Mass spectrometry method

Abstract

A mass spectrometry method for analyzing sample ions of a sample across a mass-to-charge ratio (m / z) range of interest is provided. The method includes providing the sample to be analyzed with a target list of mass-to-charge ratios of fragments of interest. The method includes ionizing the sample to form sample ions when the sample is eluted from the chromatography system. The method also includes performing a plurality of MS2 analyses on the sample ion across the m / z range of interest. Performing each MS2 analysis includes mass selecting the sample ions using an isolation window having a first m / z width, and fragmenting the sample ions within the isolation window to form fragmented ions. For each MS2 analysis of the plurality of MS2 analyses, a center m / z of the isolation window is updated such that the plurality of MS2 analyses cover the m / z range of interest. Performing each MS2 analysis also includes mass analyzing the fragment ion and determining an m / z associated with each spectral peak of the fragment ion, and comparing the m / z of the spectral peak to the m / z of the fragment of interest of the target list. Upon detecting a match between m / z of a set of one or more spectral peaks and m / z of a set of one or more fragments of interest of the target list, the method further includes performing a triggered MS2 analysis of the sample ion, where the triggered MS2 analysis has a higher sensitivity than a sensitivity of each MS2 analysis of the plurality of MS2 analyses.

Description

Technical Field

[0001] The present disclosure relates to mass spectrometry. In particular, the present disclosure relates to a mass spectrometry method involving fragmentation of ions. Background Art

[0002] Liquid chromatography mass spectrometry (LCMS) analysis of biomolecules such as proteins can be performed using one of several analytical techniques.

[0003] Generally speaking, LCMS analysis can be performed in one of several areas. In MS1 analysis, the sample ions (or precursor ions) are mass analyzed without intentionally fragmenting the ions. In MS2 analysis, the precursor ions (or a selection of precursor ions) are first fragmented to form fragment ions, and the fragment ions are mass analyzed. MS1 and MS2 analysis can be performed separately or in combination as the sample elutes from the chromatographic system to obtain information about the sample being analyzed.

[0004] Generally, there are several known methods to sequence MS1 and MS2 analysis for analysis of samples eluting from a chromatography system.

[0005] As explained in "Technical advances in proteomics: new developments in Data-independent acquisition" Alex Hu, William S. Noble, Alejandro Wolf-Yadlin, F1000 Research 2016, 5(F1000Faculty Rev): 419(https: / / doi.org / 10.12688 / f1000research.7042.1), data-dependent acquisition (DDA) methods utilize information from MS1 analysis (i.e., analysis of unfragmented precursor ions present in the sample) to trigger immediate MS2 analysis (analysis of fragmented precursor ions) using a relatively narrow isolation window (e.g., 1 Da, 2 Da, 3 Da, etc.). Analysis is typically triggered based on the intensity of the mass spectral peak identified in the MS1 scan (i.e., the strongest mass spectral peak is selected for analysis). Therefore, the MS2 analysis performed is "dependent" on the precursor ions identified during the MS1 analysis.

[0006] Data independent acquisition (DIA) methods typically fragment all precursor ions present in a relatively wide isolation window (e.g., 10 Da, 15 Da, 20 Da, etc.) sequentially, wherein the resulting MS2 fragmentation products are analyzed. The MS2 scan is repeated to cover a predefined mass-to-charge (m / z) range, e.g., 400 m / z to 1200 m / z. Thus, in the DIA method, an MS2 analysis is performed independently of the identified precursor ions. For the DIA method, the relatively wide m / z width of the isolation window (compared to the DDA method) makes it more likely that multiple precursor ions will be included in one or more MS2 analyses in the MS2 analysis, making the analysis of the MS2 analysis more complex.

[0007] Targeted mass spectrometry methods typically require a predefined inclusion m / z list for the markers of interest. These methods typically perform MS1 analysis across the m / z range of interest to detect the m / z of precursor ions on the predefined inclusion list. These methods then perform targeted MS2 analysis using narrow isolation windows (e.g., 0.5 Da, 0.7 Da, 1 Da, 2 Da, etc.) when the MS1 analysis scan detects a precursor ion present on the predefined inclusion list.

[0008] GB-A-2590601 discloses a mass spectrometry method in which isotopologues of a target precursor are added to a sample. The sample is eluted from a chromatographic system and ionized. The method then comprises performing mass analysis of the sample and the isotopologues using a data independent acquisition (DIA) method of a mass spectrometer, comprising performing mass analysis scans in the MS1 and MS2 domains. After identifying that the isotopologues are eluted from the chromatographic system, the method further comprises performing a target scan having a target isolation window comprising a mass-to-charge ratio representing the target precursor within the duration of the chromatographic peak of the isotopologue for at least one of identification and quantification of the target analyte, wherein the target scan is configured to provide additional quantitative data of the target analyte. Summary of the Invention

[0009] According to a first aspect of the present disclosure, a mass spectrometry method for analyzing sample ions of a sample across a mass-to-charge ratio (m / z) range of interest is provided. The method includes providing a target list of mass-to-charge ratios of fragments of interest to the sample to be analyzed. The method includes ionizing the sample to form sample ions when the sample is eluted from a chromatographic system. The method also includes performing multiple MS2 analyses on the sample ions across the m / z range of interest. Performing each MS2 analysis includes mass selecting the sample ions using an isolation window having a first m / z width, and fragmenting the sample ions within the isolation window to form fragment ions. For each MS2 analysis in the multiple MS2 analyses, the center m / z of the isolation window is updated so that the multiple MS2 analyses cover the m / z range of interest. Performing each MS2 analysis also includes mass analyzing the fragment ions and determining the m / z associated with each spectral peak of the fragment ions, and comparing the m / z of the spectral peak with the m / z of the fragments of interest in the target list. After detecting a match between the m / z of the set of one or more spectral peaks and the m / z of the set of one or more fragments of interest in the target list, the method also includes performing a triggered MS2 analysis of the sample ions, wherein the triggered MS2 analysis has a higher sensitivity than the sensitivity of each of the multiple MS2 analyses.

[0010] The method of the first aspect is to carry out mass analysis to sample across the m / z range of interest in the MS2 domain. Therefore, the method of the first aspect adopts the DIA method to collect the m / z of the fragmentation data in the MS2 domain. The inventor has recognized that, due to the relatively large isolation window (compared with, for example, the DDA method) for mass selection of sample ions before fragmentation, the known DIA method has a relatively low sensitivity. Although the width of the isolation window is narrowed to improve the sensitivity of a single MS2 analysis, this then increases the quantity of the MS2 analysis required for covering all m / z ranges of interest. Therefore, narrowing the isolation window towards the isolation window width (for example, about 2Da) for DDA will increase the duty cycle for analyzing the mass range of interest, so that it may become challenging to carry out DIA analysis on a time scale fast enough for chromatogram. For example, it is impossible to carry out multiple DIA cycles to each chromatographic peak. This then reduces quantitative accuracy.

[0011] The inventors have further recognized that a further consequence of the relatively wide isolation windows used in conventional DIA analysis is that detection of relatively low-abundance sample ions can be challenging if they are co-isolated with more abundant ions for MS2 analysis. That is, the dynamic range of each MS2 analysis in conventional DIA analysis can be limited in some cases.

[0012] The method of the first aspect provides a mass spectrometry method in which one or more additional triggered MS2 analyses are performed. By performing the triggered MS2 analysis with higher sensitivity than the MS2 analysis covering the m / z range of interest, the triggered MS2 analysis can be used to target the sample ions of interest (to the exclusion of other sample ions), thereby improving the dynamic range of the triggered MS2 scan.

[0013] The MS2 analysis of the triggering of the first aspect is carried out based on the comparison of the spectrum peak of the fragment ion with the target list of the fragment interested. Therefore, the MS2 analysis of the triggering is carried out in a limited manner during the method for the first aspect. Therefore, the duty cycle of the mass spectrometry method will not be significantly increased by adding the MS2 analysis of the triggering to the multiple MS2 scans carried out. Therefore, the method for the first aspect allows the use of the DIA method with improved dynamic range and without reducing quantitative accuracy to carry out mass analysis of the sample ion interested.

[0014] The method of the first aspect is based on the real-time data obtained from multiple MS2 analyses to perform triggered MS2 analysis. The triggered MS2 analysis is part of the MS2 workflow, so that each triggered MS2 analysis is performed in real time. Therefore, the triggered MS2 analysis data can be collected in a time period similar to the MS2 analysis that prompted the triggered MS2 analysis. In particular, the triggered MS2 analysis is performed independently of any MS1 analysis because, relative to the duration of the MS2 analysis, the MS1 analysis typically has a relatively long duration.

[0015] In some embodiments, the triggered MS2 analysis can use an isolation window having a second m / z width, wherein the second m / z width is narrower than the first m / z width, such that the triggered MS2 scan has a higher sensitivity than each of the multiple MS2 analyses. Thus, the triggered MS2 analysis can provide an MS2 analysis that reduces or eliminates co-separation of less abundant ions with more abundant ions.

[0016] In some embodiments, each MS2 analysis performed in multiple MS2 analyses can include accumulating sample ions in an isolation window for a first injection time. In some embodiments, performing a triggered MS2 analysis can include accumulating sample ions in an isolation window for a second injection time, wherein the second injection time is greater than the first injection time width, so that the triggered MS2 scan has a higher sensitivity than each MS2 analysis in multiple MS2 analyses. According to the present disclosure, reference to injection time can be understood as the ion accumulation performed in a specified time period (i.e., injection time). In some embodiments, ions can be accumulated until a predetermined amount of ions is accumulated, or a predetermined amount of charge (i.e., a plurality of ions with one or more different charge states) is accumulated, or a maximum injection time is reached. In some embodiments, an isolation window with a second m / z width can be selected so that the second injection time for the triggered MS2 analysis is greater than the first injection time for multiple MS2 analyses.

[0017] In some embodiments, the injection time (e.g., a first injection time and / or a second injection time) can be determined based on the predicted ion intensity for the ongoing analysis. For example, in some embodiments, the method can include using a mass analyzer to perform a pre-analysis (e.g., an automatic gain control (AGC) process) to estimate the intensity of sample ions across the m / z range of interest. The pre-analysis can then be used to predict the ion intensity for an ongoing MS2 analysis or a triggered MS2 analysis.

[0018] In some embodiments, for each MS2 analysis in a plurality of MS2 analyses, or for a triggered MS2 analysis, ions can be accumulated until the maximum injection time is reached. When the intensity of the sample ions in the isolation window of the corresponding MS2 analysis or the triggered MS2 analysis is relatively low, such a situation may occur. In such a case, the maximum injection time for one or more triggered MS2 analyses can be greater than the maximum injection time for each MS2 analysis in a plurality of MS2 analyses. That is to say, a first maximum injection time can be provided for each MS2 analysis in a plurality of MS2 analyses, which is lower than the second maximum injection time for the MS2 analysis of one or more triggers. For example, in some embodiments, the first maximum injection time can be approximately 3.5ms, and the second maximum injection time can be approximately 7ms or 10ms.

[0019] In some embodiments, for each of the multiple MS2 analyses, and / or for the triggered MS2 analysis, the fragment ions can be analyzed at a resolution of at least 50,000, preferably at least 60,000, 70,000, or 80,000. For example, a TOF mass analyzer, particularly an MRTOF mass analyzer, can be used to analyze the ions. Such a mass analyzer may be particularly advantageous for detecting fragment ions associated with endogenous analytes (e.g., endogenous peptides) in unlabeled samples (i.e., in the absence of an internal standard).

[0020] In some embodiments, the second injection time can be estimated based on the intensity of the spectral peak that matches the m / z of the fragment of interest on the target list. For example, the intensity associated with the fragment of interest can indicate the concentration of the analyte of interest present in the sample, as well as the rate at which the analyte ions of interest can accumulate (based on the known injection time used for the corresponding MS2 analysis). Therefore, the second injection time can be selected to ensure that a predetermined number of analyte ions of interest are present in the accumulated ions used for the triggered MS2 analysis.

[0021] In some embodiments, performing a triggered MS2 analysis includes: mass selecting the sample ions using an isolation window having a second m / z width; fragmenting the sample ions within the isolation window to form triggered fragment ions; and performing mass analysis on the triggered fragment ions, wherein the mass-to-charge ratio associated with each spectral peak of the triggered fragment ions is identified. Therefore, a triggered MS2 analysis can be performed in a manner similar to multiple MS2 analyses. For example, the same mass analyzer used to perform multiple MS2 analyses can be used to perform a triggered MS2 analysis. In some embodiments, a triggered MS2 analysis can be performed at any point while performing multiple MS2 analyses. For example, a mass spectrometry workflow can be arranged to perform multiple MS2 analyses sequentially (or in any other order) to cover the m / z range of interest. In some embodiments, a triggered MS2 analysis can be performed at any time during the workflow. For example, a triggered MS2 analysis can be appended to the end of the workflow, or can be arranged to be performed immediately. That is, after determining that a triggered MS2 analysis is to be performed, the workflow can be updated (or interrupted) so that the next analysis performed is a triggered MS2 analysis. By updating the workflow to schedule a triggered MS2 analysis, a triggered MS2 scan can be performed at a similar time as the MS2 analysis that prompted the triggered MS2 scan.

[0022] In some embodiments, when the m / z of the spectrum peak is within ± 20 parts per million (ppm) or ± 10ppm of the m / z of the fragment of interest, the m / z of the spectrum peak can be determined to match the m / z of the fragment of interest of the target list. Therefore, in some embodiments, when the m / z of the spectrum peak is within a predetermined range of the fragment of interest of the target list, matching can be determined. In some embodiments, the accuracy standard can be the percentage of the m / z of the fragment of interest of the target list. In other embodiments, the accuracy standard can be a fixed range, such as ± 0.1Da. Therefore, the method of the first aspect can be carried out with a specified accuracy.

[0023] In some embodiments, detecting a match between the m / z of the set of one or more spectral peaks and the m / z of the set of one or more fragments of interest of the target list comprises identifying that the analyte (ion) of interest is present in the sample ions. Thus, a target list can be provided to identify one or more predetermined analytes of interest in a sample.

[0024] In some embodiments, after identifying the analyte ion of interest as present in the sample ions, the method further comprises performing a plurality of triggered MS2 analyses, wherein each triggered MS2 analysis has an isolation window selected to mass select for a different charge state of the analyte of interest. Thus, the method of the first aspect can be used to identify and / or quantify the analyte of interest based on the plurality of MS2 measurements.

[0025] In some embodiments, after identifying that the analyte ion of interest is present in the sample ion, the method also includes performing a triggered MS2 analysis, the triggered MS2 analysis including utilizing a first isolation window with a second m / z width to mass select the sample ion, the first isolation window being centered on the m / z of the first charge state of the analyte ion of interest to produce a first mass-selected sample ion. A second isolation window with a second m / z width can also be utilized to mass select the sample ion, the second isolation window being centered on the m / z of the second charge state of the analyte ion of interest to produce a second mass-selected sample ion, wherein the first mass-selected sample ion and the second mass-selected sample ion are accumulated together. In some embodiments, the first isolation window and the second isolation window can be utilized simultaneously to mass select the sample ion (i.e., multiplex triggered MS2 scans). The accumulated first mass-selected sample ions and the second mass-selected sample ions can be fragmented to form triggered fragment ions. The triggered fragment ions can be mass analyzed, wherein the mass-to-charge ratio associated with each spectral peak of the triggered fragment ions is identified. Therefore, the triggered MS2 scan can analyze fragment ions of different charge states derived from the analyte of interest. By multiplexing triggered MS2 analysis, the mass spectrometry method of the first aspect can be performed in a more time efficient manner.

[0026] In some embodiments, the sample ions to be analyzed include multiple isotope-labeled versions of the analyte ion of interest. According to the present disclosure, the intentional isotope "labeled" version of the analyte of interest and the "unlabeled" version of the analyte of interest (i.e., each atom of the analyte of interest has an expected number of neutrons) are considered to be different isotope-labeled versions of the analyte of interest. In some embodiments, the target list of the m / z of the fragments of interest includes the m / z of the isotope-labeled fragments of interest based on multiple isotope-labeled versions of the analyte ion of interest. In some embodiments, one or more triggered MS2 analyses can be performed after a match between the m / z of a set of one or more spectral peaks and the m / z of a set of one or more fragments of interest associated with one of the multiple isotope-labeled versions of the analyte ion of interest in the target list is detected. Therefore, when one or more versions of the analyte ion of interest are detected, the triggered MS2 analysis can be triggered. Therefore, the triggered MS2 analysis can be performed when, for example, an endogenous analyte of interest is expected to be present in the sample ion (based on the presence of the isotope-labeled version of the analyte ion of interest detected). Therefore, the triggered MS2 analysis can be performed at the appropriate time. Isotopic labeling can be particularly advantageous when the analyte ions of interest have relatively low abundance or are otherwise difficult to detect in the sample ions.

[0027] In some embodiments, multiple triggered MS2 analyses can be performed, one for each of multiple isotopically labeled versions of the analyte ion of interest. Thus, triggered MS2 analyses can be performed in an attempt to increase the number of analyte ions of interest analyzed. In some embodiments, the method can include performing multiple repeated triggered MS2 analyses, wherein the first isolation window and the second isolation window are used to mass select for different charge states of the analyte of interest.

[0028] It should be understood that isotope labeling can be implemented in various ways. In some embodiments, different isotope-labeled versions of the analyte ion of interest can include a light isotope-labeled version of the analyte ion of interest and a heavy isotope-labeled version of the analyte ion of interest. In some embodiments, multiple isotope-labeled versions of the analyte ion of interest can be prepared by stable isotope labeling (SILAC) with amino acids in cell culture. In some embodiments, multiple isotope-labeled versions of the analyte ion of interest can be prepared by stable isotope dimethyl labeling. In some embodiments, multiple isotope-labeled versions of the analyte ion of interest can be prepared by stable isotope labeling (SIL).

[0029] In some embodiments, after identifying a match between the m / z of a spectral peak and the m / z of an isotopically labeled fragment of interest from a target list, the method may further include performing a triggered MS2 analysis, wherein the triggered MS2 analysis includes mass selecting the sample ions using a first isolation window having a second m / z width, the first isolation window centered on the m / z of the isotopically labeled version of the analyte ion of interest, to produce a first mass-selected sample ion. The triggered MS2 analysis may also include mass selecting the sample ions using a second isolation window having a second m / z width, the second isolation window centered on the m / z of a different isotopically labeled version of the analyte ion of interest, to produce a second mass-selected sample ion. In some embodiments, the first mass-selected sample ions and the second mass-selected sample ions may be accumulated together. In some embodiments, the accumulated first mass-selected sample ions and the second mass-selected sample ions may be fragmented to form triggered fragment ions. The triggered fragment ions may be mass analyzed, wherein the mass-to-charge ratio associated with each spectral peak of the triggered fragment ions is identified. Thus, in some embodiments, different isotopically labeled versions of the analyte ion of interest may be analyzed separately to improve dynamic range and / or sensitivity. In some embodiments, differently isotopically labeled versions of an analyte ion of interest can be analyzed in parallel to improve the signal-to-noise ratio and improve the efficiency of the workflow.

[0030] In some embodiments, sample to be analyzed can not be isotopically labeled (i.e., internal standard molecule can not be provided in combination with sample molecule).In such embodiments, target list can include the list of the m / z of the fragment ion of sample molecule, particularly the list of the m / z of the fragment ion of endogenous analyte (i.e., the spectral library of endogenous analyte fragment).Therefore, each MS2 analysis includes fragment ion being mass analyzed and determining the m / z associated with each spectral peak of fragment ion, wherein the list of the m / z of the fragment ion of the endogenous analyte of the m / z of spectral peak and target list is compared.Then the MS2 analysis of the triggering of sample ion can be carried out based on the matching between the list of the m / z of the fragment ion of the m / z of the set of one or more spectral peaks and the endogenous analyte.Therefore, the MS2 analysis of the higher sensitivity triggering of the first aspect can be advantageously carried out, for not using the experiment of internal table standard, to improve the dynamic range of experiment, particularly with respect to the endogenous analyte (such as endogenous peptide) of relatively low intensity.In some embodiments, time-of-flight mass analyzer can be used, preferably multiple reflection time-of-flight (MRTOF) mass analyzer is used to carry out such experiment. The TOF mass analyzer (particularly MRTOF mass analyzer) allows to carry out MS2 analysis and triggered MS2 analysis at a relatively high speed, for example, with a frequency of at least 150Hz, 200Hz or 250Hz. Another advantage of using the TOF mass analyzer is that it can analyze with relatively high resolution and frequency. For example, each MS2 analysis can be carried out with a resolution of at least 50,000, preferably at least 60,000, 70,000 or 80,000. Each MS2 analysis can also be carried out with a first m / z width of not more than 20Da, 15Da, 10Da, 8Da or 5Da. The higher sensitivity of the TOF mass analyzer and its faster repetition rate have improved the dynamic range of the DIA method, because narrower MS2 isolation window (reducing the possibility of a plurality of precursor ions of the abundance that separate together in the same MS2 isolation window thus) can be used, while still covering whole interested m / z scope during the DIA cycle with the duration that is less than chromatographic peak. In turn, the improved dynamic range means that triggering of triggered MS2 analysis can be based on the detection of fragments of the endogenous analyte (rather than relying on the detection of isotopically labeled analyte ions), thereby eliminating the need to spike a certain amount of isotopically labeled internal standard into the sample and significantly reducing experimental complexity, time and cost.

[0031] For example, in some embodiments, the target analyte (e.g., an endogenous analyte, preferably an endogenous peptide) can be monitored (i.e., without having to physically prepare an isotope-labeled version of the target) by including the m / z of one or more fragment ions in its computer-predicted (or database-derived) fragment ions (optionally with associated predicted / database-derived intensity and / or chromatographic retention time window) in the target list. This also means increased experimental flexibility because more target analytes can be included in the target list. Therefore, the method of the first aspect can perform instant matching of m / z peaks with endogenous analytes on the target list so as to perform additional triggered MS2 analysis in real time (i.e., the endogenous analyte detected simultaneously still exists). Therefore, the dynamic range of the DIA method can be improved by performing triggered MS2 analysis with relatively high resolution and frequency (using a TOF mass analyzer).

[0032] In some embodiments, mass spectrometry can be repeated once or many times. Each circulation of the method can be carried out with a duration (cycle time) of no more than about 2 seconds, 1.5 seconds, 1 second, 0.7 second or 0.5 second. Therefore, mass spectrometry can be carried out with a duration suitable with the duration of the chromatographic peak of sample. Especially, the cycle time of the method can allow multiple circulations to be carried out in the duration of chromatographic peak, so that the chromatographic peak can be more accurately characterized by the method.

[0033] In some embodiments, multiple MS2 analyses can be performed using a time-of-flight (TOF) mass analyzer at a frequency of at least 150 Hz. Thus, multiple MS2 analyses can be performed at a relatively high frequency, such that for a given cycle time, the number of MS2 analyses performed can be increased (e.g., MS2 analyses can be performed with a narrower isolation window).

[0034] In some embodiments, the first m / z width can be no more than 20Da, 15Da, 10Da, 8Da or 5Da. In some embodiments, the second m / z width can be no more than 5Da, 3Da or 2Da. In some embodiments, the second m / z width can be less than the first m / z width. Therefore, the MS2 analysis of relatively narrow width can be for analyte ion interested via the MS2 scan of triggering, while using the MS2 analysis of relatively wide (first width) to analyze all mass ranges of interest. Therefore, the available cycle time of the method for the first aspect can provide a balance between the more targeted MS2 analysis of analyzing all mass ranges of interest and analyte ion interested in a data-independent manner.

[0035] In some embodiments, providing a targeted list of m / z values for fragments of interest can include receiving data indicative of at least one analyte of interest, determining a plurality of fragment ions based on the analyte of interest using a computational model, and generating a targeted list of m / z values for fragments of interest based on the plurality of fragment ions. In some embodiments, the targeted list of m / z values for fragments of interest can be obtained from one or more databases. In some embodiments, the targeted list of m / z values for fragments of interest can be obtained based on the computational model and the one or more databases.

[0036] According to a second aspect of the present disclosure, there is provided a controller configured to control a mass spectrometry system that analyzes a sample received from a chromatography system across a mass-to-charge (m / z) range of interest. The controller is configured to:

[0037] obtaining a target list of mass-to-charge ratios of fragments of interest for a sample to be analyzed;

[0038] causing the mass spectrometry system to ionize the sample to form sample ions as the sample elutes from the chromatographic system;

[0039] Causing the mass spectrometry system to perform multiple MS2 analyses of sample ions across an m / z range of interest, wherein performing each MS2 analysis comprises:

[0040] a controller causing the mass spectrometry system to mass select the sample ions using an isolation window having a first m / z width and to fragment the sample ions within the isolation window to form fragment ions, wherein for each MS2 analysis in a plurality of MS2 analyses, a center of the isolation window is updated such that the plurality of MS2 analyses covers an m / z range of interest;

[0041] The controller causes the mass spectrometry system to perform mass analysis on the fragment ions and determine a mass-to-charge ratio associated with each spectral peak of the fragment ions; and

[0042] The controller compares the mass-to-charge ratio of the peak with the mass-to-charge ratios of the fragments of interest in the target list.

[0043] Wherein, upon detecting a match between the m / z of a set of one or more spectral peaks and the m / z of a set of one or more fragments of interest in a target list, the controller is configured to cause the mass spectrometry system to perform a triggered MS2 analysis of the sample ions, wherein the triggered MS2 analysis has a higher sensitivity than the sensitivity of each of the multiple MS2 analyses.

[0044] The controller of the second aspect may be configured to cause the mass spectrometry system to perform the method of the first aspect.Thus, the controller of the second aspect may incorporate any of the optional features of the first aspect and any associated advantages.

[0045] According to a third aspect of the present invention, there is provided a mass spectrometry system for analyzing sample ions received from a chromatography system across a mass-to-charge (m / z) range of interest. The mass spectrometry system comprises:

[0046] The controller according to the second aspect;

[0047] an ionization source configured to receive sample molecules from the chromatography system and generate sample ions;

[0048] a mass selector configured to mass select the sample ions to produce mass selected sample ions;

[0049] a fragmentation chamber configured to fragment the mass-selected sample ions to produce fragment ions; and

[0050] A mass analyzer is configured to perform mass analysis on the fragment ions.

[0051] The mass spectrometry system of the third aspect may be configured to perform the method of the first aspect. Thus, the mass spectrometry system of the third aspect may incorporate any of the optional features of the first aspect and any associated advantages.

[0052] According to a fourth aspect, there is provided a computer program comprising instructions for causing the mass spectrometry system of the third aspect or the controller of the second aspect to perform the method according to the first aspect.

[0053] According to a fifth aspect, there is provided a computer readable medium having stored thereon the computer program of the fourth aspect.

[0054] The computer program and computer readable medium of the fourth and fifth aspects, respectively, can be used to cause the mass spectrometry system according to the third aspect to perform the method of the first aspect. Therefore, the fourth and fifth aspects can combine any one of the optional features of the first aspect and any associated advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Embodiments of the present disclosure will now be described with reference to the following non-limiting drawings, in which:

[0056] - Figure 1 is a schematic diagram of a mass spectrometry system according to an embodiment of the present disclosure;

[0057] - Figure 2 is a flow chart of a mass spectrometry method according to an embodiment of the present disclosure;

[0058] - Figure 3 is a diagram of a first mass spectrometry method that can be performed according to embodiments of the present disclosure;

[0059] - Figure 4 is a diagram of a second mass spectrometry method that can be performed according to embodiments of the present disclosure;

[0060] - Figure 5 is a diagram of a third mass spectrometry method that can be performed according to embodiments of the present disclosure; and

[0061] and

[0062] - Figure 6 is a diagram of a fourth mass spectrometry method that can be performed according to embodiments of the present disclosure. DETAILED DESCRIPTION

[0063] In the present disclosure, reference is made to the resolution of a mass analyzer. The skilled person will understand that all resolutions mentioned in this disclosure refer to the resolution of a mass analyzer at a mass-to-charge ratio (m / z) equal to 200 amu (m / z=200 amu). The skilled person will understand that the m / z ratio on which the resolution of a given mass analyzer is based only indicates the resolution at that m / z value of the mass analyzer and is not limited to the m / z range over which the mass analyzer is scanned according to the methods of the embodiments.

[0064] Figure 1 A schematic diagram of a mass spectrometry system 10 suitable for performing methods according to embodiments is shown. Figure 1 The mass spectrometry system schematically represents the Orbitrap from Thermo Fisher Scientific, Inc. TM Astral TM Configuration of the mass spectrometer, which is a dual mass analyzer mass spectrometer.

[0065] exist Figure 1 In the present invention, the sample to be analyzed is supplied (e.g., from an autosampler) to a chromatography system such as a liquid chromatography (LC) column ( Figure 1 One such example of an LC column is the ProSwift TM Monolithic columns provide high performance liquid chromatography (HPLC) by forcing a sample carried in a mobile phase under high pressure through a stationary phase of irregular or spherical shaped particles that constitute the stationary phase. In an HPLC column, sample molecules elute at different rates depending on the degree of their interaction with the stationary phase.

[0066] A chromatogram can be produced by measuring the number of sample molecules eluted from an HPLC column over time using a detector (e.g., a mass analyzer). Sample molecules eluted from an HPLC column will be detected as peaks above the baseline measurement on the chromatogram. When different sample molecules have different elution rates, multiple peaks on the chromatogram can be detected. Preferably, individual sample peaks are separated in time from other peaks in the chromatogram so that different sample molecules do not interfere with each other.

[0067] On the chromatogram, the presence of a chromatographic peak corresponds to the time period that the sample molecule exists at the detector. Thus, the width of the chromatographic peak is equal to the time period that the sample molecule exists 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 according to the peak. For example, the peak width can be calculated based on 4 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 may also be suitable. In some embodiments, a mass spectrometry system 10 can be used to obtain a mass chromatogram. For example, in some embodiments, the mass spectrometry system 10 can repeatedly perform mass analysis on the sample eluted from the column. In some embodiments, the mass spectrometry system 10 can perform mass analysis on the sample in the MS1 domain to obtain a mass chromatogram.

[0068] Thus, an electrospray ionization source (ESI source) 20, which may be at atmospheric pressure, may be used to ionize the sample molecules separated via liquid chromatography. Those skilled in the art will appreciate that other suitable types of ionization sources, such as atmospheric pressure chemical ionization (APCI), thermospray ionization, and the like, may be used. The sample ions then enter the vacuum chamber 22 of the mass spectrometry system 10 and are guided into the ion funnel 27 via a capillary 25. The ion funnel 27 is configured to focus the sample ions into a first quadrupole mass filter 29. A calibrant ion source 28 is also provided in the vacuum chamber 22. The calibrant ion source 28 is configured to output calibrant ions to the first quadrupole mass filter 29. Thus, the calibrant ions and / or the sample ions may be injected into the first quadrupole mass filter 29 and subsequently analyzed by the mass spectrometry system 10.

[0069] The first quadrupole mass filter 29 can be a pre-filter for the mass spectrometry system 10. Ions output from the first quadrupole mass filter 29 can be delivered to a curved flatapole ion guide 50. The curved flatapole ion guide 50 can be configured to guide (charged) ions along a curved path through the curved path. Thus, the curved flatapole ion guide 50 applies low-pass ion filtering that reduces noise by removing undesirable neutral molecules (such as entrained solvent molecules) that are not guided along the curved path and are lost.

[0070] An ion gate (TK lens) 60 is located at the distal end of the curved flatapole ion guide 50 and controls the passage of ions from the curved flatapole ion guide 50 into a downstream second quadrupole mass filter 70. The second quadrupole mass filter 70 is typically, but not necessarily, segmented and, when operated in a selective mode, acts as a bandpass filter, allowing a selected mass-to-charge ratio or a limited range of mass-to-charge ratios to pass while rejecting ions of other mass-to-charge ratios (m / z). The second quadrupole mass filter 70 can be operated to allow ions of a relatively wide range of mass-to-charge ratios (e.g., 40 m / z to 2,500 m / z) to pass, which is useful for analyzing a wide range of m / z spectra.

[0071] The second quadrupole mass filter 70 can be operated to carry out mass selection (or isolation) to the precursor ion using the isolation window. Typically, the second quadrupole mass filter 70 can utilize the isolation window with a width not greater than about 1200amu to carry out mass selection to the ion. Relatively wide isolation window can be particularly suitable for MS1 analysis. The second quadrupole mass filter 70 can also utilize the isolation window with a width of at least 0.4amu to carry out mass selection to the ion. This type of relatively narrow isolation window may be particularly relevant for the selection of the precursor ion analyzed in the MS2 domain. The second quadrupole mass filter 70 can be configured to output ions to a curved trap (C trap) 100.

[0072] like Figure 1 As shown, ions output from the second quadrupole mass filter 70 can pass through a quadrupole exit lens / separation lens arrangement and a charge detector 80 on their way to the C-trap 100. The C-trap 100 has a longitudinally extending arcuate electrode supplied with an RF voltage and an end cap electrode supplied with a DC voltage to provide a potential barrier at the end of the C-trap 100. The result is a potential well extending along the curved longitudinal axis of the C-trap 100. In a first operating mode, a DC end cap voltage is set on the C-trap so that ions arriving from the second quadrupole mass filter 70 are trapped in the potential well of the C-trap 100, where the ions are cooled. The injection time (IT) of ions into the C-trap determines the number of ions (ion groups) that are subsequently ejected from the C-trap. Although in Figure 1 The C-trap 100 is used in the mass spectrometry system 10 of FIG. 1 , but in other embodiments, for example, where a different type of mass analyzer is used, a different ion storage device may be used instead, such as a linear trap having straight rather than curved electrodes. Figure 1 As shown, the C-trap 100 is configured to eject ions orthogonally toward the orbital capture mass analyzer 110. The C-trap 100 can also eject ions axially toward the fragmentation chamber 120.

[0073] In some experiments, the cooled ions trapped in the C-trap 100 may be ejected orthogonally from the C-trap 100 toward the orbital capture mass analyzer 110. The orbital capture mass analyzer may be a commercially available instrument sold by Thermo Fisher Scientific. Mass Analyzer. The orbital trapping mass analyzer 110 has an off-center injection aperture, through which ions are injected as coherent packets into the orbital trapping mass analyzer 110. The ions are then trapped within the orbital trapping mass analyzer 110 by a superlogarithmic electric field and reciprocate in the longitudinal direction while orbiting around an internal electrode.

[0074] The axial (z) component of the motion of the ion packets in the orbital trapping mass analyzer 110 is defined (more or less) 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. Thus, over time, the ions separate according to their mass-to-charge ratio.

[0075] This can be achieved by using an image current detector ( Figure 1 Ions are detected in the orbital trapping mass analyzer 110 using an image galvanometer (not shown). The image galvanometer generates a "transient" in the time domain containing information about all ion species as they pass through the image galvanometer. This transient is then subjected to a fast Fourier transform (FFT), resulting in a series of peaks in the frequency domain. From these peaks, a mass spectrum representing abundance / ion intensity versus m / z can be generated.

[0076] In the above configuration, sample ions (more specifically, a subset of sample ions within the m / z range of interest selected by the second quadrupole mass filter 70) are analyzed without fragmentation by the orbitrap mass analyzer 110. The resulting mass spectrum is denoted as MS1.

[0077] MS2 analysis (or more generally, MS n ) can also be passed Figure 1 The mass spectrometry system 10 is used. To achieve this, precursor sample ions are generated and delivered to the second quadrupole mass filter 70, where a secondary mass range is selected. The ions leaving the second quadrupole mass filter 70 are guided (axially) through the C-trap 100 to the fragmentation chamber 120. Figure 1 In the mass spectrometry system 10, the fragmentation chamber 120 is a higher energy collisional dissociation (HCD) device to which a collision gas is supplied. The potential applied to the fragmentation chamber 120 causes the precursor ions reaching the fragmentation chamber 120 to have sufficient energy so that their collision with the collision gas molecules causes the precursor ions to fragment into fragment ions.

[0078] The fragment ions are then ejected from the fragmentation chamber 120 back toward the C-trap 100, where they are again trapped in the potential well and cooled. Finally, the fragment ions captured in the C-trap are ejected orthogonally toward the orbital trapping device 110 for analysis and detection. The resulting mass spectrum of the fragment ions is denoted as MS2.

[0079] Despite Figure 1 An HCD fragmentation chamber 120 is shown in FIG, but other fragmentation devices employing methods such as collision induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), photodissociation, etc. may be used instead.

[0080] Figure 1 The fragmentation chamber 120 can be configured as a "fly through" configuration, wherein precursor ions enter the fragmentation chamber from one axial end of the fragmentation chamber 120 and fragmented ions exit the fragmentation chamber 120 from the opposite axial end. The fragmented ions can also be ejected from the fragmentation chamber 120 back to the C-trap 100 for analysis in the orbital capture mass analyzer 110. The fragmented ions can be ejected from the fragmentation chamber 120 for analysis in the MS2 domain. Alternatively, the precursor ions can be transported axially through the fragmentation chamber 120 without fragmentation, as discussed below.

[0081] like Figure 1 As shown, ions (fragmented ions or unfragmented precursor ions) may be ejected from the fragmentation chamber 120 to the ion guide 124 . Figure 1 The ion guide 124 shown in FIG is an RF multipole ion guide. The ion guide 124 is configured to transport ions from the fragmentation chamber 120 to the extraction trap 130. The extraction trap 130 is configured to accumulate ions received from the fragmentation chamber 120 for injection into a time-of-flight (TOF) mass analyzer. Figure 1 In the embodiment of the invention, the TOF mass analyzer is a multiple reflection time of flight (MRTOF) mass analyzer 140, but other types of mass analyzers can also be used. For example, US-B-10699888 and US-B-10593525 have been described in detail. Figure 1 Further information on the general instrument layout shown in .

[0082] In some embodiments, the fragmented ions can be transferred from the fragmentation chamber 120 to the extraction trap 130 (via the ion guide 124) for injection into the MRTOF mass analyzer 140. The ions (e.g., fragmented ions) are accumulated by the extraction trap 130 for injection into the MRTOF 140. The accumulated ions can be injected into the MRTOF 140 as coherent packets.

[0083] In some embodiments, the extraction trap 130 may include a high pressure region 132 followed by a low pressure region 134. A first trapping electrode having a first RF voltage may be provided in the low pressure region 134, and a second trapping electrode having a second RF voltage may be provided in the high pressure region 132. In the low pressure region 134, vacuum pumping may be provided at a rate of approximately 20 L / s. In the high pressure region 132, a capillary ( Figure 1 A N2 buffer gas may be provided (not shown). A membrane may be provided between the low pressure region 134 and the high pressure region 132. Such an extraction trap is further described in GB-A-2613439.

[0084] Thus, ions can be transferred in an axial direction from the ion guide 124 to a high pressure region 132 of the extraction trap 130. Ions trapped in the high pressure region 132 can then be transferred to a low pressure region 134 of the extraction trap. The ions can then be cooled in the low pressure region 134 and ejected into the MRTOF 140. In order to increase the frequency of MS2 analysis performed using the MRTOF 140, ion packets can be processed in parallel using the quadrupole mass filter 70, the fragmentation chamber 120, the ion guide 124, the extraction trap 130, and the MRTOF 140. In particular, ion packets can be transferred from the fragmentation chamber 120 to the high pressure region 132 of the extraction trap 130 in parallel with the ejection of ions from the low pressure region 134 of the extraction trap 130 into the MRTOF 140. The parallelization of the stages is further described in US-A-20190103263 by pre-trapping ions in the high pressure region 132 of the extraction trap 130 while the low pressure region completes its sequence (ion cooling, high pressure boost, extraction to MRTOF 140).

[0085] As described above, in some embodiments, the (unfragmented) precursor ions can be transferred from the quadrupole mass filter 70 through the fragmentation chamber 120 to the extraction trap 130 without undergoing fragmentation. Where MS2 analysis is desired, in some embodiments, the precursor ions can be fragmented in the high pressure region 132 of the extraction trap 130. For example, the fragmentation of the precursor ions in the high pressure region 132 can be controlled by controlling the pressure of the high pressure region 132 and applying a suitable DC voltage to induce fragmentation.

[0086] The MRTOF 140 includes a first converging ion mirror 141 and a second converging ion mirror 142. The first converging ion mirror 141 and the second converging ion mirror 142 are arranged relative to each other so as to define ion trajectories involving multiple reflections between the first converging ion mirror 141 and the second converging ion mirror 142. Figure 1As further shown, ions are input from the extraction trap 130 into the MRTOF 140. The ions travel from the extraction trap 130 through the ion optical assembly 144 before traveling between the converging ion mirror 141 and the converging ion mirror 142. The ion optical assembly 144 may include one or more focusing lenses and / or ion deflectors configured to focus the ion packets ejected from the extraction trap 130 into the MRTOF 140. Figure 1 As shown, when the ions ejected from the extraction trap 130 are reflected between the first converging ion mirror 141 and the second converging ion mirror 142, the ions travel in the axial direction of the MRTOF 140. The converging ion mirrors 141 and 142 cause the ions to reverse their axial direction of travel, so that the ions then travel back along the converging ion mirrors 141 and 142, where they are captured by the ion detector 147. Figure 1 In the MRTOF 140, a pair of correction strip electrodes 146 are provided between the converging ion mirror 141 and the converging ion mirror 142. Further details of the MR-TOF 10a can be found in at least US-B-9136101.

[0087] The mass spectrometry system 10 is under the control of a controller (not shown) that is configured, for example, to control the timing of ejection from the trapping assembly, to set appropriate potentials on electrodes of a quadrupole mass filter, etc., to focus and filter ions, thereby capturing mass spectrometry data from the orbital capture mass analyzer 110 and the MRTOF 140, to control the sequence of MS1 scans and MS2 scans, etc. It should be understood that the controller may include a computer that is operable according to a computer program containing instructions for causing the mass spectrometer to perform method steps according to an embodiment.

[0088] It should be understood that Figure 1 The specific arrangement of the components shown in is not necessary for the method described subsequently. In fact, other arrangements for carrying out the method of the embodiment are also suitable. For example, an additional time-of-flight mass analyzer or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer can be used to replace the orbital capture mass analyzer 110.

[0089] Now refer to Figure 2 and 3 Describe an embodiment of the method.

[0090] The sample to be analyzed contains sample molecules. In some embodiments, the composition of the sample can be completely unknown. In some embodiments, the sample can include or be expected to include at least one analyte of interest. The sample can be supplied from a liquid chromatography (LC) column connected to the mass spectrometry system 10 (e.g., Figure 1 shown).

[0091] In some embodiments, sample to be analyzed can include one or more isotope-labeled versions of the analyte molecule of interest, although isotope labeling is not essential. That is, the isotope-labeled version (i.e., isotopologue) of the analyte molecule can be intentionally added to the sample. The isotope-labeled version of the analyte molecule is a molecule with the same chemical structure as the parent molecule (i.e., the analyte molecule of interest) and the difference from its parent molecule is that at least one atom of the isotope-labeled version has a different number of neutrons. In some embodiments, the analyte of interest can only be present in the sample (even if present) with relatively low abundance (e.g., endogenous peptides), making it challenging to detect using conventional DIA methods. Isotope labeling can help detect such relatively low abundance compounds.

[0092] In particular, for a given sample, before carrying out the DIA method, the retention time of the analyte of interest may not be known. Since the analyte of interest may only be present in a small amount in the sample, it may be challenging to determine the retention time using conventional methods. Therefore, the isotope-labeled version of the analyte of interest can be added to the sample with the amount sufficient to enable the isotope-labeled version of the added to be reliably detected by mass analysis. Therefore, it can be considered that the sample comprises multiple isotope-labeled versions of the analyte of interest (that is, the isotope-labeled version of the added and the unlabeled analyte of interest are considered to be different isotope-labeled versions of the analyte of interest). Since the isotope-labeled version of the added analyte of interest has the same chemical structure as the unlabeled analyte of interest, the retention time of the isotope-labeled analyte of interest added will be similar or identical to the analyte of interest.

[0093] In some embodiments, different isotopic labels can be applied to an analyte of interest. Each isotopically labeled version of an analyte of interest can have a different isotopic label but be chemically identical. Different isotopically labeled analytes of interest can be added to a sample in different known amounts. For example, in some embodiments, a sample can contain a "light" isotopically labeled version of an analyte of interest (i.e., an endogenous version of the analyte of interest). In some embodiments, a "heavy" isotopically labeled version of an analyte of interest can be added to a sample. Relative to the light isotopically labeled analyte of interest, the "heavy" isotopically labeled version of an analyte of interest typically contains a greater number of atoms, including additional neutrons (e.g., 13 C. 15 N, deuterium, etc.).

[0094] In some embodiments, multiple isotopically labeled versions of an analyte of interest can be prepared by stable isotope labeling with amino acids in cell culture (SILAC). In the SILAC process, an isotope label is incorporated into one or more proteins in vivo. In some embodiments, the SILAC process can be used to label the relatively "light" or relatively "heavy" forms of an amino acid (e.g., with deuterium, 13 C or 15 In one embodiment, an isotopically labeled version of an analyte of interest can be prepared by stable isotope dimethyl labeling. By using a combination of several isotopomers such as formaldehyde or cyanoborohydride, multiple different isotopic labels (e.g., at least 2 different isotopic labels, or at least 3 different isotopic labels) can be prepared for a single analyte of interest. The mass of each isotopic label can differ by at least 2 Da, preferably at least 4 Da, from that of the adjacent isotopic label to allow for simplified analysis.

[0095] exist Figure 2 and Figure 3 In an embodiment of the present invention, sample molecules can be supplied from an LC column so that data about the sample molecules and any isotopically labeled versions of the analyte molecules of interest (when included) are obtained according to the methods of the embodiments as they elute from the chromatographic system. It will be appreciated that during the experiment, depending on the retention times of the sample molecules and the isotopically labeled molecules, the LC column may sometimes supply only sample molecules, only isotopically labeled molecules, or both sample molecules and isotopically labeled molecules to the mass spectrometer. In the present disclosure, reference to the supply of sample molecules and / or isotopically labeled molecules from an LC column will be considered to include any of the above situations.

[0096] According to the methods of the present disclosure, mass spectrometry system 10 analyzes sample molecules and isotopically labeled analyte molecules of interest (when present) according to a method including multiple MS2 analyses. In some embodiments, one or more MS1 analyses may also be performed. Figure 2 1 is a flow chart illustrating a mass spectrometry method 100 for analyzing a sample across a mass-to-charge ratio (m / z) range of interest according to an embodiment of the present disclosure. Method 100 indicates steps that can be performed in a single cycle. In some embodiments, multiple cycles can be performed as the sample elutes from the chromatography system.

[0097] The m / z range of interest may be predetermined before starting the method. For example, the m / z range of interest may be selected by a user. Alternatively, the mass spectrometry system 10 may be configured to analyze ions across a predetermined m / z range of interest. For example, Figure 1 The mass spectrometry system 10 may be configured to analyze ions with m / z ranging from 400 m / z to 1100 m / z.

[0098] In step 101, method 100 includes providing a target list of m / z values of fragments of interest for a sample to be analyzed. Thus, the target list provides a list of one or more m / z values, each indicating the m / z value of a known ion fragment. Method 100 can take subsequent action (performing a triggered MS2 scan) upon detecting a peak having an m / z value corresponding to an m / z value on the target list. The target list can be populated with the m / z values of the fragments of interest according to various methods.

[0099] In some embodiments, the target list of m / z of fragments of interest can be a predetermined list of m / z of fragments of interest provided by the mass spectrometry system 10. For example, in some embodiments, the target list can contain the m / z of one or more known fragments of an analyte (molecule) of interest. Therefore, in some embodiments, the mass spectrometry system 10 can receive data indicating at least one analyte of interest, such as a user selection. In some embodiments, the user can also input the m / z of one or more known fragments of an analyte of interest. In some embodiments, the mass spectrometry system 10 can utilize a computational model to determine one or more fragment ions based on the analyte of interest. The mass spectrometry system 10 can then fill in the target list of m / z of the fragments of interest based on the determined fragment ions. Various computer computational models for determining fragment ions from precursor ions (i.e., analyte ions of interest) are known to technicians.

[0100] In some embodiments in which the sample ions to be analyzed include multiple isotopically labeled versions of the analyte ions of interest, the target list can be updated based on the isotopic labeling. Thus, the target list of m / z for fragments of interest can include the m / z for isotopically labeled fragments of interest based on multiple isotopically labeled versions of the analyte ions of interest. For example, in a case where the sample includes a "light" isotopically labeled version of the analyte of interest and a "heavy" isotopically labeled version of the analyte of interest, the target list can be populated with the m / z for the "light" fragment of interest and the m / z for the "heavy" version of the same fragment of interest. In some embodiments in which a computational model is used to determine one or more fragment ions, the user can also input information about the isotopic labeling of the sample. Thus, the mass spectrometry system 10 can then populate the target list of m / z for fragments of interest based on the determined fragment ions and the one or more isotopically labeled versions of the determined fragment ions.

[0101] In step 102, method 100 includes ionizing a sample to form sample ions as the sample elutes from a chromatography system. Figure 1In a system, sample ions can be ionized by an electrospray ionization source 20. As described above, method 100 can be performed multiple times as the sample elutes from the chromatography system. Thus, different sample molecules can elute from the chromatography system at different points during the experiment. In particular, the retention time of one or more analytes of interest may be unknown prior to the experiment.

[0102] In step 103, the method includes performing multiple MS2 analyses of sample ions across an m / z range of interest, eg, m / z 400 to 1200. Performing each MS2 analysis includes mass selecting the sample ions using an isolation window.

[0103] According to the present disclosure, each isolation window has width (that is, first width) and is centered on specific m / z. Therefore, each isolation window spans from the first m / z value to the second m / z value, and the difference between the first m / z value and the second m / z value is limited by (first) width of the isolation window. The first m / z value and the second m / z value can be equally spaced apart from the center m / z value of the isolation window. The first m / z value and the second m / z value / center m / z of the isolation window can be updated for each MS2 analysis in multiple MS2 analyses so that multiple MS2 analyses cover the m / z range of interest. For example, the center m / z value can be increased progressively by the first width for each continuous MS2 analysis so that multiple MS2 analyses include all sample ions in the mass range of interest. Although multiple MS2 scans can be performed in the ascending order of the center m / z value in some embodiments, in other embodiments, multiple MS2 scans can be performed in descending order (of the center m / z value) or in random order.

[0104] exist Figure 1 In a system such as the one described above, sample ions can be transferred to a second quadrupole mass filter 70 for mass selection. The second quadrupole mass filter 70 can then filter out any sample ions with m / z values outside the isolation window. As described above, the center m / z value of the isolation window can be updated for successive MS2 analyses.

[0105] In order to carry out MS2 analysis, the first width of the isolation window can be relatively narrow (compared with MS1 analysis). For each MS2 analysis in a plurality of MS2 analyses, the first width of the isolation window can be identical. For example, in some embodiments, the width of the isolation window can be no more than 20Da, 15Da, 10Da, 8Da or 5Da. As the first width of the isolation window decreases, the MS2 analysis may be more selective. That is to say, the quantity of the different types of sample ions included in the isolation window can decrease as the width of the isolation window decreases. Reducing the first width of the isolation window increases the number of MS2 analyses required for covering a given mass range of interest, which in turn increases the cycle time of method 100. Therefore, in some embodiments, the width of the isolation window can be at least: 2Da, 3Da, 4Da or 5Da.

[0106] When performing each of the multiple MS2 analyses, sample ions can be accumulated within the isolation window for a certain period of time (the first injection time). Figure 1 In some embodiments, mass-selected ions from the quadrupole mass filter 70 can be accumulated in the fragmentation chamber 120 (without undergoing fragmentation), although the ions can be accumulated in any other suitable ion trap of the mass spectrometry system 10. In some embodiments, the ions can be accumulated for a predetermined amount of time (a first injection time). In some embodiments, the ions can be accumulated until a predetermined amount of ions are accumulated, until a predetermined amount of charge is accumulated, or until a first maximum injection time is reached.

[0107] In some embodiments, the accumulated sample ions can be fragmented in the fragmentation chamber 120. Figure 1 In an embodiment of the present invention, ion packets are accumulated in the fragmentation chamber 120 in parallel with further processing of downstream ion packets in the extraction 130. The accumulated ion packets can be transferred from the fragmentation chamber 120 to the high pressure region 132 of the extraction trap 130. The sample ions can then undergo fragmentation in the high pressure region 132 before being transferred to the low pressure region 134. The fragmented ions are injected into the MRTOF 140 as coherent ion packets from the low pressure region 134 of the extraction trap 130. The MRTOF 140 measures the time of flight of each ion within the injected ion packet and determines the m / z associated with each spectral peak of the fragment ion. Thus, the MRTOF 140 can generate a set of one or more spectral peaks with associated m / z for use in the MS2 analysis being performed.

[0108] For each MS2 analysis performed, the controller of the mass spectrometry system 10 compares the m / z of the spectrum peak set from the MS2 analysis with the m / z of the fragment of interest from the target list. When a match is detected, the method 100 continues with one or more triggered MS2 analyses (step 104). In some embodiments, when the m / z of the spectrum peak is within, for example, about ± 20 ppm or ± 10 ppm of the m / z of the fragment of interest, the m / z of the spectrum peak is determined to match the m / z of the fragment of interest from the target list. Although in some embodiments, the matching range can be based on a percentage of m / z, in other embodiments, the matching range can be an absolute range, for example, about ± 0.1 Da, ± 0.05 Da, ± 0.03 Da, ± 0.02 Da, or ± 0.01 Da.

[0109] In step 104, the method includes performing one or more triggered MS2 analyses of the sample. The triggered MS2 analysis can be performed after detecting a match between the m / z of a set of one or more spectral peaks and the m / z of a set of one or more fragments of interest from a target list. In some embodiments, detecting a single match between the m / z of a spectral peak and an m / z of an interesting fragment can be sufficient to trigger the triggered MS2 analysis. In other embodiments, method 100 may require at least two matches between two spectral peaks and corresponding fragments of interest from a target list. Therefore, in order to reduce or eliminate false positive triggered MS2 analyses, the triggered MS2 analysis can be performed only when the set of spectral peaks matches at least two fragments associated with the analyte of interest present in the fragmented ions.

[0110] According to step 104, the triggered MS2 analysis is an MS2 analysis having a higher sensitivity than the sensitivity of each of the multiple MS2 analyses (i.e., the MS2 analysis performed in step 103). The triggered MS2 analysis of the sample can be performed based on one or more matches between the m / z of the set of spectral peaks and the m / z of the target list. Thus, the one or more matches can indicate an analyte of interest, wherein the triggered MS2 analysis performed can be a relatively high-sensitivity MS2 analysis of the analyte of interest. For example, the triggered MS2 analysis can utilize an isolation window centered around the m / z associated with the indicated analyte of interest.

[0111] according to Figure 2In an embodiment of the present invention, performing triggered MS2 analysis includes mass selecting the sample ions using an isolation window having a second m / z width. The sample ions can be mass selected using a second quadrupole mass filter 70. The mass filtered sample ions can then be transferred to a fragmentation chamber 120 where the sample ions are fragmented to form triggered fragment ions. The triggered fragmented ions can then be mass analyzed using an MRTOF 140, where the m / z associated with each spectral peak of the triggered fragment ions is identified.

[0112] In some embodiments, the second width of the isolation window used to mass select sample ions for the triggered MS2 analysis can be narrower than the first width of the isolation window used for the multiple MS2 analyses (step 103). Because the second width is narrower than the first width, the triggered MS2 analysis can have a higher sensitivity than each of the multiple MS2 analyses. For example, the second width can be no greater than 3 Da, 2 Da, 1 Da, or 0.5 Da. In some embodiments, the second width can be no greater than 70%, 60%, or 50% of the first width.

[0113] When performing a triggered MS2 analysis, sample ions can accumulate within the isolation window for the second injection time. In some embodiments, the sensitivity of the triggered MS2 analysis can be increased by increasing the duration of the second injection time so that it is greater than the first injection time. For example, by narrowing the second width of the second isolation window, the intensity of the sample ions can be lower, so that the filling rate of ions for the triggered MS2 analysis is slower than the filling rate of ions for multiple MS2 analyses. In some embodiments, when the intensity of the sample ions within the isolation window is relatively low, ions can accumulate up to the second maximum injection time. In some embodiments, the second maximum injection time can be greater than the first maximum injection time. For example, in some embodiments in which the first maximum injection time is about 3.5ms, the second maximum injection time can be about 5ms. Therefore, by adjusting the second maximum injection time, the triggered MS2 analysis can provide a triggered MS2 analysis with an injection time longer than each MS2 analysis in multiple MS2 analyses.

[0114] In some embodiments, method 100 may include performing a preliminary analysis (e.g., an automatic gain control (AGC) process) using a mass analyzer (orbitrap mass analyzer 110 or MRTOF mass analyzer 140) to estimate the intensity of sample ions across the m / z range of interest. The preliminary analysis may then be used to predict ion intensities for an ongoing MS2 analysis or a triggered MS2 analysis.

[0115] Figure 3A first schematic workflow of the mass spectrometry method according to the present invention is shown. Figure 3 The workflow can be Figure 1 The system is carried out. Figure 3 As shown, multiple MS2 analyses are carried out across the mass range of interest (400m / z to 1200m / z). Each MS2 analysis is carried out with an isolation window of 8Da, so that 100 MS2 analyses are carried out to cover the m / z range of interest. Each MS2 analysis uses a maximum injection time of 3.5ms, and the overhead time of approximately 1.5ms means that each MS2 analysis takes an average of approximately 5ms to complete. Therefore, multiple MS2 analyses are carried out by MRTOF 140 at a frequency of approximately 200Hz. Therefore, multiple MS2 analyses can be carried out across the entire mass range of interest in approximately 0.5s.

[0116] like Figure 3 As shown, MS1 analysis is also performed in parallel with multiple MS2 analyses. The MS1 analysis uses an orbital capture mass analyzer 110 to mass analyze sample ions in the MS1 domain across the mass range of interest (400 m / z to 1200 m / z). Figure 3 As shown, an MS1 analysis can be performed in about 0.5 seconds at a resolution of about 240,000. Therefore, it will be appreciated that the duration of an MS1 analysis is comparable to the duration of multiple MS2 analyses.

[0117] like Figure 3 As shown, the method also includes performing one or more triggered MS2 analyses. When the peak from the MS2 analysis matches the m / z of the set of one or more fragments of interest in the target list, the triggered MS2 analysis is triggered. The set of one or more fragments of interest in the target list can correspond to one or more analytes of interest. For example, in some embodiments, the target list can include a list of m / z of fragments of endogenous analytes.

[0118] In the embodiment that target list comprises the list of the m / z of the fragment of endogenous analyte and sample does not provide internal standard, can preferably use TOF mass analyzer (such as MRTOF 140) to carry out MS2 analysis and triggered MS2 analysis.The higher sensitivity (such as, compared with such as ion trap mass analyzer) of TOF mass analyzer and its faster repetition rate have improved the dynamic range of DIA method, because can use narrower MS2 isolation window (reduced the possibility of separating together the multiple precursor ions of significant different abundance in identical MS2 isolation window thus), still cover whole interested m / z scope during DIA cycle when duration is less than chromatographic peak simultaneously.For example, in some embodiments, the first width for carrying out the isolation window of multiple MS2 analysis can be no more than 10Da, 8Da, 5Da or 3Da.TOF mass analyzer 140 can also be with relatively high resolution, such as at least 50,000 resolution, preferably at least 60,000,70,000 or 80,000 resolution operation.

[0119] When the target list only identifies an analyte of interest, the triggered MS2 analysis can be a single ion monitoring (SIM) analysis of the analyte of interest. For example, an isolation window with a second width of no more than 2Da can be utilized to perform the triggered MS2 analysis. The isolation window can be centered around the m / z of the analyte ion of interest. In order to further increase the sensitivity of the SIM analysis, the second injection time for the triggered MS2 analysis can be increased, for example, the second injection time can be at least 8.5ms. Therefore, the duty cycle of the method can be increased by approximately 10ms for each triggered MS2 analysis.

[0120] When two or more analytes of interest are identified in the same MS2 analysis by a target list, a separate triggered MS2 scan can be performed for each analyte of interest (i.e., a separate SIM analysis can be performed for each analyte of interest). Thus, the isolation window of each triggered MS2 scan can be focused on mass selection for only one analyte of interest. Thus, by performing separate SIM scans, the dynamic range for analyzing each analyte of interest in the MS2 domain can be improved by isolating the analytes of interest separately from each other.

[0121] therefore, Figure 3The mass spectrometry method shown in can carry out one or more triggered MS2 analyses, depending on the number of analytes of interest present in the sample during the method. For example, in the case where three analytes of interest are co-eluted from a chromatographic system, three triggered MS2 scans can be carried out within a duty cycle. Therefore, three triggered MS2 scans increase the duty cycle from 0.5s (for 100 MS2 analyses) to 0.530s (500ms+3×10ms). Therefore, it should be understood that including a limited number of triggered MS2 analyses in the method does not produce a significantly longer duty cycle. Therefore, the duty cycle duration is typically kept aligned with the duration of the MS1 analysis, so that an MS2-based method can be effectively performed in parallel with the MS1 analysis.

[0122] Although Figure 3 The embodiment of FIG. 5 illustrates one possible implementation of triggered MS2 analysis, but triggered MS2 analysis may be performed in different ways depending on the nature of the experiment being performed and the desired data to be obtained.

[0123] Figure 4 A second schematic workflow of the mass spectrometry method according to the present invention is shown. Figure 4 In the method, one or more triggered MS2 analyses can be performed on a single analyte of interest having different charge states. That is, after identifying that the analyte ion of interest is present in the sample ion, one or more triggered MS2 analyses with an isolation window can be performed, and the isolation window mass selects the different charge states of the analyte of interest (for example, the +2, +3 and +4 charge states of the analyte ion of interest). By using triggered MS2 analysis to analyze the different charge states of the analyte of interest, the method can increase the amount of the analyte ion of interest analyzed by the method. In particular, for the analyte ion of interest of relatively low abundance, analyzing multiple charge states can improve the signal-to-noise ratio of the analysis. For example, in an embodiment where the target list includes a list of m / z of fragments of an endogenous analyte and the sample does not provide an internal standard, it may be advantageous to analyze multiple charge states of the endogenous analyte of interest to improve the signal-to-noise ratio.

[0124] In some embodiments, similar to the above description of Figure 3 The described SIM analysis may analyze each of the different charge states in a separate triggered MS2 analysis (eg, in a separate SIMms2 analysis).

[0125] In some embodiments, different charge states can be analyzed together in a single triggered MS2 analysis (i.e., parallel reaction monitoring (PRM) analysis). That is, sample ions can be mass selected simultaneously using different isolation windows (each isolation window corresponding to a different charge state). Figure 4 In the method, after identifying that the analyte ions of interest are present in the sample ions, a triggered MS2 analysis is performed, which includes mass selecting the sample ions using a first isolation window having a second m / z width (i.e., a first mass selection step). The first isolation window can be centered on the m / z of the first charge state of the analyte ions of interest to produce a first mass-selected sample ion. The mass selection step can be performed by a second quadrupole mass filter 70. The first mass-selected sample ions can be accumulated in the second quadrupole mass filter 70, and / or transferred to the C-trap 100 as an intermediate step after mass selection. Similar to the above-mentioned triggered MS2 analysis, the second m / z width can be relatively narrow, for example no more than 5Da, 3Da or 2Da.

[0126] The triggered MS2 analysis also includes mass selecting the sample ions using a second isolation window having a second m / z width (i.e., a second mass selection step), the second isolation window being centered on the m / z of the second charge state of the analyte ion of interest to produce the second mass selected sample ions. The second mass selection step can be performed sequentially with the first mass selection step, wherein the first mass selected sample ions and the second mass selected sample ions are accumulated together in the C trap 100. Alternatively, in some embodiments, the second quadrupole mass filter 70 can apply a comb mass filter that applies the first isolation window and the second isolation window in parallel. Therefore, the mass filter can simultaneously mass select the sample ions whose m / z falls within the first isolation window or the second isolation window, so that the first mass selected sample ions and the second mass selected sample ions are accumulated in the second quadrupole mass filter 70. Other methods for mass selecting the first mass selected sample ions and the second mass selected sample ions can also be suitable for accumulating sample ions of different charge states corresponding to the analyte ions of interest.

[0127] While the above description relates to mass selection of two different charge states of the analyte ions of interest, it will be appreciated that the same principles can be applied to mass selection of, for example, at least three different charge states of the analyte ions of interest (e.g., Figure 4 ).

[0128] Once accumulated, the first mass selected sample ions and the second mass selected sample ions can be transferred to the fragmentation chamber 120. Within the fragmentation chamber 120, the accumulated first mass selected sample ions and the second mass selected sample ions are fragmented to form triggered fragment ions. The fragmentation process can be performed as described above with respect to other embodiments of the present disclosure. The resulting triggered fragment ions can then be transferred to the MRTOF 140. The triggered MS2 analysis can then include mass analysis of the triggered fragment ions, wherein the m / z associated with each spectral peak of the triggered fragment ions is identified.

[0129] exist Figure 4 In the embodiment of FIG. 1 , the m / z range of interest (400 m / z to 1200 m / z) is analyzed in the MS2 domain using 100 MS2 analyses, each with a first m / z width of 8 Da and a first injection time of 3.5 ms (total MS2 analysis duration of 5 ms). Sample ions can also be analyzed in parallel in the MS1 domain using the orbitrap mass analyzer 110 (similar to Figure 3 ).like Figure 4 As shown, three triggered MS2 analyses were performed. The first triggered MS2 analysis was performed on the analyte of interest having a 2+ charge state, the second triggered MS2 analysis was performed on the analyte of interest having a 3+ charge state, and the third triggered MS2 analysis was performed on the analyte of interest having a 4+ charge state. Figure 4 In an embodiment, the analyte ions of each charge state are mass selected separately, wherein each charge state has a second injection time of about 5 ms. The set of three mass-selected sample ions can be co-fragmented and co-analyzed in the MS2 domain (i.e., PRM process) or analyzed separately (i.e., SIM process). Performing an additional triggered MS2 analysis increases the duty cycle by about 15 ms, corresponding to the additional injection time introduced to accumulate the different charge states of the analyte ions of interest.

[0130] As discussed above, in some embodiments, the sample ions may include one or more isotopically labeled versions of the analyte ions of interest. In some embodiments, the triggered MS2 analysis may be performed based on the identification of one or more isotopically labeled versions of the analyte ions of interest. Figure 5 A third schematic workflow for a mass spectrometry method according to the present disclosure is shown, wherein an isotopically labeled version of an analyte ion of interest is detected.

[0131] like Figure 5 As shown, upon detecting a match between the m / z of a spectral peak and the m / z of an isotopically labeled fragment of interest from a target list, one or more triggered MS2 analyses are performed. Figure 5As shown, the triggered MS2 analysis can be performed after any version of the analyte ion of interest is detected. That is, the identification of an isotope-labeled analyte ion (e.g., provided as part of an internal standard) or an unlabeled version (e.g., an endogenous analyte) can result in a triggered MS2 analysis. In some embodiments, only the first identification of the analyte of interest (either version) can result in one or more triggered MS2 analyses. Therefore, in the case where the isotope labeling results in the detection of a version of the analyte of interest in two or more analyses in multiple MS2 analyses, only the first identification can result in a triggered MS2 analysis to avoid performing additional measurements of the same analyte of interest in a single cycle.

[0132] In the case where an isotopically labeled analyte of interest is added to the sample, the user can input information about the isotopic labeling into the mass spectrometry system 10 before performing the method. Thus, after identifying at least one of the multiple isotopically labeled versions of the analyte of interest in the MS2 data, the mass spectrometry system 10 can identify multiple isolation windows for mass selecting the different isotopically labeled versions of the analyte of interest. Figure 5 The method further includes performing one or more triggered MS2 analyses, wherein each triggered MS2 analysis utilizes one or more isolation windows, each isolation window being used to mass select a different isotopically labeled version of the analyte ion of interest.

[0133] according to Figure 5 In one embodiment, the sample includes both "light" and "heavy" isotope-labeled versions of the analyte of interest. Figure 5 In an embodiment of the invention, performing a triggered MS2 analysis includes mass selecting the sample ions using a first isolation window having a second m / z width. The first isolation window is centered on the m / z of a lightly isotopically labeled version of the analyte ion of interest to produce first mass selected sample ions. After mass selection, the first mass selected sample ions can be accumulated in the second quadrupole mass filter 70 or accumulated in the C trap 100. The first mass selected sample ions can be accumulated for a second injection time. Preferably, the second injection time is longer than the first injection time used for multiple MS2 analyses. For example, in Figure 5 In the embodiment of the present invention, the first injection time is 3.5 ms and the second injection time is 5 ms.

[0134] The triggered MS2 scan also includes mass selection of the sample ions using a second isolation window having a second m / z width. The second isolation window is centered on the m / z of the heavy isotope labeled version of the analyte ion of interest to produce a second mass-selected sample ion. For each triggered MS2 scan, the second m / z width may be no greater than 5Da, 3Da, or 2Da. After mass selection, the second mass-selected sample ions may be accumulated in the second quadrupole mass filter 70 or accumulated in the C-trap 100. Thus, the first mass-selected sample ions and the second mass-selected sample ions may be accumulated together. In some embodiments, a comb mass filter may be applied by the quadrupole mass filter to perform the first mass selection and the second mass selection simultaneously.

[0135] The accumulated first mass-selected sample ions and the second mass-selected sample ions can then be transferred to the fragmentation chamber 120, wherein the accumulated first mass-selected sample ions and the second mass-selected sample ions are fragmented to form triggered fragment ions. The triggered fragment ions can then be transferred to the MRTOF 140. The MRTOF 140 can then perform mass analysis on the triggered fragment ions, wherein the mass-to-charge ratio associated with each spectral peak of the triggered fragment ions is identified.

[0136] Thus, as shown in the workflow of Figure 5, sample ions in the m / z range of interest (400 m / z to 1200 m / z) are analyzed in the MS2 domain using 100 MS2 analyses. Each MS2 analysis has a first m / z width of 8 Da and a first injection time of 3.5 ms (total MS2 analysis duration is 5 ms). Optionally, the sample ions can also be analyzed in parallel in the MS1 domain using an orbital capture mass analyzer 110 (similar to Figure 3 and Figure 4 ).like Figure 5 As shown, after detecting one version of the analyte of interest (heavy or light), two triggered MS2 analyses are performed. The first triggered MS2 analysis is performed on the light isotope labeled version of the analyte of interest, and the second triggered MS2 analysis is performed on the heavy isotope labeled version of the analyte of interest. Figure 5In one embodiment, the light isotope labeled version and the heavy isotope labeled version are mass selected respectively, wherein the light isotope labeled version and the heavy isotope labeled version each have a second injection time of about 5ms. The collection of sample ions selected by the two masses can be co-fragmented and co-analyzed in the MS2 domain (i.e., PRM process) so that at least a portion of the triggered MS2 analysis is multiplexed together. In other embodiments, each triggered MS2 analysis can be performed completely separately (i.e., SIM process). Performing additional triggered MS2 analysis increases the duty cycle by about 10ms, corresponding to additional injection times introduced to accumulate different charge states of the analyte ions of interest.

[0137] As discussed above, in some embodiments, multiple different isotopic labels (e.g., at least 2 isotopic labels, or at least 3 isotopic labels) can be prepared for a single analyte of interest. Thus, in some embodiments, the sample ions can contain at least three different isotopic labeled versions of an analyte ion of interest (i.e., three different isotopic labels for a single analyte ion of interest). The mass of each isotopic label can differ from the mass of the adjacent isotopic label by at least 2 Da, preferably at least 4 Da, to allow for simplified analysis. Figure 6 A fourth schematic workflow of a mass spectrometry method according to the present invention is shown, wherein the sample ions contain at least three isotopically labeled versions of the analyte ion of interest. Figure 5 In the embodiment of the present invention, the three triggered MS2 analyses can be performed as a SIM analysis, or can be multiplexed and analyzed in parallel (ie, PRM analysis).

[0138] Although Figures 3 to 6 While the workflow 100 illustrates performing one or more triggered MS2 scans after detecting a version of an analyte of interest in multiple MS2 scans, it should be understood that the methods according to the present disclosure are not limited to detecting a single analyte of interest. That is, the target list can include m / z information associated with multiple different analytes of interest. Thus, in the case where multiple different analytes of interest are simultaneously present in a sample, method 100 can include performing one or more triggered MS2 analyses for each of the detected analytes of interest.

[0139] Similarly, it should be understood that in some embodiments, the sample molecules can include isotopic labels for multiple different analytes of interest. Thus, the sample ions can include a first plurality of isotopically labeled versions of a first analyte of interest and a second plurality of isotopically labeled versions of a second analyte of interest. After detecting a version of each of the first analyte of interest and the second analyte of interest, one or more triggered MS2 analyses can be performed.

[0140] Therefore, according to an embodiment of the present disclosure, a mass spectrometry method for analyzing a sample is provided. It should be understood that the controller that can be a processor or a computing device can be configured to control mass spectrometry system 10 so that the mass spectrometry system performs a method according to the present disclosure. Controller (e.g., processor or computing device) can perform a method according to the present disclosure according to a computer program comprising instructions. The instruction can make mass spectrometry system 10 perform method 100 according to the present disclosure when executed. In addition, the computer program can be stored on a computer-readable medium. The computer-readable medium can be any suitable computer memory provided as a part of the controller, for example, or can be a removable computer-readable medium that can be physically separated from the controller.

Claims

1. A mass spectrometry method for analyzing a sample across a mass-to-charge ratio (m / z) range of interest, the method comprising: A target list of mass-to-charge ratios of fragments of interest is provided for the sample to be analyzed; ionizing the sample to form sample ions as the sample elutes from the chromatographic system; performing a plurality of MS2 analyses of the sample ions across the m / z range of interest, wherein performing each MS2 analysis comprises: mass selecting the sample ions using an isolation window having a first m / z width and fragmenting the sample ions within the isolation window to form fragment ions, wherein for each MS2 analysis in the plurality of MS2 analyses, a center m / z of the isolation window is updated such that the plurality of MS2 analyses covers the m / z range of interest; performing mass analysis on the fragment ions and determining a mass-to-charge ratio associated with each spectral peak of the fragment ions; and comparing the mass-to-charge ratio of the spectral peak with the mass-to-charge ratio of the fragment of interest in the target list, Wherein, after detecting a match between the m / z of a set of one or more spectral peaks and the m / z of a set of one or more fragments of interest in the target list, the method further comprises performing a triggered MS2 analysis of the sample, wherein the triggered MS2 analysis has a higher sensitivity than the sensitivity of each of the multiple MS2 analyses.

2. The method according to claim 1, wherein The triggered MS2 analysis uses an isolation window having a second m / z width, wherein the second m / z width is narrower than the first m / z width, such that the triggered MS2 scan has a higher sensitivity than each of the plurality of MS2 analyses.

3. The method according to claim 1 or claim 2, wherein Performing each of the plurality of MS2 analyses includes accumulating sample ions within the isolation window for a first injection time; and Performing the triggered MS2 analysis includes accumulating sample ions within the isolation window for a second injection time, wherein the second injection time is greater than the first injection time such that the triggered MS2 scan has a higher sensitivity than each of the plurality of MS2 analyses.

4. The method according to claim 3, wherein The second injection time is calculated based on the intensity of the spectral peak matching the fragment of interest on the target list.

5. The method according to any one of claims 1 to 4, wherein Triggered MS2 analysis includes: mass selecting the sample ions using an isolation window having the second m / z width; fragmenting the sample ions within the isolation window to form triggered fragment ions; as well as The triggered fragment ions are mass analyzed, wherein the mass-to-charge ratio associated with each spectral peak of the triggered fragment ions is identified.

6. The method according to any one of claims 1 to 5, wherein When the m / z of a spectrum peak is within ±20 ppm or within ±10 ppm of the m / z of the fragment of interest, the m / z of the spectrum peak is determined to match the m / z of the fragment of interest of the target list.

7. The method according to any one of claims 1 to 6, wherein Detecting a match between the m / z of the set of one or more spectral peaks and the m / z of the set of one or more fragments of interest of the target list includes identifying that an analyte of interest is present in the sample ions.

8. The method according to claim 7, wherein After identifying the analyte ion of interest as being present in the sample ions, the method further includes performing a plurality of triggered MS2 analyses, wherein each triggered MS2 analysis has an isolation window selected to mass select for a different charge state of the analyte of interest.

9. The method according to claim 7, wherein After identifying the analyte ions of interest as being present in the sample ions, the method further includes performing a triggered MS2 analysis, the triggered MS2 analysis comprising: mass selecting the sample ions using a first isolation window having the second m / z width, the first isolation window centered on the m / z of the first charge state of the analyte ions of interest to produce first mass-selected sample ions; mass selecting the sample ions using a second isolation window having a second m / z width, the second isolation window centered on the m / z of the second charge state of the analyte ions of interest to produce second mass-selected sample ions, wherein the first mass-selected sample ions and the second mass-selected sample ions are accumulated together; fragmenting the accumulated first mass-selected sample ions and second mass-selected sample ions to form triggered fragment ions; as well as The triggered fragment ions are mass analyzed, wherein the mass-to-charge ratio associated with each spectral peak of the triggered fragment ions is identified.

10. The method according to claim 9, wherein The first isolation window and the second isolation window are simultaneously used to perform mass selection on the sample ions.

11. A method according to any preceding claim, wherein The sample ions to be analyzed contain multiple isotopically labeled versions of the analyte ion of interest; and The target list of m / z of fragments of interest contains m / z of isotopically labeled fragments of interest based on the plurality of isotopically labeled versions of the analyte ion of interest. 12 . The method of claim 11 , wherein the plurality of isotopically labeled versions of the analyte ion of interest comprises a light isotopically labeled version of the analyte ion of interest and a heavy isotopically labeled version of the analyte ion of interest.

13. The method according to claim 11 or claim 12, wherein The sample ions comprising multiple isotopically labeled versions of the analyte ion of interest are prepared by: Stable isotope labeling with amino acids in cell culture (SILAC); or Stable isotope dimethyl labeling; or Stable isotope labeling (SIL).

14. The method according to any one of claims 11 to 13, wherein Triggered MS2 analysis is performed upon detecting a match between the m / z of the set of one or more spectral peaks and the m / z of the set of one or more fragments of interest associated with one of the multiple isotopically labeled versions of the analyte ion of interest in the target list.

15. The method according to any one of claims 11 to 14, wherein Triggered MS2 analysis includes: mass selecting the sample ions using a first isolation window having the second m / z width, the first isolation window centered on the m / z of an isotopically labeled version of the analyte ion of interest to produce first mass-selected sample ions; mass selecting the sample ions using a second isolation window having a second m / z width, the second isolation window centered on the m / z of a different isotopically labeled version of the analyte ion of interest to produce second mass-selected sample ions, wherein the first mass-selected sample ions and the second mass-selected sample ions are accumulated together; fragmenting the accumulated first mass-selected sample ions and second mass-selected sample ions to form triggered fragment ions; as well as The triggered fragment ions are mass analyzed, wherein the mass-to-charge ratio associated with each spectral peak of the triggered fragment ions is identified.

16. A method according to any preceding claim, wherein The plurality of MS2 analyses are performed using a time-of-flight (TOF) mass analyzer at a frequency of at least 150 Hz.

17. A method according to any preceding claim, wherein The first m / z width is no greater than 20 Da, 15 Da, 10 Da, 8 Da or 5 Da.

18. A method according to any preceding claim, wherein The second m / z width is no greater than 5 Da, 3 Da or 2 Da.

19. A method according to any preceding claim, wherein The target list providing the m / z of the fragments of interest includes: receiving data indicative of at least one analyte of interest; using a computational model to determine a plurality of fragment ions based on the analyte of interest; as well as The target list of m / z of fragments of interest is generated based on the plurality of fragment ions.

20. A method according to any preceding claim, wherein The target list of mass-to-charge ratios of fragments of interest for the sample to be analyzed comprises a list of m / z fragments of endogenous analytes of interest, and The triggered MS2 analysis of the sample ions is performed based on a match between the m / z of the set of detected one or more spectral peaks and the list of m / z of fragments of endogenous analytes.

21. A controller configured to control a mass spectrometry system that analyzes a sample received from a chromatography system across a mass-to-charge (m / z) range of interest, the controller configured to: obtaining a target list of mass-to-charge ratios of fragments of interest for the sample to be analyzed; ionizing the sample with the mass spectrometry system to form sample ions as the sample elutes from the chromatography system; causing the mass spectrometry system to perform a plurality of MS2 analyses of the sample ions across the m / z range of interest, wherein performing each MS2 analysis comprises: the controller causing the mass spectrometry system to mass select the sample ions using an isolation window having a first m / z width and fragment the sample ions within the isolation window to form fragment ions, wherein for each MS2 analysis in the plurality of MS2 analyses, a center of the isolation window is updated such that the plurality of MS2 analyses cover the m / z range of interest; The controller causes the mass spectrometry system to perform mass analysis on the fragment ions and determine a mass-to-charge ratio associated with each spectral peak of the fragment ions; and The controller compares the mass-to-charge ratio of the spectrum peak with the mass-to-charge ratio of the fragment of interest in the target list, Wherein, upon detecting a match between the m / z of a set of one or more spectral peaks and the m / z of a set of one or more fragments of interest in the target list, the controller is configured to cause the mass spectrometry system to perform a triggered MS2 analysis of the sample ions, wherein the triggered MS2 analysis has a higher sensitivity than the sensitivity of each of the multiple MS2 analyses.

22. A mass spectrometry system for analyzing sample ions received from a chromatography system across a mass-to-charge (m / z) range of interest, the mass spectrometry system comprising: The controller according to claim 21; an ionization source configured to receive sample molecules from the chromatography system and generate sample ions; a mass selector configured to mass select the sample ions to produce mass-selected sample ions; a fragmentation chamber configured to fragment the mass-selected sample ions to produce fragment ions; and A mass analyzer is configured to perform mass analysis on the fragment ions.

23. A computer program comprising instructions for causing the mass spectrometry system according to claim 22 or the controller according to claim 21 to perform the method according to any one of claims 1 to 20.

24. A computer-readable medium having stored thereon the computer program according to claim 23.

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