproteins, oligonucleotides, oligonucleotides, DNA, DNA fragments, cDNA, cDNA fragments, RNA, RNA fragments, mRNA, mRNA fragments, tRNA, tRNA fragments, monoclonal antibodies, polyclonal antibodies, ribonucleases, enzymes, metabolites, and / or steroids. The sample may include at least 2, 5, 10, 20, 50, 100, 500, 1000, or 5000 different molecules.

[0060] Chromatography can be generated by measuring the number of sample molecules eluted from an HPLC column over time using a detector (e.g., a mass spectrometer). The sample molecules eluted from the HPLC column are detected as peaks above the baseline measurement on the chromatogram. When different sample molecules have different retention times, multiple peaks can be detected on the chromatogram. Preferably, individual sample peaks are temporally staggered from other peaks in the chromatogram so that different sample molecules do not interfere with each other. On the chromatogram, the presence of a chromatographic peak corresponds to the time period during which the sample molecule is present at the detector. Thus, the width of the chromatographic peak is equal to the time period during which the sample molecule is present at the detector. Preferably, the chromatographic peak has a Gaussian shape distribution, or it can be assumed to have a Gaussian shape distribution. Therefore, the width of the chromatographic peak can be determined based on multiple standard deviations calculated from the peak. For example, the peak width can be calculated based on four standard deviations of the chromatographic peak. Alternatively, the peak width can be calculated based on the width at half the maximum height of the peak. Other methods known in the art for determining peak width are also suitable.

[0061] The sample molecules separated by liquid chromatography are then ionized using an electrospray ionization source (ESI source) 20 at atmospheric pressure. The sample ions then enter the vacuum chamber of the mass spectrometer 10 and are guided by a capillary 25 to a lens 30, which can be implemented as an RF-only S-lens 30 or a lens 30 applying a combination of RF and DC, propelling the ions forward. The lens 30 may use a series of rings with gradually decreasing diameters, on which a positive driving DC potential can be applied. Ions are focused by the lens 30 onto an implantation multipole 40 that implants ions into a bent multipole 50 having an axial field. The bent multipole 50 guides the (charged) ions along a curved path, while unwanted neutral molecules (e.g., entrained solvent molecules) are not guided along the curved path and are lost.

[0062] An ion gate (TK lens) 60 is located at the distal end of the bent multipole 50 and controls the passage of ions from the bent multipole 50 into a downstream mass selector in the form of a quadrupole mass filter 70. The quadrupole mass filter 70 is typically, but not necessarily, segmented and functions as a bandpass filter, allowing a selected mass number or a limited mass range to pass through while excluding ions of other mass-to-charge ratios (m / z). The filter can also operate in RF-only mode, where it is not mass-selective, i.e., it transmits substantially all m / z ions. For example, the quadrupole mass filter 70 can be controlled by a controller 195 to select a range of mass-to-charge ratios of precursor ions allowed to pass through while filtering out other ions in the precursor ion stream. Alternatively, the lens 30 can operate as an ion gate, and the ion gate (TK lens) 60 can be an electrostatic lens.

[0063] although Figure 1 A quadrupole mass filter is shown, but those skilled in the art should understand that other types of mass selection devices are also suitable for selecting precursor ions within a target mass range. For example, the ion separator described in US-2015287585, the ion trap described in WO-A-2013076307, the ion migration separator described in US-A-2012256083, the ion gate mass selection device described in WO-A-2012175517, or the charged particle trap described in US799223, the disclosures of which are incorporated herein by reference in their entirety. Those skilled in the art should understand that other methods for selecting precursor ions based on ion mobility, differential mobility, and / or transverse modulation are also suitable.

[0064] A method called simultaneous precursor scanning (SPS) can also be used to perform isolation of multiple ions of different masses or mass ranges in the ion trap. Furthermore, in some embodiments, more than one ion selection device or mass selection device may be provided. For example, another mass selection device may be provided downstream of the fragmentation chamber 120. In this way, MS3 or MSn scans (typically using a TOF mass analyzer 150 for mass analysis) can be performed if necessary.

[0065] Ions then pass through the quadrupole exit lens / tangent lens arrangement 80 and enter the first transfer multipole 90. The first transfer multipole 90 guides the filtered ions from the quadrupole mass filter 70 into the curved linear ion trap (C-trap) 100. To measure the ion current, an electrometer (not shown) can be positioned near (downstream or upstream) the first transfer multipole 90. For this purpose, the electrometer can sample a predetermined proportion of ions passing through the first transfer multipole 90. Alternatively, the electrometer can be positioned near the entry point of the ions into the C-trap 100. In another alternative, the electrometer can be positioned near the exit point of the ions leaving the C-trap 100. Preferably, the electrometer is positioned upstream of the fragmentation chamber 120, as described below. Using an electrometer positioned upstream of the fragmentation chamber 120 allows for the measurement of the precursor ion current. The C-trap (first ion storage trap) 100 has a longitudinally extending curved electrode supplied with an RF voltage and an axial trapping electrode supplied with a DC voltage. The result is a potential trap extending along the curved longitudinal axis of the C-trap 100. In the first operating mode, a DC axial trapping voltage is applied to the C-well, causing ions arriving from the first transfer multipole 90 to be trapped in the potential well of the C-well 100, where they are cooled. The injection time (IT) of ions into the C-well determines the number of ions (ion swarm) subsequently ejected from the C-well into the mass analyzer.

[0066] The cooled ions are trapped at the bottom of the potential well, forming a cloud, and then orthogonally ejected from the C-well toward the first mass analyzer 110. For example... Figure 1 As shown, the first mass analyzer is an orbit trap mass analyzer 110, such as the Orbitrap® mass analyzer sold by Thermo Fisher Scientific. The orbit trap mass analyzer 110 has an off-center injection port, and ions are injected into the orbit trap mass analyzer 110 as a coherent group through the off-center injection port. The ions are then trapped within the orbit trap mass analyzer by a superlogarithmic electric field, causing the ions to move back and forth in the longitudinal direction as they orbit around the inner electrode.

[0067] The longitudinal or axial (z) component of the movement of an ion group in an orbital trap mass analyzer is more or less defined as simple harmonic motion, where the angular frequency in the z-direction is related to the square root of the mass-to-charge ratio of a given ion species. Therefore, over time, ions separate according to their mass-to-charge ratio.

[0068] Ions in the orbital trapping mass analyzer are detected using an image detector (external electrode). This image detector generates a "transient" in the time domain containing information about all ion species as they pass through it. This transient is then subjected to a Fast Fourier Transform (FFT), producing a series of peaks in the frequency domain. Based on these peaks, a mass spectrum representing abundance / ion intensity relative to m / z can be generated. A controller 195 controls the mass analyzer 110 to acquire the detector transient signal, process the transient via FFT, and generate the mass spectrum.

[0069] In the above configuration, sample ions are analyzed via an orbital trap mass analyzer (more specifically, the mass range of sample ions within a target mass range selected by a quadrupole mass filter) in the absence of fragmentation. The resulting mass spectrum is denoted as MS1. According to this teaching, the MS1 spectrum is analyzed to identify intensity peaks, which can be used to control the number of fragmented ions in each precursor mass range, as in... Figure 2 In the method described, preferably, the first quality analyzer performs an MS1 ​​scan at a quality resolution of at least 15,000, at least 30,000, at least 50,000, or more preferably at least 75,000, at least 100,000, at least 150,000, or at least 200,000 (resolution at 200 m / z). The MS1 scan is preferably performed with a quality accuracy of 5 ppm, 4 ppm, 3 ppm, or 2 ppm or better (e.g., 1-2 ppm). Preferably, the first quality analyzer performs the MS1 scan at a frequency of at least 0.5 Hz (scans / second), at least 1 Hz, at least 2 Hz, at least 3 Hz, or at least 4 Hz.

[0070] In the second operating mode of C-well 100, ions entering C-well 100 through the quadrupole exit lens / tangent lens arrangement 80 and the first transfer multipole 90 can continue their path through C-well and enter fragmentation chamber 120. Thus, C-well effectively acts as an ion guide in the second operating mode. Alternatively, ions cooled in C-well 100 can be ejected axially from C-well into fragmentation chamber 120. Figure 1 In the mass spectrometer 10, the fragmentation chamber 120 is a high-energy collisional dissociation (HCD) device to which collision gas is supplied. Precursor ions arriving in the fragmentation chamber 120 collide with collision gas molecules, thereby fragmenting the precursor ions into fragment ions.

[0071] Despite Figure 1 The HCD fragmentation chamber 120 is shown, but alternatively other fragmentation devices employing such methods as collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), photodissociation, etc., may be used.

[0072] Fragmented ions can be ejected or transported from fragmentation chamber 120 to C-trap 100 at opposite axial ends. The ejected fragmented ions enter a second transfer multipole 130. The second transfer multipole 130 guides the fragmented ions from fragmentation chamber 120 to extraction trap (second ion trap) 140. Extraction trap 140 is a radio frequency voltage-controlled trap containing a buffer gas. For example, a suitable buffer gas is argon with a pressure in the range of 5 x 10⁻⁴ mbar to 1 x 10⁻² mbar. The extraction trap is capable of rapidly cutting off the applied RF voltage and applying a DC voltage to extract the captured ions. Suitable planar extraction traps, also known as rectangular ion traps, are further described in US9548195 (B2). Alternatively, C-trap is also suitable as a second ion trap.

[0073] An extraction trap 140 is provided to form an ion bank of fragmented ions prior to the ion bank implantation time-of-flight (TOF) mass analyzer 150. Alternatively, or in addition to the electrometer described above with respect to the multipole 90 and / or C-trap 100, an electrometer may be provided to measure the flow of fragmented ions entering the extraction trap 140. For this purpose, the electrometer can sample a predetermined proportion of the fragmented ions entering the extraction trap 140. Such an electrometer may be positioned between the fragmentation chamber 120 and the extraction trap 140 to sample the flow of fragmented ions entering the extraction trap 140. Using any of the above-described electrometers or each electrometer, the ion flow rate can be measured, and the implantation time and / or number of pulses of fragmented ions for MS2 analysis in each precursor mass segment can be determined based on the ion flow measured by the electrometer. For example, the injection time and / or number of pulses for fragmented ions used for MS2 analysis in precursor mass segments with specific intensity values ​​(e.g., relatively high intensity values ​​or grades) can be determined based on ion current measured by an electrometer to analyze a predetermined number of ions (e.g., 10,000 ions) in each mass segment. In other precursor mass segments (e.g., mass segments with relatively low remaining intensity values ​​or grades, particularly the lowest intensity grades), the injection time and / or number of pulses for fragmented ions used for MS2 analysis can be a predetermined time (e.g., 2 ms per mass segment) or the remaining analysis time divided by the number of remaining mass segments. To form an ion pool, extraction trap 140 accumulates fragmented ions before implanting fragmented ions into time-of-flight mass analyzer 150 to perform an MS2 scan of the ions. Preferably, the TOF mass analyzer performs MS2 scans at a mass resolution of at least 15,000, at least 30,000, at least 50,000, or more preferably at least 60,000, at least 75,000, at least 100,000, at least 150,000, or at least 200,000 (resolution at 200 m / z). MS2 scans are preferably performed with a mass accuracy of 5 ppm or better (e.g., 1-2 ppm). Preferably, the TOF mass analyzer performs MS2 scans of the precursor mass segment at a frequency of at least 100 Hz (scans / second), at least 150 Hz, or at least 200 Hz.

[0074] The amount of fragmented ions within the ion group of the extraction trap 140 can be controlled by controlling the injection time used to accumulate fragmented ions within the extraction trap 140. For example, increasing the injection time for accumulating fragmented ions within the extraction trap 140 will increase the amount of accumulated fragmented ions and thus increase the amount of fragmented ions in the ejected ion group.

[0075] Therefore, the implantation time for accumulating fragmented ions can be calculated or determined based on the ionic strength value of the corresponding precursor mass range, such as by combining... Figure 2 The method described in the text is described in more detail.

[0076] The controller 195 is controllably connected to the mass filter 70, C-trap 100, mass analyzer 110, fragmentation chamber 120, extraction trap 140, and second mass analyzer 150 in combination as described below. Figure 2 The described mass spectrometry analysis method operates as follows. Controller 195 includes a computer having at least one processor and associated control electronics. Controller 195 controls the voltages applied to these components of the mass spectrometer. Controller 195 interfaces with the detectors of each mass analyzer 110, 150 to receive signals from the detectors in response to detected ions. The computer of the controller processes the detector signals to generate a mass spectrum (e.g., in the MS1 or MS2 domain). The controller includes a data storage unit (memory) for storing, for example, data from the detectors, mass spectrometers, etc. The computer of controller 195 runs a computer program product provided as software or firmware, which includes program instructions that, when loaded into memory and executed by one or more processors of the computer, cause the computer (and associated control electronics) to control the mass spectrometer 10 to perform the methods described below. The computer program product includes a computer program stored on a computer-readable medium. The controller also includes a user interface (e.g., a display screen, keyboard, and / or mouse) to allow a user to input information to the controller, such as the selection of an operating method and / or parameter settings for the operating method (e.g., target mass range, mass resolution of MS1 and MS2 scans, precursor mass segment width, etc.).

[0077] Despite Figure 1 The embodiments shown depict an extraction trap (ion trap), but those skilled in the art will understand that other methods for forming ion clusters of fragmented ions are equally applicable to the present invention. For example, the aggregation of ions can be influenced by the relatively slow transfer of ions through a multipole, and the ions can then be ejected as a single cluster into a TOF mass analyzer. Alternatively, orthogonal displacement of ions using an orthogonal accelerator can be used to form one or more ion pulses. The amount of fragmented ions can be controlled by controlling the number of ion pulses, as will be discussed below. Figure 2 A more detailed description is provided below. Further details of these alternatives can be found in US20030001088 A1, which describes the traveling wave ion aggregation method and whose disclosure is incorporated herein by reference in its entirety.

[0078] exist Figure 1The time-of-flight quality analyzer 150 shown is a multiple reflection time-of-flight quality analyzer (mr-TOF) 150. However, it should be understood that in other embodiments, any other suitable type of TOF quality analyzer can be used alternatively, such as a linear TOF quality analyzer, a single reflection, or a reflective TOF. The mr-TOF 150 is constructed around two opposing ion mirrors 160, 162 that extend in the drift direction. Mirrors 160, 162 are opposite each other in a direction orthogonal to the drift direction. An extraction trap 140 injects ions into the first mirror 160, and the ions then oscillate between the two mirrors 160, 162. The ejection angle of the ions from the extraction trap 140 and the additional deflectors 170, 172 allows control over the ion energy in the drift direction, such that the ions are guided along the length of the mirrors 160, 162 during oscillation, thereby producing a Z-shaped trajectory. Thirty mirrors 160, 162 are tilted relative to each other, creating a potential gradient that slows the drift velocity of ions and causes them to be reflected back along the drift dimension and concentrated onto detector 180. The tilting of the mirrors generally has the negative side effect of altering the time period of ion oscillation as ions travel along the drift dimension. This is corrected using a strip electrode 190 (as a compensation electrode) that alters the flight potential of a portion of the inter-mirror space that varies along the length of the relative mirrors 160, 162. The combination of the varying width of the strip electrode 190 and the varying distance between mirrors 160, 162 allows for ion reflection and spatial concentration onto detector 180, as well as maintaining good temporal concentration. A suitable MR-TOF 150 for use in this invention is further described in US 2015028197 (A1), the disclosure of which is incorporated herein by reference in its entirety. It should be understood that alternative types of mr-TOF mass analyzers can be used instead of the elongated relative mirror type used in the TOF mass analyzer 150, such as the type disclosed in US8395115 (Thermo Fisher Scientific).

[0079] In another embodiment, MS1 mass spectra can be acquired on the mr-ToF mass analyzer 150 instead of the first mass analyzer 110. In this embodiment, the voltage difference between the fragmentation chamber 120 and the upstream components of the system (including the C trap 100) is regulated by the controller 195 such that the collision energy of the ions does not cause fragmentation in the fragmentation chamber 120, but rather the unfragmented precursor ions enter the second transfer multipole 130 from the fragmentation chamber 120 and from there into the extraction trap (second ion trap) 140. Therefore, it should be understood that the first mass analyzer 110 may not be necessary in some embodiments. A single time-of-flight (ToF) mass analyzer (e.g., the mr-ToF analyzer 150) or two ToF mass analyzers (one in place of the orbital trap mass analyzer 110) can be used instead.

[0080] Figure 2Mass spectrometry analysis methods for samples within a target mass range, according to this teaching, are described. The target mass range can be defined by the characteristics of the sample or by the requirements for obtaining the mass spectrum. The target mass range is typically a wide or 'panoramic' range (e.g., a range with a width of 400 Da or more, 600 Da or more, 800 Da or more, 1000 Da or more, etc.). The target mass range can be at least 20x, 40x, 60x, 80x, 100x, 200x, 300x, or 400x the width of the precursor mass segment. Figure 2 The method can be combined Figure 1 , Figure 4 or Figure 5 Any mass spectrometer described is used to implement this.

[0081] The method begins with step 201, which involves ionizing a sample using an ionization source 20 to generate a plurality of precursor ions. Prior to the sample ionization step may be a step of separating sample molecules using a chromatographic system, and the sample may be ionized as it elutes from the chromatographic system.

[0082] On the precursor ion, a precursor ion mass scan or MS1 scan (i.e., without fragmentation) is performed in step 202. This can be performed using a first mass analyzer 110 (or an mr-TOF analyzer 150 in an alternative embodiment). The precursor MS1 ion scan can be a panoramic scan in the sense of obtaining a complete spectrum within a specific target mass range. Step 202 of performing the MS1 scan includes mass analysis of the precursor ion within the target mass range to obtain an MS1 ​​mass spectrum of the precursor ion in the MS1 domain, and evaluating (i.e. determining) the ion intensity values ​​within the target mass range in the MS1 spectrum (step 203), which may include identifying multiple intensity peaks and their intensities and / or may include integrated ion intensities within the MS1 spectrum, particularly within the intervals of the MS1 spectrum. The identified intensity peaks may be local maxima of the MS1 spectrum and / or regions of the MS1 spectrum above a minimum intensity threshold. Each intensity peak can be identified by a pair of values: a mass (m / z) value representing the location of the peak center and an intensity value representing the peak height (and the number of ions (i.e., intensity)).

[0083] According to the method of this teaching, multiple sub-ranges of mass can be selected within the target mass range in step 204. In one embodiment, the target mass range can be divided into multiple sub-ranges of mass. The target mass range can be continuous or discontinuous, for example, consisting of multiple sub-ranges of mass, where one or more other sub-ranges of mass are excluded from the target mass range. Precursor mass segments can be predefined according to the DIA method, i.e., defined before performing an MS1 ​​scan or after performing an MS1 ​​scan. The mass spectrometer controller can define the precursor mass segments, for example, according to preset instructions or user input instructions. The controller can control the mass selector of the mass spectrometer to sequentially select ions in each precursor mass segment, then fragment and mass analyze the ions as described, and then control the mass selector to select ions in the next precursor mass segment, and so on. The parameters of the precursor mass segments, i.e., their widths, carry information about the possible range of sample ions within the precursor mass segments.

[0084] For each precursor mass segment, the intensity value is determined by MS1 mass spectrometry. The intensity value can be a peak intensity value and can be based on the maximum intensity value of the largest peak within the precursor mass segment in the MS1 spectrum. The peak intensity value can be based on the peak intensity value of a single peak or the integral or cumulative intensity value within the precursor mass segment. The intensity value of each precursor mass segment can be a measure of the number of precursor ions detected in the MS1 spectrum within said precursor mass segment. The intensity value of a precursor mass segment can be an intensity value within the precursor mass segment or within adjacent mass segments, such as neighboring mass segments. For example, a sub-mass range can be defined within a target mass range, each sub-mass range comprising multiple adjacent precursor mass segments, such that all precursor mass segments within a sub-mass range are assigned the same intensity value.

[0085] The multiple precursor mass segments may or may not have substantially the same mass range. However, preferably, the multiple precursor mass segments have substantially the same mass range. The mass range of the precursor mass segments may be 20 Da (Daltons) or less, 15 Da (Daltons) or less, 10 Da (Daltons) or less, 8 Da (Daltons) or less, 6 Da (Daltons) or less, 4 Da (Daltons) or less, 3 Da (Daltons) or less, or 2 Da (Daltons) or less.

[0086] Multiple precursor mass segments typically span the entire target mass range. Identified precursor mass segments can be arranged on a common mass segment grid, such that the mass segments are adjacent to each other but do not overlap. Alternatively, mass segments can overlap and / or be placed around or centered around identified peaks. Each precursor mass segment may or may not include one or more identified peaks.

[0087] The method continues to perform one or more MS2 scans or fragmented ion scans, each MS2 scan or fragmented ion scan being performed on a corresponding precursor mass segment. A mass selector 70 can be used to select ions within each precursor mass segment.

[0088] For each MS2 scan, selected precursor ions within each precursor mass segment are fragmented into a set of fragmented ions 205. Precursor ions within each precursor mass segment can be fragmented in fragmentation chamber 120.

[0089] For each MS2 scan of fragmented ions, step 206 determines the amount of fragmented ions or ions to be fragmented for the precursor mass segment, wherein the control of the amount of fragmented ions or ions to be fragmented is based on intensity values ​​previously determined for the precursor mass segment. The amount of fragmented ions can be based on peak intensity values, or, if no peak is identified for the precursor mass segment, it can be a default value (which can be the lowest value) or an integrated intensity value. Step 206 can be performed before or after precursor ion fragmentation within the precursor mass segment.

[0090] The amount of fragmented ions is converted into ion groups or one or more ion pulses and provided to a mass analyzer to perform an MS2 scan 207 for mass analysis of the fragmented ions 208. A TOF mass analyzer 150 can be used to perform an MS2 scan. The amount of fragmented ions includes ion groups or one or more ion pulses.

[0091] The amount of ions can be controlled by allowing fragmented ions to enter the extraction trap (e.g., Figure 1 The injection time of the extraction trap (140) is controlled, in which ions accumulate before being provided as an ion mass to the mass analyzer. For this purpose, an ion mass from fragmented ions can accumulate within the extraction trap before mass analysis in the mass analyzer. Performing an MS2 scan can then include ejecting the ion mass from the extraction trap into the mass analyzer to perform the MS2 scan.

[0092] For example, the injection time into the extraction trap can be determined such that the total charge or the number of fragmented ions is substantially equal for the ion set generated for each precursor mass segment.

[0093] Alternatively, the amount of ions can be controlled by controlling the number of pulses generated by an injector, preferably an orthogonal accelerator injector, from fragmented ions within the precursor mass segment, wherein the pulses generated by the injector are injected into a time-of-flight mass analyzer to generate an MS2 scan / fragmented ion scan. Each pulse entering the time-of-flight mass analyzer generates a separate MS2 scan, and the separate MS2 scans are then summed to provide the MS2 scan of the precursor mass segment.

[0094] The above method relies on using intensity values ​​determined in the MS1 domain to control the amount of fragmented ions to be analyzed in order to perform an MS2 scan of the precursor mass segment.

[0095] Within the target mass range, the intensity of precursor ions can vary by several orders of magnitude. This presents a problem: when using a fixed ion implantation time or a fixed number of ion pulses for an MS2 scan, peaks in some precursor mass segments may saturate the detector, while peaks in others may not be recorded. Ion clusters with low ion numbers in certain precursor mass segments may be particularly underestimated due to their low intensity. The present invention addresses this problem by adjusting the number of fragmented ions in each precursor mass segment used for an MS2 scan, for example, by adjusting the ion implantation time or the number of ion pulses used based on the intensity value determined according to the MS1 mass spectrum of the precursor mass segment. Thus, for an MS2 scan, a longer ion implantation time or more ion pulses are used for the fragmented ions of a precursor mass segment corresponding to a lower intensity in the MS1 mass spectrum compared to a precursor mass segment corresponding to a higher intensity in the MS1 mass spectrum. The amount of fragmented ions to be analyzed for performing the MS2 scan can thus be controlled to be more equal, preferably substantially the same, in different precursor mass segments. In one embodiment, to use ionic intensity values ​​to control the amount of fragmented ions, multiple intensity levels can be defined for the intensity values, where similar intensity values ​​or intensity values ​​within a defined range are grouped into a common intensity level and given a common intensity value for said level. Each precursor mass segment can then be assigned to one of the multiple intensity levels, and the amount of fragmented ions can be determined or controlled based on the assigned intensity level, for example, such that each intensity level can be used to determine the injection time of ions into the extraction trap, or to determine the number of pulses from the orthogonal accelerator injector for an MS2 scan in a TOF mass analyzer, said pulse number being associated with a fixed amount of fragmented ions or ions to be fragmented.

[0096] Mass spectrometers must process precursor mass segments in a specific order to perform MS2 scans. For this purpose, the order in which precursor mass segments are processed can be defined for intensity levels. In one such embodiment, precursor mass segments assigned to intensity levels associated with higher peak intensities are processed earlier or before precursor mass segments assigned to intensity levels associated with lower peak intensities, which can then be processed later. In another embodiment, the reverse processing order can be used. For this purpose, all precursor mass segments within the target mass range can be processed, or only those with higher intensity levels can be processed, or only those with lower intensity levels can be processed. If necessary, one or more precursor mass segments can be skipped (omitted) from the MS2 scan sequence, for example, if any such mass segments have been placed in an exclusion list. Mass segments can be placed in an exclusion list, for example, based on earlier mass analyses of said mass segments.

[0097] Adjacent precursor mass segments within a target mass range can be grouped into a common mass subrange, such that all precursor mass segments within the subrange are assigned the same amount of fragmented ions for mass analysis within the MS2 domain. Therefore, all precursor mass segments within a subrange can be assigned the same intensity value, for example, representing the intensity level of the subrange's intensity range. Thus, multiple mass subranges can be defined within the target mass range, each comprising multiple adjacent precursor mass segments grouped based on their intensity values.

[0098] The amount of ions can be controlled by controlling the injection time of fragmented ions that accumulate as an ion mass in the extraction trap and by ejecting the ion mass from the extraction trap into a mass spectrometer for performing an MS2 scan.

[0099] Alternatively, the amount of ions can be controlled by controlling the number of ion pulses generated by the orthogonal accelerator injector from fragmented ions in the precursor mass segment and injected into the time-of-flight mass spectrometer for performing the MS2 scan.

[0100] In the mass spectrometry analysis method described above, the same mass analyzer can be used to perform the MS1 scan and the MS2 scan. Preferably, the mass analyzer used to perform the MS1 scan and then the MS2 scan can be a time-of-flight mass analyzer.

[0101] Alternatively, in the above-described mass spectrometry analysis method, an MS1 ​​scan can be performed using a first mass analyzer operating according to a first mass analysis principle, and an MS2 scan can be performed using a second mass analyzer operating according to a second mass analysis principle. For example, the first mass analyzer can be a Fourier transform mass analyzer, such as an Orbitrap mass analyzer, and the second mass analyzer can be a time-of-flight mass analyzer, such as a multiple reflection time-of-flight mass analyzer.

[0102] Figure 3 A schematic mass spectrum of MS1 scans from precursor ions of the sample is depicted (where the horizontal axis represents m / z and the vertical axis represents ion intensity). The precursor mass segments are divided into sub-mass ranges in the upper and lower halves of the figure, which are used for subsequent MS2 scans of fragmented ions obtained by fragmenting the precursor ions within the respective precursor ion mass segments.

[0103] MS1 scans can be performed, for example, using a Fourier transform quality analyzer or a time-of-flight quality analyzer, such as... Figure 1 As shown. MS1 scans can be obtained as a full spectrum (survey spectrum), which is acquired using a fixed total number of ions or a fixed implantation time to accumulate ions for the MS1 scan. On a time-of-flight mass analyzer, the acquisition time for an MS1 ​​scan can be on the order of a few microseconds, or it can be longer or shorter than a few microseconds. For high-resolution MS1 ​​scans using a Fourier transform mass analyzer, the acquisition time is typically longer than this.

[0104] MS1 mass spectrometry indicates spectral regions with high-intensity peaks, low-intensity peaks, and no observable peaks at all. MS1 mass spectra consist of multiple intensity peaks, which can be grouped into peak intensity levels. Each intensity level encompasses a range of intensity values. Any number of intensity levels can be defined. Figure 3 In the MS1 scan depicted in the upper half of the image, three intensity levels are defined, which are represented by a dark gray band indicating high-intensity peaks, a medium gray band indicating medium-intensity peaks, and a white band indicating the absence of peaks or peaks above a predefined threshold (low intensity). It should be understood that more than three intensity levels may be used in other embodiments.

[0105] exist Figure 3 In the lower half, the precursor mass spectrum is depicted as sub-ranges of mass based on the peak intensities (i.e., intensity levels) present in the MS1 scan. Sub-ranges of mass containing high-intensity peaks are labeled or classified as intensity level "1", and sub-ranges of mass containing medium-intensity peaks are labeled or classified as intensity level "2". Other sub-ranges of mass between those depicted as "1" and those described as "2", i.e., sub-ranges of mass without identified peaks or without peaks above a certain threshold, are depicted as intensity level "3".

[0106] The mass subranges with high intensity peaks (classified as intensity level "1") are typically relatively narrow. These mass subranges are divided into multiple narrower precursor mass segments, preferably each having a fixed width of 2-20 Da or less (preferably 2 Da or less). The precursor mass segments within these mass subranges have the shortest injection time or the fewest pulses for their fragmented ions.

[0107] Mass subranges with lower (maximum) intensity peaks (classified as intensity level "2") are typically wider than high-intensity subranges. These mass subranges are also divided into multiple narrower precursor mass segments, preferably each with a fixed width of 2–20 Da or less (preferably 2 Da or less). Compared to the precursor mass segments in the high-intensity mass subranges, the precursor mass segments in these mass subranges have longer implantation times or more pulse numbers for their fragmented ions.

[0108] The process is repeated for mass subranges with even lower intensity peaks (e.g., intensity levels "3", "4", ..., "n"). The final mass subrange with no visible peaks or only peaks below a certain threshold is denoted as "n". Figure 3 In this context, it indicates that these mass subranges have no visible peaks or only peaks below a certain threshold, denoted as "3". Similarly, these mass subranges are also divided into multiple narrower precursor mass segments, preferably each having a fixed width of 2-20 Da or less (preferably 2 Da or less). The precursor mass segments within these mass subranges preferably have the longest injection time or the most pulses for their fragmented ions.

[0109] In some embodiments, instead of using the maximum peak intensity in a mass subrange to classify the subrange, the integrated intensity within the mass subrange may also be used. Therefore, in some embodiments, the method may include a first group (“1”) of identifying one or more mass subranges within a target mass range based on the MS1 spectrum, wherein each of the one or more mass subranges in the first group includes one or more peaks or integrated intensities in the MS1 spectrum with an intensity higher than a first intensity threshold (corresponding to intensity level 1). The method may further include a second group (“2”) of identifying one or more mass subranges within a target mass range based on the MS1 spectrum, wherein each of the one or more mass subranges in the second group does not include one or more peaks or integrated intensities in the MS1 spectrum with an intensity higher than the first intensity threshold. In such cases, the one or more mass subranges in the second group may include one or more peaks with an intensity higher than a second intensity threshold (corresponding to intensity level 2). The method may further include a third group (“3”) (corresponding to intensity level 3) of identifying one or more mass subranges within a target mass range based on the MS1 spectrum, wherein each of the one or more mass subranges in the third group does not include one or more peaks with an intensity higher than the second intensity threshold. In some embodiments, more than three groups of mass subranges (corresponding to individual intensity levels) may be used. Therefore, embodiments may further include identifying additional [specific information] based on the MS1 spectrum. n One or more mass sub-ranges of the group ( n (It is an integer). Typically, in the... i Within the group of mass sub-ranges, each sub-range includes those with strengths higher than the first.i One or more peaks or integral intensities above the intensity threshold, but excluding those above the threshold. i-1 One or more peak or integrated intensities of the intensity threshold. Preferably, the width of the mass sub-range is: width of mass sub-range 1 < width of mass sub-range 2 < width of mass sub-range 3 < width of mass sub-range n.

[0110] The target quality range is divided into a series of narrow precursor quality segments for MS2 scanning. Each of the quality sub-ranges, labeled with intensity levels 1, 2, and 3, comprises multiple precursor quality segments, some of which are within... Figure 3 The lower half of the left side of the first three mass sub-ranges is marked as " in the magnified view". s The precursor mass segments typically have a fixed width, ranging from 2 to 20 Da, or even lower or higher, as further described below. The depicted precursor mass segments are adjacent to each other but do not overlap. Precursor mass segments may be arranged on a common mass segment grid. Alternatively, mass segments may overlap and / or be placed or centered around the identified peak. Each precursor mass segment including the identified peak may include one or more identified peaks. Precursor mass segments may or may not have the same or substantially the same mass range width. In some embodiments, precursor mass segments have the same or substantially the same mass range width (referred to herein as a 'fixed' width). The mass range width of the precursor mass segments may, for example, be preset by the mass spectrometer controller. The mass range width may be 20 Da or less, 15 Da or less, 10 Da or less, 8 Da or less, 6 Da or less, 4 Da or less, 3 Da or less, or 2 Da (Daltons) or less. In some embodiments, precursor mass segments are defined as having a fixed width (e.g., a fixed width in the range of 20 Da to 2 Da or less) and collectively covering the target mass range. For example, an 800 Da wide target mass range of 400–1200 (Da or m / z) can be divided into 200 consecutive 4 Da wide precursor mass segments (400–404 Da, 404–408 Da, 408–412 Da, …, 1196–1200 Da). Alternatively, such a target mass range can be divided into 400 precursor mass segments with a width of 2 Da, or into 100 precursor mass segments with a width of 8 Da, or into 40 precursor mass segments with a width of 20 Da, etc. Using narrower mass segment widths (e.g., about 2 Da or less) allows database searches to be used as an alternative to or supplement to using spectral libraries to identify molecular species in a sample.

[0111] For each precursor mass segment, the precursor ions of the mass segment are fragmented in a fragmentation step (e.g., in...). Figure 1In the fragmentation chamber 120, the fragmented ions are processed to obtain the MS2 spectrum of the fragmented ions originating from the specific precursor mass segment.

[0112] Therefore, the peak intensity values ​​of the precursor mass segment in the MS1 scan are converted into the amount of fragmented ions or ions to be fragmented, and the MS2 scan is analyzed to obtain the fragmented ions. Thus, the amount of fragmented ions can be determined based on the aforementioned peak intensity levels.

[0113] The amount of ions can be controlled by controlling the injection time of the ion pack used to accumulate fragmented ions or by controlling the number of ion pulses for fragmented ions.

[0114] If the implantation time used for accumulating the ion pool is controlled, the implantation time IT can be determined using the following method:

[0115] A fixed injection time IT is determined for each precursor mass segment within each mass subrange (“1” to “n-1”), with shorter injection times for high-intensity mass subranges: IT1 < IT2 < ... < ITn-1. Suitable injection times can be determined through calibration or from previous experiments. A fixed injection time can be determined for each mass subrange to provide an appropriate number of ions for the MS2 scan (e.g., without saturating the detector but providing measurable intensity). The remaining mass subranges “n” can be used as injection times by dividing the remaining acquisition time of the MS2 scan by the number of remaining precursor mass segments (e.g., 2–20 Da width segments) in those mass subranges “n”. This is based on the characteristic that DIA cycles (i.e., cycles comprising a full set of MS2 scans across the entire target mass range) typically have a maximum or defined amount of time to provide sufficient sampling points on the chromatographic peak. Therefore, maximizing the available IT in the low-intensity region is advantageous. Thus, the present invention can improve the dynamic range of DIA analysis by spending different (longer) amounts of time on portions of the mass spectrum with lower intensities.

[0116] The cycle time of the DIA method according to the invention preferably consists essentially of the time for performing MS1 scans and a full set of MS2 scans (i.e., for all precursor mass segments within the target mass range). The DIA cycle is preferably performed at least 2, 3, 4, 5, 6, 7, or 8 times across the peak width (peak base width). A single DIA cycle may require 1-5 seconds (e.g., 2-4 or about 3 seconds).

[0117] Alternatively, the injection time IT can be determined using a fixed injection time IT for each mass subrange (“1” to “n”), i.e., a fixed injection time for all subranges, where the injection time for the high-intensity mass subrange is shorter: IT1 < IT2 < ... < ITn.

[0118] The processing of precursor mass segments for a mass spectrometer used to perform MS2 scans can be initiated by first processing the precursor mass segments in the high-intensity ("1") mass subrange, and then continuing to process the precursor mass segments in the next highest-intensity ("2") mass subrange, until the lowest-intensity ("n") mass subrange.

[0119] Using the methods according to this teaching, in addition to MS1 mass spectra within the target mass range, it is possible to independently acquire precursor mass segments across the entire target mass range as a set of MS2 mass spectra. Information on the precursor ion mass from peaks identified in the MS1 mass spectra and the corresponding fragmented ion mass from peaks identified in the MS2 mass spectra can be used for database searches or spectral libraries to identify the types of precursor molecules present in the sample. Using narrow (e.g., 2 Da or less) precursor mass segments, database searches can be performed with or without MS1 peak information. In one approach, as described above, MS1 spectra obtained with high resolution and high quality accuracy can be used to generate a candidate list of precursor ions using a standard non-fragmented ion database. Computer-simulated (in-silico) fragmentation of the candidates in the list can generate a library within a narrow mass range, and the fragmented spectra are compared to the library and / or quantized as known in the art to provide identification of the precursor ions.

[0120] Alternative embodiments of mass spectrometers suitable for use in conjunction with the methods embodying the present invention are described below. Figure 4 It is illustrated schematically. Figure 4 In this embodiment, the ion source 10 is coupled to the mass selection device 20. This can be achieved through methods such as... Figure 1 The embodiment shows an ESI ion source 20 and its corresponding coupling with a quadrupole mass filter 70 to provide such an arrangement. Figure 4 As shown, the output of mass selection device 20 is coupled to C-trap (curved linear ion trap) 40. In the first operating mode, precursor ions can be collected in C-trap 40 for subsequent emission into mass analyzer 50 to perform an MS1 ​​scan. The MS1 scan can be analyzed for the identification of intensity peaks and / or the identification and classification of mass subranges, as described above regarding... Figure 2 and Figure 3 The method described herein. The mass analyzer 50 may be a Fourier transform mass spectrometry (FTMS) analyzer 50, which may be embodied as an orbital trapping mass analyzer, a Fourier transform ion cyclotron resonance (FTICR) mass analyzer, or any other type of Fourier transform mass analyzer. Mass analyzers such as orbital trapping mass analyzers and ion cyclotron resonance mass analyzers may also be used in this invention even when other types of signal processing besides Fourier transform are used to obtain mass spectrometry information from transient signals (see, for example, WO2013 / 171313, Thermo Fisher Scientific).

[0121] According to the second operating mode, the precursor ions are further transported by C-trap 40 to fragmentation chamber 80, such as a collision / transport cell, where they are fragmented. The mass range of the precursor ions to be transported to the transport / collision cell 80 can be determined according to the above description. Figure 2 and Figure 3 The method described is used for selection. Fragmented ions are then accumulated in the RF (radio frequency controlled) trap 70, where the implantation time for accumulating fragmented ions can be determined according to the above description. Figure 2 and Figure 3 The method described is for control. The fragmented ion mass accumulated in the RF trap 70 is then ejected into the mass analyzer 70 to perform an MS2 scan, which can be embodied as a time-of-flight mass analyzer, such as a multi-reflection time-of-flight mass analyzer.

[0122] In another alternative embodiment, it can be, for example, by Figure 5 The branch path arrangement shown in the embodiments is used to provide a mass spectrometer according to the invention. Figure 5 In one embodiment, the ion source 200 is coupled to the mass selection device 210. This can be achieved, for example... Figure 1 The embodiment shows an ESI ion source 200 and its corresponding coupling with a quadrupole mass filter 70 to provide such an arrangement. Figure 5 As shown, the output of mass selection device 210 is coupled to branched ion path 220. The branched ion path guides ions output from the mass selection device along one of two paths. First path 222 guides ions to C-trap 230, where ions are collected for analysis in the MS1 domain by a Fourier transform mass analyzer, such as orbital trap mass analyzer 240, to obtain an MS1 ​​scan. The MS1 scan can be analyzed based on the identification of intensity peaks and / or the identification and classification of mass subranges, as described above regarding... Figure 2 and Figure 3 The method described herein. The second path 224 guides ions to the fragmentation chamber 250 for ion fragmentation and subsequent mass analysis in the MS2 domain. The branched ion path can use an RF voltage to guide ions along either the first path 222 or the second path 224. The branched ion path can be a branched RF multipole. Suitable for use with Figure 5 The branched ion pathways in the embodiments are further described in US 7420161.

[0123] according to Figure 5 In an alternative embodiment, the branched ion path can be used to direct ions to C trap 230 for MS1 analysis or to fragmentation chamber 250 for MS2 analysis. Fragmented ions ejected from fragmentation chamber 250 can be accumulated in ion extraction trap 260 prior to being injected into the mr-TOF analyzer 270 as a group. This can be understood according to the above description regarding... Figure 2 and Figure 3 The described method controls the injection time for accumulating fragmented ion clusters in the ion extraction trap 260. Thus, it is possible to control the injection time as follows: Figure 1 A similar arrangement is described in the text to provide the arrangement of the fragmentation chamber 250, the ion trap 260, and the mr-TOF 270.

[0124] Therefore, according to Figure 5 In an alternative embodiment, ions can be guided to empty the C-trap 230 of the MS1 orbital trap mass analyzer 240 for MS2 analysis. This configuration allows for increased parallelism of MS1 and MS2 scans. Consequently, a larger proportion of the peak duration is available for performing the MS2 scan.

[0125] By utilizing a branched path arrangement, C-trap 230 is no longer located in the path supplying ions to the fragmentation chamber for performing MS2 scans. Therefore, C-trap 230 according to the branched path embodiment can be loaded using multiple smaller fill steps over an extended time period. This makes the ions accumulated in C-trap 230 more representative of chromatographic peaks.

[0126] Figure 6 The figure above shows the chromatographic peaks of the sample as it eluted from the chromatographic apparatus. The dots superimposed on the peaks represent examples of the time interval at which the MS1 scan began. The figure below shows a magnified portion of the chromatographic peaks, where the vertical lines represent the time intervals during which a separate MS2 scan was performed on the precursor mass fraction.

Claims

1. A mass spectrometry method for analyzing samples within a target mass range, comprising the following steps: -Ionize the sample to generate multiple precursor ions; - Perform an MS1 ​​scan on the precursor ion, including mass analysis of the precursor ion within the target mass range to obtain the MS1 mass spectrum of the precursor ion; - Determine the ion intensity values ​​within the MS1 mass spectrum; - Select multiple precursor mass segments within the target mass range, wherein the multiple precursor mass segments span the entire target mass range, wherein each precursor mass segment has a mass range, and for each precursor mass segment: To fragment the precursor ions within the precursor mass segment; as well as The MS2 scan of the fragmented ions was performed in the following manner: - The amount of fragmented ions in the precursor mass segment is controlled based on the intensity value of the precursor mass segment derived from the MS1 mass spectrometer; as well as - Perform mass analysis on the amount of the fragmented ions; The control of the amount of fragmented ions is achieved by: (i) controlling the injection time of the fragmented ions to accumulate an ion mass from the fragmented ions within the extraction trap, wherein performing the MS2 scan includes ejecting the ion mass from the extraction trap into a mass analyzer for performing the MS2 scan; or (ii) controlling the number of pulses generated by the ion implanter from the fragmented ions within the precursor mass segment, the pulses being injected by the ion implanter into a time-of-flight mass analyzer for generating the MS2 scan; and Compared to the precursor mass segment corresponding to higher intensities in the MS1 mass spectrum, the precursor mass segment corresponding to lower intensities in the MS1 mass spectrum uses a longer ion implantation time or more ion pulses for its fragmented ions.

2. The method of claim 1, wherein the injection time is determined such that the total charge or the number of fragmented ions is substantially equal for each ion group of the precursor mass segment.

3. The method of claim 1, wherein the ion implanter is an orthogonal accelerator implanter.

4. The method according to any one of the preceding claims, wherein a plurality of intensity levels are defined for the intensity value, and wherein each of the precursor mass segments is assigned to one of the plurality of intensity levels, and wherein the amount of the fragmented ions is controlled based on the assigned intensity level.

5. The method of claim 4, wherein the order of processing the precursor mass segments is defined based on the strength level, such that precursor mass segments assigned to strength levels associated with higher strength values ​​are processed before precursor mass segments assigned to strength levels associated with lower strength values.

6. The method of claim 1, wherein a plurality of mass sub-ranges are defined within the target mass range, each mass sub-range comprising a plurality of adjacent precursor mass segments, such that all the precursor mass segments within the mass sub-range are assigned the same intensity value.

7. The method of claim 1, wherein an intensity peak is identified within the target mass range, and wherein the intensity value includes the peak intensity value of the identified intensity peak.

8. The method of claim 1, wherein each of the plurality of precursor mass segments has the same mass range, and wherein the mass range is 20 Da (Daltons) or less.

9. The method of claim 1, wherein the precursor mass segments comprise precursor mass segments that are adjacent to each other but do not overlap.

10. The method of claim 1, wherein the mass ranges of the precursor mass segments overlap.

11. The method of claim 1, wherein the same quality analyzer is used for performing the MS1 scan and for performing the MS2 scan, and wherein the quality analyzer is a time-of-flight quality analyzer.

12. The method of claim 1, wherein the MS1 scan is performed using a first quality analyzer, and wherein the MS2 scan is performed using a second quality analyzer, wherein the first quality analyzer is a Fourier transform quality analyzer, and wherein the second quality analyzer is a time-of-flight quality analyzer.

13. The method of claim 12, wherein the Fourier transform quality analyzer is an Orbitrap quality analyzer.

14. The method of claim 12, wherein the time-of-flight quality analyzer is a multi-reflection time-of-flight quality analyzer.

15. A mass spectrometer for performing mass spectrometric analysis of a sample within a target mass range, the mass spectrometer comprising: An ionization source, said ionization source being used to generate a plurality of precursor ions from said sample provided by the chromatographic system; A mass selector for selecting a mass range of the precursor ions; A mass analyzer for performing an MS1 ​​scan on the precursor ions in the MS1 domain; Fragmented devices; A mass analyzer for performing MS2 scans on fragmented ions in the MS2 domain; Ion array or ion pulse forming equipment; and a controller, the controller being configured to: (i) causing the mass selector to select precursor ions within the target mass range; (ii) The mass analyzer used to perform the MS1 scan performs an MS1 ​​scan on the precursor ions within the target mass range to obtain the MS1 mass spectra of the precursor ions in the MS1 domain; (iii) Determine the ion intensity values ​​in the MS1 mass spectrometer; (iv) The mass selector selects a plurality of precursor mass segments within the target mass range, wherein the plurality of precursor mass segments span the entire target mass range, and each precursor mass segment has a mass range. (v) The amount of fragmented ions in the precursor mass segment is controlled based on the intensity value of each precursor mass segment derived from the MS1 mass spectrometer; (vi) The fragmentation device fragments the precursor ions within each precursor mass segment; (vii) The ion group or ion pulse forming device forms the amount of fragmented ions into one or more ion groups or ion pulses, and injects the ion group or ion pulse of the fragmented ions into the mass analyzer to perform an MS2 scan. and (viii) The mass analyzer used to perform the MS2 scan performs mass analysis on the amount of fragmented ions in the one or more ion groups or ion pulses, wherein the amount of fragmented ions is controlled by: (i) controlling the injection time of the fragmented ions to accumulate ion groups from the fragmented ions in the extraction trap, and wherein performing the MS2 scan includes ejecting the ion groups from the extraction trap into the mass analyzer for performing the MS2 scan; or (ii) controlling the number of pulses generated by the ion implanter from the fragmented ions in the precursor mass segment, the pulses being injected by the ion implanter into the time-of-flight mass analyzer for generating the MS2 scan; and Compared to the precursor mass segment corresponding to higher intensities in the MS1 mass spectrum, the precursor mass segment corresponding to lower intensities in the MS1 mass spectrum uses a longer ion implantation time or more ion pulses for its fragmented ions.

16. The mass spectrometer for performing mass spectrometry analysis according to claim 15, wherein the controller is adapted to perform the method according to any one of claims 1 to 14.

17. A computer program product comprising program instructions that, when loaded into the memory of a controller, cause the controller of a mass spectrometer to perform the method according to any one of claims 1 to 14.

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