Triggered high resolution mass spectrometer analysis of isobarically labeled reporter ions

Abstract

A method of mass analysis of analytes in a sample mixture, wherein all analytes of each sample of the mixture are comprised of the same isotopically labeled marker, and wherein each respective sample is associated with a different unique isotopically labeled marker, the method comprising: within the mass spectrometer, fragmenting a reporter ion species from analyte ions of the mixture, wherein the reporter ion species comprises all isotopically labeled markers from each different labeled sample; mass analyzing the reporter ion species at a first mass spectral resolution; comparing an observed intensity pattern of the mass analyzed reporter ion species to a predetermined intensity pattern; and if the observed intensity pattern does not match the predetermined intensity pattern, mass analyzing the reporter ion species at a second mass spectral resolution higher than the first mass spectral resolution.

Description

Technical Field

[0001] This application generally relates to mass spectrometers and mass spectrometry analysis. Specifically, this application relates to the analysis of isotopically labeled samples, and more specifically, to the analysis of samples having isotopic isotopic labels. Background Technology

[0002] A mass spectrometer is a sensitive instrument that can detect, identify, and / or quantify molecules based on their mass-to-charge ratio (m / z). A mass spectrometer typically includes an ion source for generating ions from components contained within a sample, a mass analyzer for separating the ions based on their m / z, and an ion detector for detecting the separated ions. The mass spectrometer can be connected to a computer-based software platform that uses data from the ion detector to construct a mass spectrum showing the relative abundance of each detected ion as a function of m / z. The m / z of the ions can be used to detect and quantify molecules in both simple and complex mixtures. Separation devices such as liquid chromatography (LC) or gas chromatography (GC) can be connected to a mass spectrometer (MS) in a combined system (e.g., an LC-MS or GC-MS system) to separate components contained in a sample, which are then introduced into the mass spectrometer.

[0003] Even with targeted acquisition, selective detection and quantification of specific target analytes in complex mixtures are often extremely challenging. For example, in proteomics studies, target peptides may be contained within complex biological matrices composed of mixtures of tens of thousands of peptides with abundances spanning numerous orders of magnitude. Tandem mass spectrometry (MS / MS or MS2) can be used to quantify molecules in complex mixtures. For instance, in a modified targeted acquisition technique called internal standard triggered parallel reaction monitoring (IS-PRM), a sample containing a peptide target can be spiked with a known amount of a corresponding internal standard (IS) (e.g., a synthetic peptide with the same amino acid sequence but containing a stable heavy isotope) and scanned by the instrument in a dual-mode combined single-stage and tandem mass spectrometry. Detection of the internal standard triggers the mass spectrometer to monitor the specific endogenous target peptide.

[0004] Use identical mass labels such as Tandem Mass Tag ® (TMT) ® Reagents (manufactured by Electrophoretics Limited and available from Thermo Fisher Scientific, Waltham, Massachusetts) and / or isotopic labels (iTRAQ) for relative and absolute quantification. ® Multiplexing (AB Sciex Pte. Ltd.) can improve sample throughput. Isotopic mass tags are compounds that react with and bind to an analyte (such as a peptide). For example... Figure 1As shown, isotopic mass tags have a "reporter region," a "mass equilibrium region," and reactive groups (e.g., amine reactive groups, cysteine ​​reactive groups, or carbonyl reactive groups). Several versions of isotopic mass tags have been created, all having the exact same total mass of the reporter region plus the equilibrium region, but using various different combinations and positions of stable isotopes (e.g., 13 C and 15 (N isotopes), the quality of the reported region and the quality of the equilibrium region are different for each version.

[0005] In proteomics research, the use of isotopic mass tags is advantageous because it enables single-sample mass analysis instead of multiple multiplex mass analyses (where each sample is analyzed individually). Multiple individual samples can be analyzed by tagging the analytes (e.g., peptides) in each sample with different versions of isotopic mass tags, pooling all samples together, and analyzing the pooled samples in a single experiment using LC-MS or GC-MS. According to isotopic labeling, the peptide or protein analytes in each sample are tagged with the corresponding isotopic label before preparing the mixture. Although each tagged analyte gains mass due to the labeling procedure, the mass gain is the same across all samples because any mass change in the report region due to isotopic substitution is offset by the corresponding mass reduction in the "mass equilibrium region." Therefore, during the detection (and potential separation) of specific analyte ions in a mass analysis, all similar analytes from different samples can be detected and / or separated simultaneously because they all contain the same m / z value, regardless of their origin.

[0006] Fracturing basis Figure 1 The bonds defined by the dashed lines release each reporter ion. Therefore, a mass spectrometry experiment based on isotopic mass tags can determine the relative amount of an analyte in each of several different samples. For this purpose, the analyte in each sample (including any possible reference samples) is labeled with an isotopic mass tag. Most reporter ions have m / z values ​​in the range of 126–135 Th. Labeling reagents for generating isotopic tags are available, which require high-energy collisional dissociation (HCD) to release various mass reporter ions. Before releasing the mass reporter ions, protein fragment ions or peptide fragment ions with m / z values ​​searchable to a database can be generated using a low-energy procedure (such as resonance-excited collisional dissociation), and the structure of the analyte, targeting proteins or peptides, can be identified by standard MS / MS analysis. Subsequently, the reporter ions can be cleaved using HCD, and the protein fragment ions or peptide fragment ions can be analyzed by MS3. The relative intensity ratios of the various reporter ions thus released at their various m / z values ​​indicate the relative concentration of the analyte among the individual samples.

[0007] Recently, Thermo Fisher Scientific, located in Waltham, Massachusetts, introduced a new suite of tandem mass tagging (TMT) methods that include deuterated substances that differ from previously available TMT markers by up to 3 mTh. These new TMT reagents roughly double the total number of reporter ions that can be used as markers in a sample set (from 18 to 32). For example, Figure 2A The m / z values ​​for various reporter ions in the TMTpro™ 32-cell series are shown (note the various breakpoints along the m / z axis). The signals for multiple reporter ions are only resolvable when using a high-resolution mass analyzer such as the Orbitrap™ mass analyzer. For comparison, Figure 2B The format of various reporter ion peaks is shown when using a standard resolution mass analyzer (note that there are no breakpoints on this x-axis). Using a D20Orbitrap running at 4 kV, a 192 ms Orbitrap transient signal is required to fully resolve all these TMT reporter ions, twice the duration of the transient signal required to fully resolve the previous 18-unit TMT reagent set. Clearly, when the full set of labels is applied to 32 different samples, this longer transient signal will reduce the MSn acquisition rate and limit the LC-MS sampling depth of the mass analyzer. The method described here aims to mitigate the impact of these longer transient signals by limiting the number of acquisitions that require these slower, high-resolution mass spectrometer acquisitions. Summary of the Invention

[0008] According to a first aspect of this disclosure, a method for mass analysis of analytes in a sample mixture is provided, wherein all analytes in each sample generating the mixture contain the same isotopic label, and wherein each respective sample is associated with a different unique isotopic label, the method comprising: Within the mass spectrometer, reporter ions are cleaved from the analyte ions of the mixture, wherein the cleaved reporter ions contain all isotopic labels from each of the different labeled samples; Mass analysis of the reporter ion was performed at a first mass-to-charge ratio (m / z) resolution; The observed intensity variation patterns of reported ionic substances with mass analysis were compared with the predetermined intensity variation patterns with mass. If the observed intensity pattern does not match the predetermined intensity pattern within the tolerance range, the reported ion substance is subjected to mass analysis at a second mass-to-charge ratio (m / z) resolution greater than the first mass-to-charge ratio (m / z) resolution.

[0009] According to a first aspect of this disclosure, the distribution of analytes among the samples can be determined based on mass analysis of the group of reporter ions at a second mass-to-charge ratio (m / z) resolution. In various embodiments, the time required to perform mass analysis of the group of reporter ions at the second mass-to-charge ratio (m / z) resolution is longer than the time required to perform mass analysis of the group of reporter ions at the first mass-to-charge ratio (m / z) resolution. In various embodiments, the mass spectrometer system includes a first mass analyzer and a second mass analyzer, wherein the first mass analyzer performs mass analysis of the group of reporter ions at the first mass-to-charge ratio (m / z) resolution, and the second mass analyzer performs mass analysis of the group of reporter ions at the second mass-to-charge ratio (m / z) resolution. In various embodiments, the mass-to-charge ratio difference between at least some of the reporter ions may be less than 0.01Th.

[0010] According to some embodiments, the method according to the first aspect may further include: Tandem mass analysis of each analyte introduced into the mass spectrometer; and Based on the tandem mass analysis, the structure and / or composition of the analyte are identified; In such embodiments, each isotope label may include a respective reporter ion portion and a respective mass normalization region, such that the sum of the masses of the reporter ion portion and the mass normalization region is the same for all said isotope labels. Attached Figure Description

[0011] Figure 1 The general chemical structures of various known tandem mass tag parts are shown.

[0012] Figure 2A It is a set of mass spectra of reporter ions with 32 different mass tags.

[0013] Figure 2B The results observed during analysis using a standard resolution mass spectrometer system are shown. Figure 2A Report the mass spectrometry peak morphology of the ions.

[0014] Figure 2C This is an example of a predicted intensity pattern for the relative intensity of the reporter ions, which can be seen in a low-resolution mass spectrum of a mixture of individually isotopically labeled sample samples where the signals of the individual reporter ions are not resolved.

[0015] Figure 3 This is a functional block diagram of an exemplary LC-MS system.

[0016] Figure 4 This is a functional block diagram of an exemplary embodiment of a mass spectrometer system.

[0017] Figure 5 This is a flowchart of a method for mass spectrometry analysis of isotropically labeled samples according to this teaching.

[0018] Figure 6 This is a schematic diagram of a first exemplary mass spectrometer system that can implement the methods of this teaching.

[0019] Figure 7 This is a schematic diagram of a second exemplary mass spectrometer system that can implement the methods of this teaching.

[0020] Figure 8 This is a functional block diagram of an exemplary MS control system. Detailed Implementation

[0021] As used in this application and claims, the singular forms “a” and “the” include plural references unless the context clearly specifies otherwise. Additionally, the term “comprising” means “including”. Furthermore, the term “coupled” does not exclude the existence of intermediate elements between coupled items.

[0022] The systems, apparatuses, and methods described herein should not be construed as limiting in any way. Rather, this disclosure relates to all novel and non-obvious features and aspects of the various disclosed embodiments, whether they exist individually or in various combinations and sub-combinations with each other. The disclosed systems, methods, and apparatuses are not limited to any particular aspect, feature, or combination thereof; and the disclosed systems, methods, and apparatuses are not required to have any one or more particular advantages, nor are they required to solve any one or more particular problems. Any operational theory is intended to facilitate explanation, but the disclosed systems, methods, and apparatuses are not limited to such operational theory.

[0023] While some operations of the disclosed methods are described in a specific chronological order for ease of description, it should be understood that this descriptive approach encompasses rearrangements of the order unless specific terminology given below requires a particular order. For example, operations described sequentially may be rearranged in some cases, or performed in parallel. Furthermore, for the sake of brevity, the accompanying drawings may not show various ways in which the disclosed systems, methods, and apparatus are used in conjunction with other systems, methods, and apparatuses. Additionally, terms such as “produce” and “provide” are sometimes used in this specification to describe the disclosed methods. These terms are high-level abstractions of the actual operations performed. The actual operations corresponding to these terms will vary depending on the specific implementation and will be readily identified by those skilled in the art.

[0024] In some examples, numerical values, procedures, or devices are referred to as “lowest,” “best,” “smallest,” “greater than,” “less than,” “equal to,” or similar expressions. It should be understood that such descriptions are intended to indicate that a choice can be made among many functional alternatives used, and that such a choice is not necessarily better, smaller, or otherwise superior to the other choices.

[0025] Quantitative multiplex mass spectrometry analyses are typically performed using separation-mass spectrometry (LC-MS) systems. Therefore, LC-MS systems are described below. The LC-MS systems described are exemplary and not limiting. The methods and systems described herein can be operated as part of, in combination with, or in conjunction with any other suitable separation-mass spectrometry system, such as a high-performance liquid chromatography-mass spectrometry (HPLC-MS) system, a gas chromatography-mass spectrometry (GC-MS) system, or a capillary electrophoresis-mass spectrometry (CEMS) system. The methods and systems described herein can also be operated in conjunction with any other continuous-flow sample source, such as a flow injection mass spectrometry (FI-MS) system, in which the analyte is injected into the mobile phase (without column separation) and enters the mass spectrometer with an intensity that varies over time (e.g., a Gaussian-like peak). The methods and systems described herein can also be operated on a continuous-flow sample source that provides a stable and consistent sample quantity, such that the sample measured by the mass spectrometer does not change over time.

[0026] Figure 3 A functional block diagram of an exemplary LC-MS system 100 is shown. The LC-MS system 100 includes a liquid chromatograph 102, a mass spectrometer system 104, and a controller 106. The liquid chromatograph 102 is configured to separate analytes within a sample 108 injected into the liquid chromatograph 102 over time. The sample 108 may include, for example, chemical analytes (e.g., molecules, ions, etc.) and / or biological analytes (e.g., metabolites, proteins, peptides, lipids, etc.) for detection and analysis by the LC-MS system 100. The liquid chromatograph 102 can be implemented by any liquid chromatograph suitable for a particular implementation. In the liquid chromatograph 102, the sample 108 is injected into a mobile phase (e.g., a solvent) that carries the sample 108 through a chromatographic column 110 containing a stationary phase (e.g., an adsorbent packing material). As the mobile phase passes through the column 110, the analytes in the sample 108 elute from the column 110 at different times based on their (e.g.) size, affinity for the stationary phase, polarity and / or hydrophobicity.

[0027] A detector (e.g., an ion detector assembly, ion-electron converter, and electron multiplier of mass spectrometry system 104) measures the relative intensity of a signal modulated by the separated analyte from eluent 112 of column 110. The data generated by this detector can be represented as a chromatogram, plotting retention time on the x-axis and a signal representing relative intensity on the y-axis. The retention time of the analyte is typically measured as the time interval from injection of sample 108 into the mobile phase to the appearance of the maximum relative intensity peak after chromatographic separation. In some examples, the relative intensity may be correlated with or represent the relative abundance of the separated analyte. Data generated by liquid chromatograph 102 is output to controller 106.

[0028] In some cases, especially when analyzing complex mixtures, multiple different analytes in sample 108 may co-elute from column 110 at approximately the same time, and therefore may have the same or similar retention times. Therefore, determining the relative intensity of individual analytes within sample 108 requires further separation of the signals attributed to each individual analyte. For this purpose, liquid chromatograph 102 directs the analytes contained in eluent 112 to mass spectrometry system 104 for further separation, identification, and / or quantification of the analytes.

[0029] Mass spectrometry system 104 ionizes ions from the analyte received from liquid chromatograph 102 and sorts or separates the resulting ions based on their m / z. Mass spectrometry system 104 can be implemented using a multistage mass spectrometer or a tandem mass spectrometer, wherein the multistage mass spectrometer is configured to perform multistage mass spectrometry analysis (also referred to as MSn, where n is 2 or greater), and the tandem mass spectrometer is configured to perform tandem mass spectrometry analysis (a form of multistage mass spectrometry analysis referred to as MS / MS or MS2 (where n is 2)). The detector in mass spectrometry system 104 measures the signal intensity produced by the ions. As used herein, “intensity” or “signal intensity” refers to the detector’s response and can represent absolute abundance, relative abundance, ion count, intensity, relative intensity, ion current, or any other suitable ion detection measure. Data acquired by mass spectrometry system 104 is output to controller 106. The data generated by the detector can be represented by a mass spectrum, which plots the intensity of the observed signal as a function of the m / z of the detected ions.

[0030] Figure 4This is a functional block diagram of an exemplary embodiment of a mass spectrometer system 104. The mass spectrometer system 104 includes an ion source 202, a first mass analyzer 204-1, a collision cell 204-2, a second mass analyzer 204-3, and a controller 206. The mass spectrometer system 104 may also include any additional or alternative components (e.g., ion optics, filters, ion storage devices, autosamplers, detectors, etc.) that may be required depending on the specific embodiment.

[0031] Ion source 202 ionizes the analyte received from chromatographic column 110 to generate an ion stream 208 and delivers the ions to first mass analyzer 204-1. Ion source 202 can employ any suitable ionization technique, including but not limited to electron ionization, chemical ionization, matrix-assisted laser desorption / ionization, electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, inductively coupled plasma, etc. Ion source 202 may include various components for generating ions from the analyte contained in sample 108 and delivering the ions to first mass analyzer 204-1.

[0032] The first mass analyzer 204-1 receives the ion stream 208, isolates precursor ions in a selected m / z range, and delivers the precursor ion beam 210 to the collision cell 204-2. The collision cell 204-2 receives the precursor ion beam 210 and generates product ions (e.g., fragment ions) through a controlled dissociation process. The collision cell 204-2 guides the product ion beam 212 to the second mass analyzer 204-3. The second mass analyzer 204-3 filters and / or performs mass analysis on the product ions.

[0033] Mass analyzers 204-1 and 204-3 isolate or separate ions based on the m / z of each ion. Mass analyzers 204-1 and 204-3 can be implemented using any suitable mass analyzer, such as quadrupole mass filters, ion traps (e.g., three-dimensional quadrupole ion traps, cylindrical ion traps, linear quadrupole ion traps, toroidal ion traps, etc.), time-of-flight (TOF) mass analyzers, electrostatic trap mass analyzers (e.g., orbital electrostatic traps, such as Orbitrap mass analyzers, Kingdon traps, etc.), Fourier transform ion cyclotron resonance (FT-ICR) mass analyzers, etc. Mass analyzers 204-1 and 204-3 do not necessarily need to be implemented using the same type of mass analyzer.

[0034] The collision cell 204-2 can be implemented by any suitable collision cell. As used herein, "collision cell" can encompass any structure or device configured to generate product ions through a controlled dissociation process, and is not limited to devices for collision-induced dissociation. For example, the collision cell 204-2 can be configured to fragment precursor ions using collision-induced dissociation, electron transfer dissociation, electron capture dissociation, photo-induced dissociation, surface-induced dissociation, ion / molecular reactions, etc.

[0035] An ion detector (not shown) detects ions at various m / z values ​​and responsively generates an electrical signal representing the ion intensity. This electrical signal is transmitted to a controller 206 for processing, such as constructing a mass spectrum of the analyzed ions. For example, a mass analyzer 204-3 can emit a separated ion emission beam to an ion detector configured to detect ions in the emission beam and generate or provide data that the controller 206 can use to construct a mass spectrum. The ion detector can be implemented using any suitable detection device, including but not limited to electron multipliers, Faraday cups, etc. In other examples, such as when a second mass analyzer 204-3 is implemented using an orbital electrostatic trap mass analyzer, the second mass analyzer 204-3 functions as both a mass analyzer and a detector.

[0036] Controller 206 is communicatively coupled to mass spectrometer system 104 and configured to control the operation of mass spectrometer system 104. For example, controller 206 may be configured to control the operation of various hardware components included in ion source 202 and / or mass analyzers 204-1, 204-3. For example, controller 206 may be configured to: control the accumulation time of ion source 202 and / or mass analyzer 204; control the oscillating voltage power supply and / or DC power supply to provide radio frequency voltage and / or DC voltage to mass analyzer 204; adjust the values ​​of radio frequency voltage and DC voltage to select the effective m / z (including the mass tolerance window) for analysis; and adjust the sensitivity of the ion detector (e.g., by adjusting the detector gain).

[0037] Controller 206 may include any suitable hardware (e.g., processor, circuitry, etc.) and / or software for a particular implementation. Although Figure 4 The display controller 206 is included in the mass spectrometer system 104, but the controller 206 may alternatively be implemented wholly or partially independent of the mass spectrometer system 104, for example, by a computing device communicatively coupled to the mass spectrometer system 104 via a wired connection (e.g., cable) and / or a network (e.g., local area network, wireless network (e.g., Wi-Fi), wide area network, Internet, cellular data network, etc.). In some examples, the controller 206 is implemented wholly or partially by the controller 106.

[0038] exist Figure 4In the example, the mass spectrometer system 104 is spatially tandem (e.g., having multiple mass analyzers) and has two stages for performing tandem mass spectrometry analysis. However, the mass spectrometer system 104 is not limited to this configuration and can have any other suitable configuration. For example, the mass spectrometer system 104 can be time-tandem. Furthermore or alternatively, the mass spectrometer system 104 can be a multi-stage mass spectrometer and can have any suitable number of mass analyzers and stages (e.g., three or more) for performing multi-stage tandem mass spectrometry analysis (e.g., MS / MS / MS). The following is combined with... Figures 6 to 7 A detailed discussion of specific mass spectrometer systems that can be used is provided.

[0039] In typical "real-world" biological experiments, peptide abundance is fairly uniform across samples. For example, most samples contain a variety of "household" proteins that do not vary significantly across different tissue or cellular states. Therefore, the abundance of these proteins does not differ significantly between samples. Thus, when protein samples are tagged with isotopic labels, it is assumed that the relative abundance of the various tagged portions at the time of labeling is substantially uniform (e.g., ...). Figure 2A As shown), the reporter ion ratio of the peptides produced by digesting these proteins will be consistently uniform across most samples. (If the relative abundance of the tagged portions is not uniform at the time of application, the relative abundance of reporter ions from these housekeeping proteins will also be non-uniform at the time of analysis, but it will still be systematic.)

[0040] These assumed uniform reporter ion intensities form the basis for establishing a testable "null hypothesis" for quantitative measurements using isotopic markers. For example, for a set of perfectly resolved TMT reporter ions ( Figure 2A ), and in the area where the label is applied evenly ( Figure 2A Under the assumption that the abundance of each protein or peptide analyte detected by mass spectrometry is uniform across all samples, the null hypothesis presupposes that the abundance of each protein or peptide is uniform across all samples. Under this assumption, it is expected that the intensity of all TMT reporter ions is uniform. Figure 2A This statement ignores the influence of isotopic impurities, but small deviations in the intensity of these reporter ions can be compensated for based on given reagent parameters. Even when TMT reporter ions are unresolved, a stable set of TMT reporter ion intensities can still be expected; although these intensities are not uniform, they still follow a predictable intensity pattern, i.e., the sum of all unresolved peaks. Figure 2CIn the case where, under all conditions, all fine isotopic differences in each group (i.e., corresponding to m / z ≈ 126Th, 127Th, ..., 135 h) collapse into a single corresponding peak, the measured ratio would be 1:3:4:4:4:4:4:4:4:3:1. This specific example assumes that 32 different commercially available isotopic isomers exist in equal proportions in the sample mixture, and that their individual mass spectra are unresolved, thus producing a 1:3:4:4:4:4:4:4:3:1 pattern. Any deviation from this expected pattern (or any other expected pattern, depending at least on the amount of label used) beyond the predetermined acceptable tolerance will overturn the null hypothesis and indicate that the TMT reporting ion intensity of one of the underlying layers has deviated from the assumed 1:1 distribution. Deviations from the expected ratio may be due to the abundance of the target analyte being higher or lower in one or more samples of the prepared mixture than in the others. This increase or decrease in abundance may be due to different treatment methods used for the samples in preparing the mixture, or due to different sample sources. Whether the observed intensity pattern matches the predetermined intensity pattern within a certain acceptable tolerance range can be determined by any suitable data comparison method. As an example, both the observed pattern and the predetermined pattern can be considered as independent vectors in the m / z contrast intensity space (or mass contrast intensity space). Through this calculation, the degree of matching between the two sets of data can be measured based on the difference vector, or based on the angle between the two vectors.

[0041] The above principle shows that by performing real-time analysis of the TMT reporter ion ratio at standard resolution, deviations from the assumed null hypothesis distribution can be identified, thereby instructing the mass spectrometer to trigger a higher resolution quantitative scan to fully resolve the entire set of TMT reporter ions. Figure 5 This is a flowchart of method 600 for implementing this process. In step 602, proteins or peptides from each of multiple samples are labeled with corresponding isotopic markers, and these samples are mixed (preferably in equal proportions) to prepare a sample mixture. In step 604, the mixture is introduced into a liquid chromatography-mass spectrometry (LC-MS) analysis system (e.g., system 100), thereby introducing the mixture into the chromatographic column 110 of chromatograph 102 for separation; wherein each analyte is introduced into a mass spectrometry system (e.g., mass spectrometry system 104, mass spectrometry system 200, or mass spectrometry system 300) as it elutes from the chromatograph at its respective elution time. Each analyte, upon introduction into the mass spectrometer, is subjected to ionization by an ion source (e.g., Figure 6 and Figure 7 The ion source 5 shown is ionized to form an ion stream of the analyte, which is then guided through and processed by the various components of the mass spectrometer system.

[0042] In step 606 of method 600, ions, whether directly introduced or generated by processing (e.g., fragmentation) the introduced ions, are directed to a mass analyzer (e.g., Orbitrap™ mass analyzer 212, linear ion trap mass analyzer 217b, or Astral mass analyzer 500) to detect the m / z value of the initially introduced ions or the resulting fragment ions. This step may optionally include identifying the analyte by recognizing characteristic m / z values ​​recorded from MS2 analysis. In step 608, various reporter ions are fragmented from their associated mass equilibrium chemical structures and released as free ions via HCD fragmentation.

[0043] In step 612, the released reporter ions are detected at standard or low mass spectrometry resolution, and the relative intensity of the reporter ions is compared to a predetermined intensity pattern, wherein the predetermined intensity pattern is consistent with the aforementioned “null hypothesis” that the abundance of the analyte protein or peptide is assumed to be homogeneous across all samples. The predetermined intensity pattern depends on the actual relative abundance of the analyte itself or the precursor molecules that produce the analyte in all samples of the mixture. In some cases, the predetermined intensity pattern may also depend on the relative abundance of the isotopic tag portion applied between the samples during labeling and / or the mixing ratio of the samples. In some cases, the “predetermined” pattern may consist of a “rolling average” of all low-resolution intensity patterns previously measured in the LC-MS experiment. Therefore, the “predetermined” pattern can be created before performing the relevant specific MS2 analysis, but not before the LC-MS experiment itself. If the low-resolution intensity pattern is found to be inconsistent with the predetermined “null hypothesis” intensity pattern in step 612, a high-resolution mass analysis is performed on the reporter ion region (e.g., m / z range 126Th to 135Th) to resolve the discrete signal of each reporter ion portion.

[0044] Figure 6 and Figure 7 This is a more detailed schematic diagram of an exemplary mass spectrometer system, which conforms to Figure 4 The system architecture shown is shown in the figure. Mass spectrometer system 200 ( Figure 6 ) and mass spectrometer system 300 ( Figure 7Both systems are commercially available hybridization systems from Thermo Fisher Scientific in Waltham, Massachusetts, USA, and utilize multiple mass analyzers. Specifically, mass spectrometry system 200 includes an ion trap mass analyzer 216 and an Orbitrap™ analyzer 212, the latter being an electrostatic trap mass analyzer. During operation of mass spectrometry system 200, electrospray ionization source 5 delivers ions from the sample to be analyzed to ion inlet aperture 207, where the ions enter a first intermediate vacuum chamber. Upon entry, the ions are captured and focused into a narrow ion beam by stacked ring ion guides 209 or by ion funnels located at the same position. A first multipolar ion guide 45 delivers the ion beam to downstream chambers of the mass spectrometer, where differential evacuation of each chamber ensures that each subsequent chamber is maintained at a lower pressure than the previous chamber. A second multipolar ion guide 45b is curved to separate neutral molecules from the main ion beam. Specifically, neutral molecules move along a straight path, while the target ions are deflected around a 90-degree angle by the drag field applied to the ion guide 45b, thereby achieving separation.

[0045] The quadrupole mass filter 208 of the mass spectrometer system 200 functions as a tunable mass filter in its conventional sense, allowing only ions within a selected narrow mass-to-charge ratio (m / z) range to pass through. A subsequent third ion guide 45c delivers the filtered ions to a curved quadrupole ion trap (“C-trap”) assembly 210. The C-trap 210 is capable of transporting ions along a path between the quadrupole mass filter 208 and the ion trap mass analyzer 216. The C-trap 210 also has the capability to temporarily collect and store ion clusters, subsequently delivering the ions in the form of pulses or ion packets to the Orbitrap™ mass analyzer 212. The transport of the ion packets is controlled by applying a potential difference between the C-trap 210 and a set of injection electrodes 211 positioned between the C-trap 210 and the Orbitrap™ mass analyzer 212. The curvature of the C-trap is designed to spatially focus the ion clusters to match the angular reception of the Orbitrap™ mass analyzer 212 inlet aperture.

[0046] Multipolar ion guide 214 (also referred to as an "ion routing multipolar bar") and ion guide 45d are used to guide ions between C-type trap 210 and ion trap mass analyzer 216. Multipolar ion guide 214 provides temporary storage for ions and can also act as a fragmentation cell. Various gate electrodes along the path between C-type trap 210 and ion trap mass analyzer 216 are controllable, allowing ions to be transported in either direction (e.g., from the multipolar ion guide toward ion trap mass analyzer 216 or toward C-type trap 210), depending on the sequence of ion processing steps required in any particular analytical method.

[0047] The ion trap mass analyzer 216 is a dual-pressure linear ion trap (i.e., a two-dimensional trap) comprising a high-pressure linear trap unit 217a and a low-pressure linear trap unit 217b; the two units are arranged adjacently and separated by a plate lens with a pinhole that allows ion transport between the two units and forms a evacuation confinement to allow different pressures to be maintained in the two traps. The environment of the high-pressure unit 217a is conducive to ion cooling and also to ion fragmentation under controlled conditions via collision-induced dissociation, electron transfer dissociation, or ion-ion reactions (e.g., proton transfer reactions). The low-pressure unit 217b is a mass analyzer because the lower pressure of unit 217b facilitates analytical scanning with high resolution and mass accuracy. The low-pressure unit includes a dual-polarity ion detector 215. The architecture of the mass spectrometer system 200 enables efficient operation because different ion packs can be processed or analyzed simultaneously in two or more of the following components: quadrupole mass filter 208, C-type trap 210, Orbitrap mass analyzer 212, ion routing multipole 214, high-pressure linear ion trap 217a, and low-pressure linear ion trap 217b.

[0048] Figure 7 This is a detailed schematic diagram of another exemplary mass spectrometer system 300, commercially available by Thermo Fisher Scientific of Waltham, Massachusetts, under the name Orbitrap™ Astral™. Compared to mass spectrometer system 200 ( Figure 6 Similar to the mass spectrometer system 200, the mass spectrometer system 300 includes an ion source 5, an ion inlet aperture 207, and multiple vacuum chambers. It also includes an Orbitrap™ mass analyzer and its associated C-trap 210 and injection optics system 211. Further similar to the mass spectrometer system 200, the mass spectrometer system 300 includes a first multipolar ion director 345a, a bent second multipolar ion director 345b, a quadrupole mass filter 308, a third multipolar ion director 345c, and an ion routing multipolar bar 314. These further components perform functions similar to those of the first multipolar ion director 45a, the second multipolar ion director 45b, the quadrupole mass filter 208, and the third multipolar ion director 45c of the mass spectrometer system 200, respectively. However, the components of system 300 are not necessarily structurally identical to their counterparts in mass spectrometer system 200.

[0049] Mass spectrometer system 300 ( Figure 7 )and Figure 6The mass spectrometer system 300 differs significantly from the system 200 in that the dual-pressure ion trap mass analyzer 216 in the latter system is replaced by an Astral™ time-of-flight mass analyzer 500, which provides a significantly higher resolution than the ion trap mass analyzer. Details of the mass analyzer 500 are described in U.S. Patent No. 9,136,102, the disclosure of which is incorporated herein by reference in its entirety. Details of the Orbitrap™ and Astral™ mass analyzer combination are described in the article “Parallelized acquisition of orbitrap and astralanalyzers enables high-throughput quantitative analysis” by Stewart, Hamish I., Dmitry Grinfeld, Anastassios Giannakopulos, Johannes Petzoldt, Toby Shanley, Matthew Garland, Eduard Denisov, et al. (published in Analytical Chemistry, Vol. 95, No. 42, 2023: 15656-15664), which is incorporated herein by reference in its entirety.

[0050] After the ions pass through the ion routing multipole 314 and the octet ion guide 347, the introduction of the ions into the mass analyzer 500 is performed by an ion processor 332, which includes a high-pressure chamber 332a and a low-pressure chamber 332b. The high-pressure chamber 332a contains a multipole ion trap within which the ions can be fragmented for MS / MS analysis. The low-pressure chamber 332b includes a set of repulsion and extraction electrodes 333 that inject ion packets into the ion inlet 507 of the mass analyzer 500 in a direction substantially orthogonal to the overall trajectory 337 of the ions as they pass through the upstream system components of the mass analyzer 500. An ion lens 335 focuses and shapes the ion beam before it enters the mass analyzer 500.

[0051] The mass analyzer 500 includes a first set of ion mirrors 501a and a second set of ion mirrors 501b, the second set of ion mirrors being non-parallel to the first set of ion mirrors 501a. After injection into the mass analyzer 500, ions oscillate repeatedly between ion mirrors 501a and 501b; simultaneously, ions drift slowly parallel to the convergence direction of the ion mirrors, this drift being caused by the initial tilt angle of the ion trajectory. The convergence of the ion mirrors causes the drift to gradually decelerate during the first 12-13 oscillations between the ion mirrors. The first set of ion foil electrodes 505a is arranged on one side of the ion oscillation plane, and the second set of ion foil electrodes 505b is arranged on the opposite side of the oscillation plane. The shapes of the ion foil electrodes 505a and 505b, when an appropriate potential is applied, are used to maintain the correct ion trajectory between ion mirrors 501a and 501b.

[0052] Ultimately, due to the tilt of the ion mirror 501b and the refraction on the ion foil, the ion drift reverses, causing the ions to drift in the opposite direction of spatial divergence from the ion mirror. During this reverse drift, the ions undergo 12-13 oscillations between the ion mirrors, resulting in a total path length exceeding 30 meters. This long drift length enables high-resolution spatial separation of ions based on their individual m / z values. The high dynamic range detector 503 detects these spatially separated ion packets. (This is related to the mass spectrometer system 200...) Figure 6 Similarly, the architecture of the mass spectrometer system 300 enables efficient operation because different ion packs can be processed or analyzed simultaneously in two or more of the following components: quadrupole mass filter 208, C-trap 210, Orbitrap mass analyzer 212, ion routing multipole 214, high-pressure chamber 332a of ion processor 332, and Astral™ mass analyzer 500.

[0053] Back to Figure 5 Method 600 and returning to the mass spectrometer system 200 ( Figure 6 ) and 300 ( Figure 7As described above, providing more than one mass analyzer in a single mass spectrometer system facilitates task parallelization, where the first mass analyzer is used for low-resolution MS1, MS2, and semi-quantitative reporter ion "survey" data acquisition, and the second mass analyzer is used for quantitatively accurate high-resolution reporter ion measurements. The actual allocation of these tasks to specific mass analyzers is quite flexible. For example, low-resolution scans can be acquired using a short transient Orbitrap™ mass analyzer, an Astral™ mass acquisition, or even an ion trap scan using the ion trap mass analyzer 217b. While high-resolution scans are likely to be longer Orbitrap™ acquisitions, they could also be multi-pass Astral™ spectra. Furthermore, although this approach was designed to limit the impact of the new 32-fold TMT reagent, the same approach can be used with currently available 18-fold TMT reagents.

[0054] Although the discussion of this invention has focused on isotopic labeling of samples using tandem mass spectrometry (TMT) tags, the mass spectrometry methods described herein are also applicable to isotopic labeling techniques known as "iTRAQ for relative and absolute quantification," which are based on the covalent labeling of N-terminal and side-chain amines from peptides derived from protein digestion with tags of varying masses. Similarly, the methods described herein are further applicable to experimental procedures known as "Stable Isotopic Labeling of Amino Acids in Cell Culture" (SILAC), in which independent cell populations are cultured using media containing isotopically labeled amino acids under different laboratory conditions or treatments. Each cell population is cultured using amino acids with a corresponding isotopic label that is different from all isotopic labels used in the culture of other cell populations. Analytes from all samples are then pooled to form a mixture. These analytes may contain native proteins from the cell populations, or alternatively, a series of peptides derived from protein digestion. Mass analysis of the analytes in the mixture is performed using various mass spectrometry techniques, including the detection of the distribution of isotopic labels in the analytes. Since different markers correspond to different cell growth conditions or treatments, the effects of these conditions or treatments can be quantified.

[0055] Refer again Figure 3 The controller 106 is communicatively coupled to the LC-MS system 100 (e.g., liquid chromatograph 102 and mass spectrometer system 104) and configured to control the operation of the system. The controller 106 may include any suitable hardware (e.g., processor, circuitry, etc.) and / or software configured to control the operation of various components of the LC-MS system 100 (e.g., liquid chromatograph 102 or mass spectrometer system 104) and / or interface with said various components.

[0056] Controller 106 and / or controller 206 may also include and / or provide a user interface configured to enable interaction between a user and LC-MS system 100 or mass spectrometer system 104. Users can interact with controller 106 and / or controller 206 via the user interface using tactile, visual, auditory, and / or other sensory types. For example, the user interface may include a display device (e.g., a liquid crystal display (LCD) screen, a touchscreen, etc.) for displaying information (e.g., mass spectra, notifications, etc.) to the user. The user interface may also include an input device (e.g., a keyboard, mouse, touchscreen device, etc.) that allows the user to provide input to controller 106 and / or controller 206. In other examples, the display device and / or input device may be separate from controller 106 and / or controller 206 but communicatively coupled to it. For example, the display device and input device may be contained in a computer (e.g., a desktop computer, a laptop computer, etc.) communicatively connected to controller 106 and / or controller 206 via a wired connection (e.g., via one or more cables) and / or a wireless connection.

[0057] The controller 106 acquires data collected over time by the LC-MS system 100. The data may include a series of mass spectra comprising ionic intensity values ​​generated by the analytes in sample 108 as a function of ion m / z. This series of mass spectra can be represented in a three-dimensional plot where elution time (e.g., retention time) is plotted along the X-axis, m / z along the Y-axis, and intensity along the Z-axis. Spectral features on the plot (e.g., Z-axis intensity peaks) represent the detection of ions generated by the various analytes contained in sample 108 by the LC-MS system 100. The X and Z axes of the plot can be used to generate elution curves (e.g., mass chromatograms) that plot the detection intensity at a selected m / z as a function of time.

[0058] As used herein, “selected m / z” refers to a specific m / z (with or without a quality tolerance window, e.g., + / - 0.5Th) or a narrow range of m / z (e.g., a separation window with a width or range such as 20Th, 10Th, 4Th, 3Th, etc.). In MS2 or MSn analyses (e.g., data-dependent MS2 analyses, selected reaction monitoring (SRM) analyses, multiple reaction monitoring (MRM) analyses, or parallel reaction monitoring (PRM) analyses), the selected m / z corresponds to the m / z of the precursor ion, which is separated and subsequently fragmented to generate a reporter ion for relative quantification.

[0059] Figure 8A functional block diagram of an exemplary MS control system 400 (“System 400”) is shown. System 400 may be implemented wholly or partially by an MS system, such as LC-MS system 100 (e.g., implemented by controller 106 and / or controller 206). Alternatively, System 400 may be implemented independently of an MS system (e.g., a remote computing system or server separate from but communicatively coupled to controller 106 and / or controller 206 of LC-MS system 100). System 400 may include (but is not limited to) a memory 402 and a processor 404 that are selectively and communicatively coupled to each other. Memory 402 and processor 404 may each include, or be implemented by, hardware components and / or software components (e.g., processor, memory, communication interface, instructions stored in memory for execution by the processor, etc.). Memory 402 and processor 404 may be distributed among multiple devices and / or multiple locations to meet the needs of a particular implementation.

[0060] Memory 402 may maintain (e.g., store) executable data used by processor 404 to perform any of the operations described herein. For example, memory 402 may store instructions 406 that can be executed by processor 404 to perform any of the operations described herein. Instructions 406 may be implemented by any suitable application, software, code, and / or other instance of executable data. Memory 402 may also maintain any data acquired, received, generated, managed, used, and / or transmitted by processor 404. For example, memory 402 may maintain LC-MS data.

[0061] Processor 404 is configured to perform (e.g., by executing instructions 406 stored in memory 402) the various processing operations described herein. It should be understood that the operations and examples described herein are merely exemplary illustrations of the many different types of operations that processor 404 can perform. Any reference to operations performed by system 400 in this specification is to be understood as an operation performed by processor 404 of system 400. Furthermore, any operation performed by system 400 in this specification is to be understood as including system 400 instructing, commanding, or directing another system or device to perform said operation.

[0062] As used herein, “acquisition” refers to a mass analysis performed at discrete time points to obtain a single mass spectrum, wherein selected m / z values ​​are separated and fragmented to generate an MS2 or MSn type acquisition. It should be recognized that in some embodiments, the acquisition is based on data-dependent analysis, while in other embodiments, the acquisition may be a targeted MS2 analysis or a data-independent analysis. In other embodiments, multiple rounds of fragmentation and m / z selection may occur, sometimes involving the simultaneous selection of multiple m / z values, all in order to perform a higher-order MSn acquisition.

[0063] Various embodiments have been described in the foregoing description. Specific configurations and details have been set forth for illustrative purposes to provide a thorough understanding of the embodiments. However, those skilled in the art will also clearly recognize that the embodiments can be practiced without these specific details. Furthermore, well-known features may have been omitted or simplified so as not to obscure the described embodiments.

[0064] Some embodiments of this disclosure include a system comprising one or more data processors and / or logic circuitry. In some embodiments, the system includes a non-transitory computer-readable storage medium containing instructions that, when executed on the one or more data processors, cause the one or more data processors to perform part or all of the methods disclosed herein and / or part or all of one or more processes and workflows. Some embodiments of this disclosure include a computer program product tangibly embodied in a non-transitory machine-readable storage medium, the computer program product containing instructions configured to cause one or more data processors to perform part or all of the methods disclosed herein and / or part or all of one or more processes.

[0065] The terminology and expressions used are descriptive rather than restrictive, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof. Rather, it should be understood that various modifications are possible within the scope of the claims. Therefore, it should be understood that although this disclosure includes specific embodiments and optional features, modifications and alterations to the concepts disclosed herein can be made by those skilled in the art, and such modifications and alterations are considered to be within the scope of the appended claims.

Claims

1. A method for mass analysis of analytes in a sample mixture, wherein, All analytes in each respective sample generating the mixture contain the same isotopic label, and each respective sample is associated with a distinct and unique isotopic label, the method comprising: Within the mass spectrometer system, a set of reporter ion substances are cleaved from the analyte ions of the mixture, wherein the reporter ion substances contain all isotopic labels from various labeled samples; Mass analysis of the reporter ion was performed at a first mass-to-charge ratio (m / z) resolution; The observed intensity variation patterns of reported ionic substances with mass analysis were compared with the predetermined intensity variation patterns with mass. If the observed intensity pattern does not match the predetermined intensity pattern within the tolerance range, then the mass analysis of the group of reporter ions is performed at a second mass-to-charge ratio (m / z) resolution greater than the first mass-to-charge ratio (m / z) resolution.

2. The method according to claim 1, characterized in that, The distribution of the analytes among the samples is determined by mass analysis of the group of reporter ions at the second mass-to-charge ratio (m / z) resolution.

3. The method according to claim 1 or claim 2, characterized in that, The time required to perform mass analysis of the group of reporter ions at the second mass-to-charge ratio (m / z) resolution is longer than the time required to perform mass analysis of the group of reporter ions at the first mass-to-charge ratio (m / z) resolution.

4. The method according to claim 1 or claim 2, characterized in that, The mass spectrometer system includes a first mass analyzer and a second mass analyzer, wherein the first mass analyzer performs mass analysis on the group of reporter ions at a first mass-to-charge ratio (m / z) resolution, and the second mass analyzer performs mass analysis on the group of reporter ions at a second mass-to-charge ratio (m / z) resolution.

5. The method according to claim 1 or claim 2, characterized in that, At least some of the reported ionic substances have a mass-to-charge ratio difference of less than 0.01Th.

6. The method according to claim 1 or claim 2, characterized in that, Also includes: Tandem mass analysis was performed on each analyte introduced into the mass spectrometer; as well as The structure and / or composition of the analyte are identified based on the tandem mass analysis.

7. The method according to claim 6, characterized in that, Each isotope label includes its own reporter ion portion and its own mass normalization region, such that the sum of the masses of the reporter ion portion and the mass normalization region is substantially the same for all the isotope labels.

8. The method according to claim 1 or claim 2, characterized in that, The samples were derived from cell cultures grown under different growth conditions or different treatments.

9. The method according to claim 1 or claim 2, characterized in that, The predetermined intensity relative to the mass pattern is determined as a rolling average of previously measured reported ion intensity patterns obtained using the first mass-to-charge ratio resolution.

10. A liquid chromatography-mass spectrometry system, comprising: A liquid chromatograph configured to receive a mixture of samples, wherein the analytes of each individual sample that produces the mixture contain the same isotopic label, and wherein each individual sample is associated with a different unique isotopic label. A mass spectrometer system including at least one mass spectrometer; and A controller electrically coupled to the liquid chromatograph and the mass spectrometer, the controller including non-transient computer-readable instructions for: The liquid chromatograph fractionates the mixture and provides the mass spectrometer system with the mixture fraction containing the analyte of the sample, thereby generating ions of the provided analyte by the mass spectrometer system; The mass spectrometer system is used to cleave multiple reporter ions from the ions of the provided analyte, wherein the reporter ions include all isotopic labels from each differently labeled sample; The mass spectrometer system is used to perform mass analysis on the reporter ion at a first mass-to-charge ratio (m / z) resolution; The observed intensity variation patterns of reported ionic substances with mass analysis were compared with the predetermined intensity variation patterns with mass. If the observed intensity mode does not match the predetermined intensity mode within the tolerance range, the mass spectrometer system performs mass analysis on the group of reporter ions at a second mass-to-charge ratio (m / z) resolution greater than the first mass-to-charge ratio (m / z) resolution.

11. The liquid chromatography-mass spectrometry system according to claim 10, characterized in that, The non-transient computer-readable instructions can also be used for: The distribution of the analytes among the samples is determined by mass analysis of the grouped reporter ions at the second mass-to-charge ratio (m / z) resolution.

12. The liquid chromatography-mass spectrometry system according to claim 10 or claim 11, characterized in that, The time required to perform mass analysis of the group of reporter ions at the second mass-to-charge ratio (m / z) resolution is longer than the time required to perform mass analysis of the group of reporter ions at the first mass-to-charge ratio (m / z) resolution.

13. The liquid chromatography-mass spectrometry system according to claim 10 or claim 11, characterized in that, The mass spectrometer system includes: First quality analyzer; and Second quality analyzer, The first mass analyzer performs mass analysis on the grouped reporter ion material at the first mass-to-charge ratio (m / z) resolution, and the second mass analyzer performs mass analysis on the grouped reporter ion material at the second mass-to-charge ratio (m / z) resolution.

14. The liquid chromatography-mass spectrometry system according to claim 10 or claim 11, characterized in that, At least some of the reported ions have a mass-to-charge ratio difference of less than 0.01Th.

15. The liquid chromatography-mass spectrometry system according to claim 10 or claim 11, characterized in that, The non-transient computer-readable instructions can also be used for: The mass spectrometer system performs tandem mass analysis on the analytes in the sample; and The structure and / or composition of the analyte are identified based on the tandem mass analysis.

16. The liquid chromatography-mass spectrometry system according to claim 10 or claim 11, characterized in that, Each isotope label includes its own reporter ion portion and its own mass normalization region, such that the sum of the masses of the reporter ion portion and the mass normalization region is substantially the same for all the isotope labels.

17. The liquid chromatography-mass spectrometry system according to claim 10 or claim 11, characterized in that, The samples were derived from cell cultures grown under their respective growth conditions or treatments.

18. The liquid chromatography-mass spectrometry system according to claim 10 or claim 11, characterized in that, The predetermined intensity relative to the mass pattern is determined as a rolling average of previously measured reported ion intensity patterns obtained using the first mass-to-charge ratio resolution.

19. A computer program product tangibly embodied in a computer-readable medium, the computer program product comprising computer-readable instructions that can be used to: To enable liquid chromatography to fractionate sample mixtures, wherein... Each sample that produces the mixture contains the same isotopic label, and each sample is associated with a different unique isotopic label. The liquid chromatograph provides a mixture fraction containing the analytes of the sample mixture to the mass spectrometer system, thereby generating ions of the provided analytes by the mass spectrometer system; The mass spectrometer system is used to lyse the ions of the provided analyte into a group of reporter ions, wherein the reporter ions include all isotopic labels from each differently labeled sample; The mass spectrometer system is used to perform mass analysis on multiple reporter ion substances at a first mass-to-charge ratio (m / z) resolution; The observed intensity variation patterns of reported ionic substances with mass analysis were compared with the predetermined intensity variation patterns with mass. If the observed intensity mode does not match the predetermined intensity mode within the tolerance range, the mass spectrometer system performs mass analysis on the multiple reporter ions at a second mass-to-charge ratio (m / z) resolution greater than the first mass-to-charge ratio (m / z) resolution.

20. The computer program product according to claim 19, characterized in that, The computer program product can also be used to determine the distribution of the analytes among the samples based on mass analysis of the group of reporter ions at the second mass-to-charge ratio (m / z) resolution.

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