Tandem mass spectrometer and method for tandem mass analysis

Abstract

To provide a method of tandem mass spectrometry for analyzing precursor ions across a mass-to-charge (m / z) range of interest.SOLUTION: The method comprises analyzing the precursor ions across the m / z range of interest in the MS1 domain using a first mass analyzer of a tandem mass spectrometer operated at a first sensitivity. The method also comprises analyzing some of the precursor ions across the m / z range of interest in the MS1 domain using a second mass analyzer of the tandem mass spectrometer operated at a second sensitivity, where the second sensitivity is higher than the first sensitivity. The analysis in the MS1 domain performed by the second mass analyzer is performed concurrently with the analysis performed in the MS1 domain by the first mass analyzer. The method also comprises combining data from the MS1 analyses performed by the first and second mass analyzers to identify and / or quantify precursor ions. The method also comprises analyzing the precursor ions in the MS2 domain using the second mass analyzer of the tandem mass spectrometer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to mass spectrometry. In particular, the present disclosure relates to tandem mass spectrometers and methods of tandem mass spectrometry. [Background technology]

[0002] Mass spectrometry is a long-established technique for identifying and quantifying often complex mixtures of large organic molecules. In particular, proteins, which contain many amino acids, typically have a significant molecular weight. Accurate identification and quantification of proteins by direct mass spectrometry measurements is therefore difficult. It is therefore well known to fragment precursor sample ions. Various fragmentation techniques are known that can result in the production of different fragment ions from precursor ions. Furthermore, the fragmentation mechanism can be affected by different applied fragmentation energies.

[0003] One known method of mass spectrometry data acquisition that involves fragmentation of precursor ions is known as data-independent analysis / acquisition (DIA) mass spectrometry.

[0004] DIA seeks to determine what is present in a potentially unknown sample. To determine the molecular structure of the sample molecules, a mass spectrometer is first used to mass analyze all sample ions (precursor ions) within the mass-to-charge ratio (m / z) range of interest. Such a scan is often referred to as an MS1 ​​scan. Selected sample ions are then fragmented and the resulting fragments are subsequently mass analyzed. A scan of the fragmented ions is often referred to as an MS2 scan.

[0005] An example of a DIA methodology is further described in U.S. Patent No. 10,699,888(B), in which a tandem mass spectrometer comprising an orbital trap mass analyzer and a time-of-flight (TOF) mass analyzer is used. The orbital trap analyzer is used to perform a series of relatively broad mass-to-charge (m / z) range MS1 scans, while the TOF performs multiple narrow m / z range MS2 scans.

[0006] Another method of mass spectrometry data acquisition that involves fragmentation of ions is known as Data-Dependent Analysis / Acquisition (DDA) mass spectrometry. DDA mass spectrometry seeks to confirm the presence of one or more species in a given sample. DDA methods identify a fixed number of precursor ion species and select them to analyze by mass spectrometry. The decision of which precursor ion species to target in DDA may be based on intensity ranking (e.g., the top 10 largest species found in peaks in the MS1 spectrum) and / or by the definition of an "inclusion list" of precursor mass spectrum peaks (e.g., by user selection) that always acquires an MS2 spectrum regardless of the intensity ranking of the peaks in the MS1 mass spectrum. Additionally, an "exclusion list" of peaks in MS1 ​​may be defined, e.g., by the user, based on prior knowledge of the expected sample content, for example. DDA methodology is further described in Hu A, Noble WS and Wolf-Yadlin A. "Technical advances in proteomics: new developments in data-independent acquisition" [version 1; peer review: 3 approved]. F1000Research 2016, 5(F1000 Faculty Rev):419.

[0007] Another data acquisition method for mass spectrometry, called "BoxCar" or "HDR", is disclosed in Meier et al, Nature Methods, 2018, 15, 440-448. This method involves splitting a wide mass range into many separate isolation windows. Each isolation window has a different fill time into the C-trap, depending on the ion current of the isolation window. This workflow efficiently redistributes the C-trap ion capacity between analytes, but it also significantly increases the time required to accumulate ions, especially when using a significant number of windows. In particular, relatively long fill times are required for isolation windows with low ion currents. Furthermore, additional time is required to switch quadrupole and ion source voltages between isolation windows. BoxCar / HDR data acquisition can therefore immediately impact the speed at which MS2 scans can be performed.

[0008] The method of mass spectrometry, which involves the analysis of precursor ions (MS1 scan) and fragment ions (MS2 scan), can be performed by a tandem mass spectrometer, an example of which is disclosed in U.S. Pat. No. 10,699,888(B).

[0009] Against this background, the present disclosure seeks to provide improved, or at least commercially useful, alternative methods of tandem mass spectrometry. Summary of the Invention

[0010] According to a first aspect of the present disclosure, a method of tandem mass spectrometry for analyzing precursor ions across a mass-to-charge (m / z) range of interest is provided. The method includes analyzing some of the precursor ions across the m / z range of interest in the MS1 domain using a first mass analyzer of the tandem mass analyzer operating at a first sensitivity. The method also includes analyzing some of the precursor ions across the m / z range of interest in the MS1 domain using a second mass analyzer of the tandem mass analyzer operating at a second sensitivity, the second sensitivity being higher than the first sensitivity. The analysis in the MS1 domain performed by the second mass analyzer is performed simultaneously with the analysis performed in the MS1 domain by the first mass analyzer. The method also includes combining data from the MS1 analysis performed by the first mass analyzer and the second mass analyzer to identify and / or quantify the precursor ions. The method also includes analyzing some of the precursor ions in the MS2 domain using the second mass analyzer of the tandem mass analyzer.

[0011] According to the method of the first aspect, a tandem mass analyzer is used to perform an analysis in the MS1 domain using a first mass analyzer and a second mass analyzer. The first mass analyzer and the second mass analyzer operate with different sensitivities. In particular, the second mass analyzer operates with a higher sensitivity than the first mass analyzer, in addition to being used to perform an analysis of precursor ions in the MS2 domain. Thus, the MS1 analysis performed by the second mass analyzer is used to complement the data from the MS1 analysis performed by the first mass analyzer. The more sensitive MS1 data acquired by the second mass analyzer can be used in particular to identify lower intensity precursor ions that may not be identified in the MS1 analysis performed by the first mass analyzer. That is, the more sensitive MS1 analysis performed by the second mass analyzer can identify said low abundance precursor ions when the dynamic range of the first mass analyzer is not capable of reliably identifying the relatively low abundance precursor ions.

[0012] By sensitivity, it is understood that the sensitivity of a mass analyzer reflects the intensity of the signal recorded by the mass analyzer for a given amount of ions. Thus, analyzing a given amount of ions with relatively high sensitivity will result in a mass peak having a higher intensity than mass analysis of the same amount of a given ion mass analyzed with lower sensitivity. It will be understood that some mass analyzers may operate at different sensitivities. Furthermore, different types of mass analyzers may be capable of operating in different sensitivity ranges. For example, a TOF mass analyzer may be capable of detecting single ions (i.e. operating at a relatively high intensity), while other mass analyzers, such as an orbital trap mass analyzer, may be capable of operating in a relatively low sensitivity range.

[0013] By performing MS1 and MS2 analysis using a second mass analyzer simultaneously with the MS1 analysis performed by the first mass analyzer, the method according to the first aspect can identify a wider range of precursor ions present in the sample in real time. For example, due to the higher sensitivity of the second mass analyzer, the MS1 data from the second mass analyzer can be used to identify a wider range of precursor ions. This data may be combined with the MS1 data from the first mass analyzer to improve the identification of precursor ions. For example, the combined data can be used to inform or update the precursor ions analyzed in the MS2 domain. For example, in some embodiments, MS1 analyses performed at different sensitivities may also have different mass accuracies and / or different peak acceptances. By combining MS1 data from two different MS1 analyses performed by two mass analyzers operating in parallel, the method of tandem mass spectrometry can provide improved identification of precursor ions, particularly low abundance precursor ions.

[0014] In some embodiments, the first mass analyzer operates with a first sensitivity and a first mass accuracy, and the second mass analyzer analyzes some of the precursor ions in the MS1 domain with a second sensitivity and a second mass accuracy, the second mass accuracy being lower than the first mass accuracy. Thus, the first mass analyzer, e.g., an orbital trap mass analyzer, can be used to perform the MS1 analysis with a relatively low sensitivity but a relatively high mass accuracy. The second mass analyzer (e.g., a TOF mass analyzer) can perform the MS1 analysis in tandem with a lower mass accuracy but a higher sensitivity than the first mass analyzer. Advantageously, by performing the second MS1 analysis with a higher sensitivity, a relatively low abundance precursor ion peak can be detected. In addition, the first MS1 analysis provides relatively high mass accuracy information of the detected precursor ions. Thus, by combining the data from the first mass analyzer and the second mass analyzer, precursor ions can be identified and / or quantified over a wide dynamic range and with improved mass accuracy.

[0015] In some embodiments, data from the MS1 analyses performed by the first and second mass analyzers may be combined to generate a combined list of precursor ion peaks in the MS1 domain. That is, the MS1 analyses performed by the first and second mass analyzers with different sensitivities may each identify a different set of precursor ion peaks in the MS1 domain. In particular, the MS1 analysis performed by the second mass analyzer with higher sensitivity may identify precursor ion peaks corresponding to precursor ions with relatively low intensity that may not have been detected in the MS1 analysis performed by the first mass analyzer. Thus, the combined list of precursor ion peaks may include more precursor ion peaks than are present in the list of precursor ion peaks based on the MS1 analysis performed by the first mass analyzer. Furthermore, the second mass analyzer may not accurately record the peak shapes of precursor ion peaks corresponding to precursor ions with relatively high intensity due to the high sensitivity of the second mass analyzer. Thus, data from the MS1 analysis performed by the first mass analyzer may provide a more accurate depiction of the relatively high intensity precursor ion peaks due to the lower sensitivity of the first mass analyzer. In particular, the combined list of precursor ion peaks in the MS1 domain may provide a more complete list of precursor ion peaks and may also have a more accurate list of mass to charge ratios of the precursor ion peaks in the MS1 domain.

[0016] In some embodiments, the precursor ion peak combination list includes a first set of precursor ion peaks identified from an MS1 ​​analysis performed by a first mass analyzer and a second set of precursor ion peaks identified from an MS1 ​​analysis performed by a second mass analyzer. The combination list may be filtered to remove any precursor ion peaks that are repeated between the first and second sets of precursor ion peaks. Thus, the precursor ion peak combination list may be based on data from MS1 analyses performed at different sensitivity and then filtered to take advantage of the advantages associated with each of the MS1 analyses.

[0017] In some embodiments, the second set of precursor ion peaks is thresholded to remove any precursor ion peaks below a first predetermined intensity level. In some embodiments, the second set of precursor ion peaks may be thresholded to remove any precursor ion peaks above a second predetermined intensity level.

[0018] The second set of precursor ion peaks may therefore be filtered to remove any low intensity peaks in the mass spectrum that may be indistinguishable from noise. The second set of precursor ion peaks may also be filtered to remove any precursor ion peaks having relatively large intensities that are expected to have been recorded by the MS1 analysis performed by the first mass analyser. The second set of precursor ion peaks may therefore be thresholded to focus on identifying precursor ion peaks associated with relatively low abundance precursor ions.

[0019] In some embodiments, the first precursor ion peak produced by the second mass analyzer and the corresponding first precursor ion peak produced by the first mass analyzer are used to calibrate the tandem mass analyzer. The calibration is used to identify any precursor ion peaks that are repeated between the first and second sets of precursor ion peaks. For example, the calibration may be used to determine a correlation between the mass-to-charge m / z ratio of the first precursor ion peak produced by the second mass analyzer and the m / z ratio of the corresponding first precursor ion peak produced by the first mass analyzer. The correlation may then be used to identify other precursor ion peaks that are repeated between the first and second sets of precursor ion peaks.

[0020] In some embodiments, the analysis performed in the MS2 domain is based on precursor ions identified by the MS1 analysis. For example, a combined list of precursor ion peaks may be used to generate a list of precursor ions that are further analyzed in the MS2 domain. It will be appreciated that by identifying a wider range of precursor ions in the MS1 domain, the precursor ions analyzed in the MS2 domain may be more representative of precursor ions present in the sample, particularly since two different mass analyzers operating at different sensitivities are used.

[0021] It will be understood that, according to the present disclosure, references to analyzing precursor ions in the MS2 domain are interpreted to mean that the precursor ions are fragmented before being analyzed by the second mass analyzer, i.e., analyzing precursor ions in the MS2 domain includes fragmenting the precursor ions to produce product ions and analyzing the product ions using the second mass analyzer.

[0022] In some embodiments, the method includes performing multiple analysis cycles using a tandem mass analyzer, each cycle including performing one analysis over the m / z range of interest in the MS1 domain using a first mass analyzer, one analysis over the m / z range of interest in the MS1 domain using a second mass analyzer, and performing an analysis of some of the precursor ions in the MS2 domain using the second mass analyzer. The analyses performed in the MS1 and MS2 domains by the second mass analyzer are performed simultaneously with one analysis in the MS1 domain performed by the first mass analyzer. Thus, the method according to the first aspect may be a tandem mass spectrometry methodology, where the analysis cycles are repeated over a period of time to analyze the precursor ions. In particular, the methodology may be particularly suitable when the precursor ions being analyzed change over time, for example because the precursor ions are provided by an ion source coupled to a chromatography device.

[0023] In some embodiments, the method includes performing multiple analysis cycles using a tandem mass analyzer, each analysis cycle including performing a single analysis of an m / z range of interest in an MS1 ​​domain using a first mass analyzer, subdividing the m / z range of interest into multiple m / z subranges and performing an analysis over each m / z subrange of interest in the MS1 domain using a second mass analyzer, and performing an analysis of some of the precursor ions in an MS2 domain using the second mass analyzer. The analysis performed in the MS1 and MS2 domains by the second mass analyzer is performed simultaneously with the single analysis in the MS1 domain performed by the first mass analyzer.

[0024] According to some embodiments, the analysis cycle may include performing multiple MS1 analyses of m / z subranges of interest using a second mass analyzer. By dividing the mass range of interest into subranges and analyzing them with higher sensitivity using a second mass analyzer, the second mass analyzer can identify precursor ions with relatively low abundance. In particular, by subdividing the mass range of interest and analyzing each subrange separately, the ion current used to populate each m / z subrange can be adjusted. That is, subdividing the m / z range of interest into multiple m / z subranges can improve the identification of precursor ions with relatively low abundance using a second mass analyzer.

[0025] In some embodiments, an analytical cycle may include performing a single analysis across the m / z range of interest in the MS1 domain using a first mass analyzer, and may also include performing multiple analyses across the m / z range of interest in the MS1 domain using a second mass analyzer.

[0026] In some embodiments, data from the MS1 analyses performed by the second mass analyzer may be averaged for each analysis cycle. Thus, an averaged MS1 mass spectrum may be generated using the second mass analyzer. Because the second mass analyzer operates with higher sensitivity than the first mass analyzer, averaging the MS1 mass spectra generated by the second mass analyzer may increase the number of precursor ion peaks detectable in the MS1 data. In particular, precursor ions of relatively low abundance (e.g., about 1-3 precursor ions present per MS1 analysis) may be more reliably detected across multiple MS1 analyses. In some embodiments, at least 5 MS1 analyses, more preferably at least 10 MS1 analyses, may be performed and averaged by the second mass analyzer.

[0027] In some embodiments, averaging the data from the MS1 analysis further includes thresholding the average data to remove precursor ion peaks below a third intensity level. For example, in some embodiments, averaging the MS1 analysis may result in many low intensity precursor ion peaks (e.g., corresponding to a single ion intensity) that may be the result of ion scattering during mass analysis. Such precursor ion peaks are not "true" peaks representing precursor ions, and the accuracy of the average data may be improved by thresholding the average data to remove "false" precursor ion peaks.

[0028] It will be understood that the MS1 analysis performed by the second mass analyzer may be performed at any time during the analysis cycle. For example, in some embodiments, the MS1 analysis performed by the second mass analyzer may be performed near the beginning of each MS1 analysis performed by the first mass analyzer. For example, the MS1 analysis performed by the second mass analyzer may be performed during an initial period of the MS1 analysis performed by the first mass analyzer. In some embodiments, the initial period may be 30% or 20% or less of the total (expected) duration of the MS1 analysis performed by the first mass analyzer. Thus, it will be understood that the initial period extends from the start of the MS1 analysis performed by the first mass analyzer to the end of the initial period (i.e., when 30% of the total (expected) duration of the MS1 analysis has elapsed).

[0029] In some embodiments, it may be advantageous to distribute the MS1 analysis performed by the second mass analyzer over the duration of the MS1 analysis performed by the first mass analyzer. Thus, in some embodiments in which multiple MS1 analyses are performed by the second mass analyzer, the MS1 analyses may be distributed evenly over the duration of the MS1 analysis performed by the first mass analyzer. That is, in some embodiments, multiple MS1 analyses performed by the second mass analyzer may be interleaved with MS2 analyses performed by the second mass analyzer. For example, at least three, five, or seven analyses may be performed in the MS1 domain by the second mass analyzer for each analysis cycle. In some embodiments, the analyses performed in the MS1 domain are interleaved evenly over the duration of the analyses performed in the MS1 domain by the first mass analyzer. By interleaving the MS1 analyses in this manner, the MS1 data obtained by the second mass analyzer can more accurately characterize the changes in the precursor ion being analyzed over time (e.g., due to the elution of a chromatographic peak from a chromatographic separator).

[0030] In some embodiments, each analysis cycle further includes performing a gain control analysis using the first mass analyzer and / or the second mass analyzer. The injection time of each MS1 analysis performed by the first and / or second mass analyzer may be adjusted based on the gain control analysis. Thus, the tandem mass analyzer may be adjusted based on the gain control analysis to control the ion current used to populate the precursor ions used for each MS1 analysis. This may be particularly suitable for the MS1 analysis performed by the second mass analyzer to improve the identification of relatively low abundance precursor ions.

[0031] In some embodiments, the precursor ions to be analyzed are filtered to remove most or all of any singly charged precursor ions. For example, the precursor ions may be filtered by a field asymmetric ion mobility spectrometer (FAIMS). By filtering out singly charged precursor ions, primarily or exclusively multiply charged precursor ions can be analyzed by the method of the first aspect. Multiply charged ions are more useful species to analyze using MS2 analysis, thereby improving the efficiency of the methodology.

[0032] In some embodiments, the precursor ions to be analyzed are provided by an ion source configured to ionize molecules provided from a chromatographic separator. For example, a sample may be provided to a chromatographic separator, where the sample elutes from the chromatographic separator. The eluted sample is then ionized by the ion source, resulting in a stream of precursor ions that are provided to a tandem mass spectrometer for analysis. It will be appreciated that sample molecules eluting from a chromatographic separator may elute at different times depending on the sample molecules present in the sample. Each of the different sample molecules may elute with its own chromatographic peak over a period of time. For example, some chromatographic peaks may have durations of about 1-20 seconds depending on the chromatographic separator and the sample molecules. Thus, the method of tandem mass spectrometry of the first aspect may be performed in some embodiments for the duration of the chromatographic peak. Preferably, the method of tandem mass analysis may be repeated multiple times over the duration of the chromatographic peak, so that the MS1 analysis performed by the first mass analyzer and the second mass analyzer is repeated over the duration of the chromatographic peak. For example, the MS1 analysis performed by the first mass analyzer may preferably be performed at least 2, 3, 5 or 7 times over the duration of the chromatographic peak. Of course, it will be understood that for some particularly fast-eluting chromatographic peaks, there may not be enough time to perform multiple repetitions of the analysis cycle according to the first aspect.

[0033] In some embodiments, data from the MS1 analysis performed by the first mass analyzer and the second mass analyzer may be used to identify a chromatographic peak eluting from the chromatographic separator. In some embodiments, the analysis performed in the MS2 domain by the second mass analyzer may be selected based on the identified chromatographic peak eluting from the mass spectrometer. Thus, the method of the first aspect can more accurately characterize the chromatographic peak eluting from the chromatographic separator.

[0034] In some embodiments, the first mass analyzer is a mass analyzer selected from the group including an orbital trap mass analyzer, a Fourier transform ion cyclotron resonance (FTICR) mass analyzer, and a time-of-flight (TOF) mass analyzer. In some embodiments, the second mass analyzer is a TOF mass analyzer. Thus, in some embodiments, the first mass analyzer and the second mass analyzer may be different mass analyzers. Alternatively, in some embodiments, the first mass analyzer and the second mass analyzer may be the same type of mass analyzer. In either case, each mass analyzer may be capable of operating at a different sensitivity range. The method according to the first aspect operates the two mass analyzers at different sensitivities in order to obtain information about relatively high abundance precursor ions and also about relatively low abundance precursor ions. That is, the method according to the first aspect seeks to obtain information in the MS1 domain about precursor ions that exist over a larger dynamic range than would be possible with the use of a single mass analyzer. For example, in some embodiments, the second mass analyzer may have a larger dynamic range than the first mass analyzer.

[0035] In some embodiments, the first mass analyzer operates in one dynamic range to perform the respective MS1 analysis, and the second mass analyzer operates in a second dynamic range larger than the first dynamic range to perform the respective MS1 analysis, i.e., in addition to the difference in sensitivity, the method according to the first aspect may also operate the mass analyzers in different dynamic ranges to obtain a more complete picture of the precursor ions in the MS1 domain.

[0036] According to a second aspect of the present disclosure, there is provided a tandem mass spectrometer for analyzing precursor ions across a mass-to-charge (m / z) range of interest. The tandem mass spectrometer comprises a first mass analyzer, a second mass analyzer, and a controller. The first mass analyzer is configured to analyze precursor ions in an MS1 ​​domain. The second mass analyzer is configured to analyze precursor ions in an MS1 ​​domain and an MS2 domain. The controller is configured to: causing a first mass analyzer to analyze some of the precursor ions across an m / z range of interest in an MS1 ​​domain at a first sensitivity; causing a second mass analyzer to analyze some of the precursor ions across an m / z range of interest in the MS1 domain at a second sensitivity, the second sensitivity being greater than the first sensitivity; a first mass analyzer analyzing the precursor ions in the MS1 domain at the same time that a second mass analyzer analyzes the precursor ions in the MS1 domain; combining data from the MS1 analyses performed by the first mass analyzer and the second mass analyzer to identify and / or quantify precursor ions; and causing a second mass analyzer to analyze some of the precursor ions in an MS2 domain.

[0037] It will therefore be appreciated that the tandem mass spectrometer of the first aspect is configured to carry out the method of the first aspect, in particular it will be appreciated that the tandem mass spectrometer may include features configured to carry out any of the features of the first aspect.

[0038] In particular, it will be appreciated that in some embodiments the tandem mass spectrometer may comprise a fragmentation device configured to fragment precursor ions to generate product ions, which may be analyzed to provide MS2 data.

[0039] In some embodiments, the tandem mass spectrometer may be configured to receive precursor ions from an ion source. The precursor ions to be analyzed may be provided by an ion source configured to ionize molecules provided from a chromatographic separator. For example, a sample may be provided to a chromatographic separator, where the sample elutes from the chromatographic separator. The eluted sample is then ionized by the ion source, resulting in a stream of precursor ions that are provided to the tandem mass spectrometer for analysis.

[0040] According to a third aspect of the present disclosure there is provided a computer program comprising instructions for causing a tandem mass spectrometer of the second aspect to carry out the steps of the method according to the first aspect.

[0041] According to a fourth aspect of the present disclosure, there is provided a computer readable medium storing the computer program of the third aspect.

[0042] Embodiments of the present disclosure will now be described with reference to the following non-limiting figures. [Brief description of the drawings]

[0043] [Figure 1] 1 shows a schematic diagram of a tandem mass spectrometer according to the present disclosure. [Diagram 2]1 shows a block diagram of a method for tandem mass spectrometry according to an embodiment of the present disclosure. [Diagram 3] 4 shows a further flow chart of a method of tandem mass spectrometry according to an embodiment of the present disclosure. [Figure 4a] 1 shows the MS1 mass spectrum of the calibration solution obtained by an orbital trap mass spectrometer operating at a resolving power of 120,000. [Figure 4b] 1 shows the MS1 mass spectrum of the calibration solution obtained by an orbital trap mass spectrometer operating at a resolving power of 120,000. [Figure 5a] 1 shows the MS1 mass spectrum of the calibration solution obtained by an orbital trap mass spectrometer operating at a resolving power of 240,000. [Figure 5b] 1 shows the MS1 mass spectrum of the calibration solution obtained by an orbital trap mass spectrometer operating at a resolving power of 240,000. [Figure 6a] The MS1 mass spectrum of the calibration solution obtained by time-of-flight mass spectrometry (1 time) is shown. [Figure 6b] The MS1 mass spectrum of the calibration solution obtained by time-of-flight mass spectrometry (1 time) is shown. [Figure 7a] 1 shows the average MS1 mass spectrum of a calibration solution obtained from ten MS1 ​​mass analyses performed with a time-of-flight mass spectrometer. [Figure 7b] 1 shows the average MS1 mass spectrum of a calibration solution obtained from ten MS1 ​​mass analyses performed with a time-of-flight mass spectrometer. [Figure 8] 1 shows a bar graph illustrating the number of distinct precursor ion peaks identified using MS1 analysis of calibration solutions for different mass analyzers and mass spectrometry techniques. [Figure 9] 1 shows a graph illustrating the number of precursor ion peaks detected using a TOF mass analyzer at varying first intensity levels (thresholds). [Figure 10] FIG. 1 shows a block diagram of a method for tandem mass spectrometry according to a further embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] FIG. 1 shows a schematic diagram of a tandem mass spectrometer 10 suitable for carrying out a method according to an embodiment of the present invention.

[0045] In FIG. 1, a sample to be analyzed is delivered (e.g., from an autosampler) to a chromatography device (not shown in FIG. 1), such as a liquid chromatography (LC) column. One such example of an LC column is the Thermo Fisher Scientific, Inc. ProSwift® monolithic column, which provides high performance liquid chromatography (HPLC) by forcing the sample with a mobile phase under high pressure through a stationary phase of irregular or spherical particles that make up the stationary phase. In an HPLC column, sample molecules elute at different rates depending on the extent of their interaction with the stationary phase.

[0046] Thus, the sample molecules separated by liquid chromatography are then ionized using an ion source to generate precursor ions, which in the embodiment of Figure 1 is an electrospray ionization source (ESI source) 20 at atmospheric pressure.

[0047] Precursor ions generated by the ESI source 20 then enter the vacuum chamber of the tandem mass spectrometer 10 and are guided by the capillary 25 to the RF-only S-lens 30. The precursor ions are focused by the S-lens 30 into the injection flatapole 40, which injects the precursor ions into the bent flatapole 50, which has an axial field. The bent flatapole 50 guides the (charged) precursor ions along a curved path through it, but unwanted neutral molecules such as entrained solvent molecules are not guided along the curved path and are lost.

[0048] An ion gate (TK lens) 60 is disposed at the distal end of the bent flatapole 50 and controls the passage of precursor ions from the bent flatapole 50 to a downstream mass selector in the form of a quadrupole mass filter 70. Alternatively, the S-lens 30 may operate as the ion gate and the ion gate (TK lens) 60 may be an electrostatic lens. The quadrupole mass filter 70 is typically, but not necessarily, segmented and acts as a bandpass filter, allowing the passage of selected mass numbers or limited mass ranges while excluding precursor ions of other mass-to-charge ratios (m / z). The mass filter may also be operated in an RF-only mode that is not mass selective, i.e., transmitting precursor ions of substantially all m / z. For example, the quadrupole mass filter 70 may be controlled by a controller (not shown in FIG. 1) to select a range of mass-to-charge ratios to pass precursor ions that are allowed to pass, while filtering out (not allowing to pass) other ions in the precursor ion stream. Thus, the mass filter 70 may filter precursor ions based on the m / z range of interest.

[0049] Although a quadrupole mass filter 70 is shown in Figure 1, those skilled in the art will appreciate that other types of mass selection devices may be suitable for selecting precursor ions within the mass range of interest, such as an ion separator as described in US Patent Publication No. 2015287585 (A), an ion trap as described in WO 2013076307 (A), an ion mobility separator as described in US Patent Publication No. 2012256083 (A), an ion gate mass selection device as described in WO 2012175517 (A), or a charged particle trap as described in US Patent Publication No. 799223, the contents of which are incorporated herein by reference in their entireties. Those skilled in the art will appreciate that other methods of selecting precursor ions according to ion mobility, differential mobility, and / or transverse modulation may also be suitable.

[0050] Separation of multiple precursor ions of different masses or mass ranges can also be performed in the ion trap using a method known as synchronous precursor scanning (SPS). Furthermore, in some embodiments, more than one ion selection or mass selection device may be provided. For example, a further mass selection device may be provided downstream of the fragmentation chamber 120. In this way, multiple mass selection devices may be provided, if desired, for MS selection. 3 or M.S. n A scan may be performed (typically using a TOF mass analyzer for mass analysis).

[0051] The tandem mass spectrometer 10 may operate in various modes of operation to perform analysis of precursor ions in the MS1 domain and / or the MS2 domain. In a first mode of operation, a first mass analyzer (the orbital trap mass analyzer 110) may be used to analyze precursor ions in the MS1 domain.

[0052] In a first mode of operation, precursor ions may pass through a quadrupole exit lens / split lens arrangement 80 and proceed to a first transfer multipole 90. The first transfer multipole 90 directs the mass filtered ions from the quadrupole mass filter 70 into a curved linear ion trap (C-trap) 100. The C-trap (first ion trap) 100 has longitudinally extending curved electrodes that are supplied with an RF voltage and end caps that are supplied with a DC voltage. As a result, a potential well is obtained that extends along the curved longitudinal axis of the C-trap 100. In the first mode of operation, a DC end cap voltage is set on the C-trap so that ions arriving from the first transfer multipole 90 are trapped in the potential well of the C-trap 100 and cooled there. The cooled precursor ions reside in a cloud near the bottom of the potential well of the C-trap 100. The injection time (IT) of the ions into the C-trap determines the number of precursor ions (ion populations) that are subsequently released from the C-trap 100. From the C-trap 100, the precursor ions may be directed to different parts of the tandem mass spectrometer 10 depending on the analysis to be performed.

[0053] When precursor ions are to be analyzed by the orbital trap mass analyzer 110 (first mass analyzer), they are ejected orthogonally from the C-trap towards the orbital trap mass analyzer 110. As shown in FIG. 1, the orbital trap mass analyzer 110 may be an Orbitrap® mass analyzer sold by Thermo Fisher Scientific, Inc. The orbital trap mass analyzer 110 has an off-center injection aperture through which precursor ions are injected as coherent packets into the orbital trap mass analyzer 110. The precursor ions are then trapped in the orbital trap mass analyzer 110 by a hyperlogarithmic electric field and undergo a longitudinal back and forth motion while orbiting around an inner electrode.

[0054] The axial (z) component of motion of an ion packet in the orbital trap mass analyzer 110 is defined as (more or less) simple harmonic motion, with the angular frequency in the z direction related to the square root of the mass-to-charge ratio of a given ion species. Thus, precursor ions separate over time according to their mass-to-charge ratio.

[0055] Precursor ions in the orbital trap mass analyzer 110 are detected using an imaging current detector (not shown), which produces a "transient" in the time domain that contains information about all ion species as they pass through the imaging current detector. The transient is then subjected to a Fast Fourier Transform (FFT), which produces a series of peaks in the frequency domain. From these peaks, a mass spectrum can be produced that represents the presence / intensity of ions versus m / z.

[0056] In the above configuration, precursor ions within the mass range of interest (selected by quadrupole mass filter 70) are analyzed without fragmentation by the orbital trap mass analyzer 110. The resulting mass spectrum is denoted MS1.

[0057] Although an orbital trap mass analyzer 110 is shown in FIG. 1, it will be understood that other mass analyzers may be used as the first mass analyzer according to embodiments of the present disclosure. For example, a Fourier transform ion cyclotron resonance (FTICR) mass analyzer may be used as the first mass analyzer to analyze precursor ions in the MS1 domain. Mass analyzers such as orbital trap mass analyzers and ion cyclotron resonance mass analyzers may be similarly used in the present invention, even if they use other types of signal processing other than Fourier transform to obtain mass spectral information from transient signals (see, for example, WO 2013 / 171313, Thermo Fisher Scientific). In some embodiments, a TOF mass analyzer may be used instead of the orbital trap mass analyzer 110 to analyze precursor ions in the MS1 domain.

[0058] In a second mode of operation of the tandem mass spectrometer 10, precursor ions may be analyzed in the MS1 domain by the TOF mass analyzer 150 (second mass analyzer). The precursor ions analyzed by the second mass analyzer may be mass filtered by the quadrupole mass filter 70. Thus, the precursor ions may be filtered to include precursor ions from an m / z range of interest, or an m / z sub-range of interest.

[0059] For the TOF mass analyzer 150 to analyze precursor ions, the precursor ions may travel a path from the quadrupole exit lens / split lens arrangement 80 and the first transfer multipole 90 to the C-trap 100 and then through the C-trap 100 into the fragmentation chamber 120. Thus, the C-trap 100 can effectively operate as an ion guide in the second mode of operation. Alternatively, the cooled ions in the C-trap 100 can be axially ejected from the C-trap into the fragmentation chamber 120. When precursor ions are analyzed in the MS1 domain, the fragmentation chamber 120 is not used to fragment the precursor ions. Thus, the precursor ions may continue through the fragmentation chamber 120 and be ejected from the fragmentation chamber 120 at the axial end opposite the C-trap 100. Thus, the fragmentation chamber 120 can also effectively operate as an ion guide in the second mode of operation.

[0060] The ejected precursor ions proceed to a second transfer multipole 130, which directs the precursor ions from the fragmentation chamber 120 to an extraction trap (second ion trap) 140. The extraction trap 140 is a radio frequency voltage controlled trap containing a buffer gas. For example, a suitable buffer gas is 5×10 -4 mBar~1×10 -2 The second ion trap is preferably nitrogen at a pressure in the mBar range. The extraction trap has the ability to quickly switch off the applied RF voltage and apply a DC voltage to extract the trapped precursor ions. A suitable planar extraction trap, also called a linear ion trap, is further described in U.S. Pat. No. 9,548,195 (B2). Alternatively, a second C trap may also be suitable for use as the second ion trap.

[0061] The extraction trap 140 is provided to form ion packets of precursor ions prior to injection into the TOF mass analyzer 150. The extraction trap 140 accumulates fragmented ions before the precursor ions are injected into the TOF mass analyzer 150.

[0062] Although an extraction trap 140 (ion trap) is shown in the embodiment of FIG. 1, those skilled in the art will appreciate that other methods of forming ion packets of precursor ions are similarly suitable for the present invention. For example, relatively slow movement of ions through a multipole can be used to affect a bunching of ions that can then be emitted as a single packet into a TOF mass analyzer. Alternatively, orthogonal displacement of precursor ions may be used to form packets. Further details of these alternative methods can be found in U.S. Patent Application Publication No. 20030001088(A), which describes a traveling wave ion bunching method, the contents of which are incorporated herein by reference.

[0063] In FIG. 1, the TOF mass analyzer 150 shown is a multi-reflecting time-of-flight mass analyzer (mr-TOF). The TOF mass analyzer 150 is built around two opposing ion mirrors 160, 162 that are elongated in the drift direction. The mirrors face each other in a direction perpendicular to the drift direction. The extraction trap 140 injects ions into the first mirror 160, which then oscillates between the two mirrors 160, 162. The ejection angle of the ions from the extraction trap 140 and additional deflectors 170, 172 allows the energy of the ions in the drift direction to be controlled so that as they oscillate, they are directed down the length of the mirrors 160, 162, creating a zigzag trajectory. The mirrors 160, 162 themselves are tilted relative to each other to create a potential gradient that slows the drift velocity of the ions and causes them to reflect in the drift dimension and focus onto the detector 180. Tilting the opposing mirrors typically has the negative side effect of changing the time of ion oscillation down the drift dimension. This is compensated for by the stripe electrodes 190 (acting as compensation electrodes) which change the flight potential in a portion of the inter-mirror space to change the length of the opposing mirrors 160, 162 to be shorter. The combination of changing the width of the stripe electrodes 190 and changing the distance between the mirrors 160, 162 allows for reflection and spatial focusing of the ions onto the detector 180, as well as maintaining good temporal focusing. An mr-TOF suitable for use in the present invention is further described in U.S. Patent Application Publication No. 2015028197(A), the contents of which are incorporated herein by reference in their entirety.

[0064] The precursor ions stored in the extraction trap 140 are injected as packets of ions into a TOF mass analyzer 150 (a second mass analyzer). The time it takes for the precursor ions to reach a detector 180 is recorded by the TOF mass analyzer 150 and used to generate an MS1 ​​mass spectrum of the precursor ions.

[0065] The MS1 analysis performed by the TOF mass analyzer 150 is performed at a second sensitivity. According to an embodiment of the present disclosure, the TOF mass analyzer performs the MS1 analysis at a second sensitivity that is higher than the first sensitivity of the MS1 analysis performed by the orbital trap mass analyzer 110. For example, the TOF mass analyzer may be configured to operate at a single ion sensitivity. That is, the TOF mass analyzer 150 may be configured to generate a signal associated with a single ion arriving at the detector of the TOF mass analyzer 150 with sufficient intensity to be distinguishable from background noise.

[0066] Those skilled in the art will be aware of the sensitivity of different mass analyzers and the effect of different operating conditions on the sensitivity of a given mass analyzer. For example, according to the present disclosure, one measure of the sensitivity of a mass analyzer may be the minimum number of ions required in an analysis to produce a peak that is distinguishable from background noise. As mentioned above, the TOF mass analyzer 150 may operate with single ion sensitivity (i.e., a single ion of a given mass may produce a corresponding peak). Other mass analyzers (e.g., the orbital trap mass analyzer 110) may require the presence of multiple ions to produce a detectable peak (see, e.g., the discussion of Figures 4a, 5b, 5a, and 5b below).

[0067] It will be appreciated that the TOF mass analyser also performs MS1 analysis with an associated second (mass) accuracy.

[0068] In a third mode of operation of the tandem mass spectrometer 10, a TOF mass analyzer 150 (a second mass analyzer) may be used to analyze precursor ions in the MS2 domain.

[0069] For analysis of the precursor ions in the MS2 domain, some of the precursor ions may be transmitted from the quadrupole mass filter 70 to the fragmentation chamber 120 in a manner similar to the second mode of operation described above. The transmitted precursor ions may be mass selected by the quadrupole mass filter 70 to include targeted precursor ion species, i.e. m / z subranges of interest, as described in more detail below.

[0070] The fragmentation chamber 120 is a higher energy collisional dissociation (HCD) device in the tandem mass spectrometer 10 of Figure 1. A collision gas may be supplied to the fragmentation chamber 120. When precursor ions are fragmented, the kinetic energy of the precursor ions may be increased so that the precursor ions that reach the fragmentation chamber 120 and collide with collision gas molecules produce fragment ions by fragmentation of the precursor ions.

[0071] Although an HCD fragmentation chamber 120 is shown in FIG. 1, other fragmentation devices using methods such as collision induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), photodissociation, etc. may be used instead.

[0072] The fragmented ions may be ejected from the fragmentation chamber 120 at the opposite axial end from the C-trap 100. The ejected fragmented ions pass through the second transfer multipole 130 and proceed to the extraction trap 140 where they are accumulated. The fragmented ions may then be injected into the TOF mass analyzer 150 as described above.

[0073] It will be appreciated that in some embodiments the first mass analyzer (the orbital trap mass analyzer 110) and the second mass analyzer (the TOF mass analyzer 150) may operate simultaneously, i.e. the tandem mass spectrometer 10 may operate in the first mode of operation simultaneously with the second or third modes of operation.

[0074] The tandem mass spectrometer 10 may be under the control of a controller configured to, for example, control the timing of ejection of trapped components, set appropriate potentials on electrodes such as the quadrupole to focus and filter ions, acquire mass spectral data from the orbital trap device 110, acquire mass spectral data from the orbital trap mass analyzer 110 and the TOF mass analyzer 150, control the sequence of MS1 and MS2 scans, etc. It will be appreciated that the controller may comprise a computer capable of operating according to a computer program including instructions that cause the tandem mass spectrometer to carry out steps of a method according to the present invention.

[0075] It should be understood that the particular arrangement of components illustrated in Figure 1 is not required for the methods described herein, and indeed other configurations are suitable for carrying out the methods of embodiments of the present invention.

[0076] An embodiment of the method will now be described with reference to Figures 2 and 3, where sample molecules are delivered from a liquid chromatography (LC) column (as shown in Figure 1) as part of the exemplary apparatus described above.

[0077] In an embodiment of the present invention, the sample molecules are delivered from an LC column. Thus, the method according to the present invention can obtain data on the sample over a duration corresponding to the duration of one or more chromatographic peaks of the sample delivered from the LC column. Thus, the controller can be configured to perform an analysis cycle according to an embodiment of the present disclosure over the duration of the chromatographic peaks. In some embodiments, an analysis cycle according to an embodiment of the present disclosure may be repeated multiple times as the sample elutes from the LC column.

[0078] As shown in FIG. 2, an orbital trap mass analyzer 110 (denoted "Orbitrap") is used to perform multiple MS1 scans across the m / z range of interest. For example, as shown in FIG. 2, the m / z range of interest to be analyzed is 300-1100 m / z. As shown in FIG. 2, for each analysis cycle, the orbital trap mass analyzer 110 is used to perform one MS1 analysis of precursor ions within the m / z range of interest (300-1100 m / z). The precursor ions to be analyzed may be fed through a quadrupole mass filter 70, which filters the precursor ions based on the m / z range of interest.

[0079] As shown in FIG. 2, the MS1 analysis performed by the orbital trap mass analyzer may be performed for a long period of time (relative to the duration of the MS1 and MS2 analyses performed by the second mass analyzer). Thus, the MS1 analysis performed by the orbital trap mass analyzer may have a relatively high mass accuracy and / or resolution relative to the MS1 analysis performed by the TOF mass analyzer 150. In some embodiments, the MS1 analysis performed by the orbital trap mass analyzer may have a resolution of at least 80,000, 100,000, 120,000, 150,000, 200,000, or 240,000. For example, in the embodiment of FIG. 2, the MS1 analysis performed by the orbital trap mass analyzer may have a duration of at least 250 ms, with the resulting MS1 spectrum having a resolution of about 120,000. In other embodiments, the MS1 analysis performed by the orbital trap mass analyzer 150 may have a duration of about 512 ms and a resolution of 240,000.

[0080] Generally, increasing the resolution at which an MS1 ​​analysis is performed reduces the noise baseline of the analysis. For example, when the orbital trap mass analyzer 110 of FIG. 1 operates at a resolution of about 120,000, the noise baseline would be equivalent to about 3.8 ions. When operated at a resolution of about 240,000, the noise baseline would be about 2.6 ions. Thus, it will be appreciated that several ions of any single analyte must be present to be detected by the orbital trap mass analyzer 110, even when the orbital trap mass analyzer operates at a relatively high resolution.

[0081] By using an orbital trap mass analyzer 110 (or any other relatively accurate mass analyzer), the MS1 analysis using the first mass analyzer is performed with high mass accuracy. Preferably, the MS1 analysis is performed with a mass accuracy of less than 5 parts per million (ppm), or more preferably less than 3 ppm. The parts per million mass accuracy Δm of the mass analyzer is determined by the measured mass m of an ion as follows: i and the actual mass of the ion m a Divide the difference between the mass and the actual mass of the ion by 10 6 can be determined as a multiplication of

[0082]

number

[0083] Examples of mass spectra obtained by the orbital trap mass analyzer 110 are shown in Figures 4a, 4b, 5a, and 5b. In each case, the sample analyzed is Pierce® FlexMix® Calibration Solution manufactured by Thermo Fisher Scientific®. This calibration solution is a mixture of a mixture of 16 highly pure ionizable components with a mass range of 50 m / z to 3000 m / z and is designed for calibration of both positive and negative ionization. Figures 4a and 4b show mass spectra obtained by the orbital trap mass analyzer 110 of Figure 1 when performing an MS1 ​​analysis of the sample (calibration solution) at a resolving power of 120,000. Figure 4a shows a relatively high abundance precursor ion peak identified in the analysis, and Figure 4b shows a zoomed-in view of the precursor ion peak of Figure 4a with a relative abundance of less than 5%.

[0084] Figures 5a and 5b show mass spectra obtained by the orbital trap mass analyzer 110 of Figure 1 when performing MS1 analysis of a sample (calibration solution) at a resolving power of 240,000. Figure 5a shows a relatively high abundance precursor ion peak identified in the analysis, and Figure 5b shows a zoomed-in view of the precursor ion peak of Figure 5a having a relative abundance of less than 5%.

[0085] It will be appreciated from Figures 4a, 4b, 5a, and 5b that by increasing the resolution of the orbital trap mass analyzer 110, more precursor ion peaks can be detected in the MS1 domain. For example, as seen in the tests of Figures 4a and 4b, when the orbital trap mass analyzer 110 was operated at a resolution of 120,000, it had sufficient sensitivity to distinguish a precursor ion peak caused by about 3.8 precursor ions from the baseline noise. At a resolution of 240,000 (Figures 5a and 5b), the orbital trap mass analyzer has the sensitivity to distinguish a precursor ion peak corresponding to about 2.6 ions from the baseline noise. It will therefore be appreciated that even when the orbital trap mass analyzer 110 is operated at a relatively high resolution and a relatively high sensitivity, several ions of any single analyte must still be present to be reliably detected by the orbital trap mass analyzer 110. That is, it is generally not practical / possible to detect precursor ion peaks associated with a single precursor ion (single ion sensitivity) using an orbital trap mass analyzer 110. To the extent that single ion sensitivity can be achieved, MS1 analysis using such an orbital trap mass analyzer 110 may require significant transient or other time-consuming ion processing steps, and the resulting MS1 analysis would not easily fit into a typical mass spectrometry workflow. For example, it would be preferable to be able to perform multiple mass analyses on a time scale that is compatible with chromatographic peaks eluting from a chromatography system.

[0086] In parallel with the MS1 analysis performed by the orbital trap mass analyzer 110, one or more MS1 scans are performed by a second mass analyzer (TOF mass analyzer 150), for example as shown in Figure 2. Multiple MS2 scans are also performed by the TOF mass analyzer 150. As shown in Figure 2, simultaneously with the MS1 analysis performed by the orbital trap mass analyzer 110, the MS1 and MS2 analyses are performed by the TOF mass analyzer 150.

[0087] To perform an MS1 ​​scan using the TOF mass analyzer 150, precursor ions are transported from the ion source 20 to the extraction trap 140 as described above.

[0088] In the embodiment of Figure 2, multiple MS1 analyses are performed by TOF mass analyzer 150, with each MS1 analysis being performed at a different m / z subrange within the mass range of interest (300-1100 m / z). Thus, the mass range of interest is divided into multiple m / z subranges. In the embodiment of Figure 2, each m / z subrange has an m / z range of 200 m / z.

[0089] To perform a single MS1 analysis of a portion of the mass range, sample molecules from the LC column (e.g., molecules of a sample solution) are ionized and injected into the tandem mass spectrometer 10 in a manner similar to the process for performing an MS1 ​​analysis using the orbital trap mass analyzer 110.

[0090] When precursor ions reach the quadrupole mass filter 70 for MS1 analysis, the quadrupole mass filter 70 is controlled by the controller to filter the precursor ions according to the m / z subrange being analyzed. In some embodiments, each precursor m / z subrange may have an m / z range of at least 20, 50, 100, 200, 300, 400, or 500. Thus, the number of m / z subranges analyzed will depend on the m / z range of each m / z subrange and the size of the m / z range of interest. For example, in some embodiments, the m / z range of interest may be divided into at least 3, 4, 5, 7, or 10 m / z subranges. The advantage of having multiple TOF MS1 analyses of different m / z subranges is to maximize the dynamic range of each MS1 analysis. Thus, the multiple MS1 analyses performed by the TOF mass analyzer 150 can detect relatively low abundance precursor ion species that cannot be detected in the MS1 analyses performed by the orbital trap mass analyzer 110 (e.g., due to the lower dynamic range of the orbital trap mass analyzer 110). That is, the TOF mass analyzer can be operated to perform MS1 analyses having a higher dynamic range than the MS1 analyses performed by the orbital trap mass analyzer.

[0091] According to the present disclosure, the dynamic range of a mass analyzer can be the ratio of the maximum detectable signal of the mass analyzer to the minimum detectable signal of the mass analyzer. One way to characterize the dynamic range of a mass analyzer can be the intensity ratio of a peak having 95% intensity of the detection range of the mass analyzer to a peak having 5% intensity of the detection range of the mass analyzer.

[0092] In some embodiments, the TOF MS1 analysis may have different sizes of precursor m / z subranges. For example, in some embodiments, the mass range of interest may be divided into a predetermined number of subranges based on a previous analysis. For example, the previous analysis may be a previous MS1 analysis or a previous gain control analysis (described below). The previous analysis may indicate one or more precursor ion species having relatively high abundance (e.g., the highest abundant precursor ion, or the top few (e.g., three) most abundant precursor ions). Thus, the TOF MS1 analysis may subdivide the m / z range of interest such that the identified precursor ion species are excluded from some or all of the precursor m / z subranges. It will be appreciated that the relatively high abundance precursor ion species are identified in the MS1 analysis performed by the orbital trap mass analyzer 110. Thus, the m / z subranges of the MS1 analysis performed by the TOF mass analyzer 150 may be directed to identifying lower abundance precursor ions. By excluding relatively high abundance precursor ion species from one or more m / z subranges, the dynamic range of the TOF mass analyzer can be further directed towards detecting low abundance precursor ion species.

[0093] The precursor ions (in the filtered m / z subrange) pass from the quadrupole mass filter 70 to the C-trap 100 as described above for the MS1 scan. The controller then controls the C-trap 100 to pass the precursor ions axially towards the fragmentation chamber 120 and into the extraction trap 140. When the precursor ions are analyzed in the MS1 domain, they are not fragmented in the fragmentation chamber 120. The precursor ions are accumulated in the extraction trap 140 prior to analysis by the TOF mass analyzer 150. The precursor ions may be accumulated in the extraction trap 140 for a predetermined time.

[0094] The precursor ions are then injected from the extraction trap 140 into the TOF mass analyzer 150. Pre-accumulation of the precursor ions in the extraction trap 140 allows the precursor ions to be injected into the TOF mass analyzer 150 as packets. The ion packets travel along the flight path of the TOF mass analyzer before being detected by the detector 180. The different arrival times of the precursor ions within the packet allow an MS1 ​​mass spectrum to be generated for the packet of precursor ions. In the case of the TOF of FIG. 1, the length of the flight path of the TOF mass analyzer 150 combined with the time resolution of the detector allows the TOF mass analyzer to perform MS1 analysis with a resolution of approximately 40,000 to 100,000 depending on the intensity of the analyte peak.

[0095] Each MS1 analysis performed by the TOF mass analyzer 150 has a duration that is largely determined by the time it takes to transport an ion from the quadrupole to the extraction trap 140 and onward to the detector (i.e., the ion's time of flight). For example, in the embodiment of FIG. 2, each TOF MS1 analysis has a duration of about 5 ms. It will thus be appreciated that each MS1 analysis performed by the second mass analyzer (TOF mass analyzer 150) may have a duration of 15% or less, preferably 10% or less, and more preferably 5% or less, of the duration of an MS1 ​​scan performed by the first mass analyzer (orbital trap mass analyzer 110).

[0096] As shown in Figure 2, the TOF mass analyzer 150 sequentially performs an MS1 ​​analysis of each m / z subrange. Following the MS1 analysis, the TOF mass analyzer then performs multiple MS2 analyses. The TOF mass analyzer 150 may also perform MS2 analyses in the remainder of the analysis cycle. The nature of the MS2 analyses performed depends on the type of analysis performed by the tandem mass spectrometer, as will be described in more detail below.

[0097] As an example, Figures 6a and 6b show graphs of mass spectra obtained over the m / z range of 380-980 m / z following MS1 analysis of a sample (calibration solution) by TOF mass analyzer 150. Figure 6a shows all peaks recorded by TOF mass analyzer 150, and Figure 6b shows a zoomed-in view of a precursor ion peak of Figure 6a having a relative abundance of less than 5%. The mass spectra of Figures 6a and 6b show all precursor ion peaks detected following one MS1 analysis ("single" analysis). It will be appreciated that, compared to the MS1 analysis performed by the orbital trap mass analyzer 110, the MS1 analysis of Figures 6a and 6b captures more low abundance analyte peaks than the 240K resolution mass spectra of Figures 5a and 5b due to the single ion level detection characteristics of the TOF mass analyzer 150.

[0098] Some of the precursor ion peaks recorded in the mass spectra of Figures 6a and 6b may be false positives resulting from, for example, random collisions of the precursor ion with gas during acceleration into the analyzer. One way to improve the mass spectrum is to average multiple mass spectra obtained using the TOF mass analyzer 150 and apply an appropriate threshold to remove noise. Thus, in some embodiments, the TOF mass analyzer 150 may be configured to perform multiple MS1 analyses of the mass range of interest. That is, the TOF mass analyzer 150 repeats the MS1 analysis of the mass range of interest multiple times. For example, the MS1 analysis may be performed at least five times, preferably at least ten times. The data from the multiple MS1 analyses performed by the TOF mass analyzer may then be averaged together to generate an average MS1 spectrum from which the precursor ion peaks may be detected.

[0099] For example, Figures 7a and 7b show graphs of average mass spectra obtained over the m / z range of 380-980 m / z by performing 10 MS1 analyses of a sample (calibration solution) using TOF mass analyzer 150. Figure 7b shows a zoomed-in view of a precursor ion peak in Figure 7a with a relative abundance of less than 5%. To show the effect of averaging the MS1 analyses, the same Pierce® FlexMix® sample is used to generate the precursor ions analyzed in Figures 6a and 6b and analyzed in Figures 7a and 7b. As can be seen from Figures 7a and 7b, the number of precursor ion peaks detected is increased compared to Figures 6a and 6b.

[0100] In addition to performing MS1 analyses using the TOF mass analyzer 150, the TOF mass analyzer can be used to perform MS2 analyses.

[0101] To perform an MS2 analysis in the TOF mass analyzer 150, sample molecules from the LC column are ionized and injected into the tandem mass spectrometer 10 in a manner similar to the process for performing an MS1 ​​analysis.

[0102] Precursor ions travel from the ion source 20 to a quadrupole mass filter 70. The precursor ions in the quadrupole mass filter 70 may be mass selected and the mass selected precursor ions are then transferred to the fragmentation chamber 120 for fragmentation. It will be appreciated that the precursor ions may be mass selected based on a variety of criteria depending on the type of analysis to be performed.

[0103] For example, in some embodiments where data-dependent analysis (DDA) is being performed, a precursor ion peak combination list (i.e., an inclusion list) may be generated based on the analysis of precursor ions performed in the MS1 domain. For each precursor ion on the precursor ion peak combination list, the precursor ions may be filtered to select precursor ions having a corresponding m / z and exclude precursor ions having a different m / z. For example, the quadrupole mass filter 70 may be configured to mass select precursor ions using a relatively narrow mass selection window of 5 amu or less, preferably 2 amu or less, and more preferably 0.7 amu or less, centered on the m / z of a precursor ion peak of interest included in the precursor ion peak combination list.

[0104] The mass-selected precursor ions are then transferred to the fragmentation chamber 120 where they are fragmented to produce fragment ions related to the precursor ion of interest. The fragment ions are then transferred to the extraction trap 140 and to a TOF mass analyzer 150 which determines the m / z of the fragment ions.

[0105] Each MS2 analysis performed by the TOF mass analyzer 150 may have a duration similar to the duration of an MS1 ​​scan performed by the TOF mass analyzer 150. For example, in the embodiment of FIG. 2, each MS2 analysis performed by the TOF mass analyzer 150 may have a duration of about 5 ms.

[0106] The tandem mass spectrometer 10 is capable of performing multiple MS2 analyses using a TOF mass analyzer in parallel with an MS1 ​​analysis performed by the orbital trap mass analyzer 110, as will be described in more detail below.

[0107] In some embodiments, the tandem mass analyzer 10 may also be configured to perform a gain control analysis (also known as automatic gain control). The gain control analysis is configured to determine a precursor ion current. For example, the amount of sample molecules eluting from a chromatography device may vary over time (e.g., by chromatographic peaks), resulting in associated fluctuations in the precursor ion current. The gain control analysis may be performed to determine the precursor ion current at a given time, and the injection time of the precursor ions into the C-trap 100 or extraction trap 140 may be adjusted by the controller. For example, the injection time may be adjusted to ensure that a relatively consistent amount of ions is analyzed in each MS1 analysis. The gain control analysis may be performed by the first mass analyzer (the orbital trap mass analyzer 110) or the second mass analyzer (the TOF mass analyzer 150). Thus, the gain control analysis may be a process to calibrate the number of precursor ions present in an MS1 ​​or MS2 analysis.

[0108] 2, a gain control analysis (referred to as an AGC prescan) may be performed by the TOF mass analyzer 150 simultaneously with the MS1 analysis performed by the first mass analyzer (the orbital trap mass analyzer). Thus, the gain control analysis may be used to adjust the accumulation time in the extraction trap 140 of the TOF mass analyzer 150 to ensure that a consistent number of precursor ions are analyzed in each subsequent MS1 analysis performed by the TOF mass analyzer 150. In some embodiments, an AGC prescan may be performed prior to performing an MS2 analysis to ensure that a consistent number of ion species are analyzed in each MS2 analysis.

[0109] In the embodiment of FIG. 2, the flow chart illustrates a method of performing a TOF gain control analysis, then a TOF MS1 analysis, and then a TOF MS2 analysis. Of course, in other embodiments, the analyses performed by the TOF mass analyzer 150 may be performed in any order. For example, the TOF mass analyzer 150 may be configured to perform a gain control analysis at any time during the method. That is, the TOF mass analyzer 150 may in some embodiments perform a gain control analysis clustered with the MS1 scan, but this need not be the case, and may be, for example, interspersed among the MS2 scan, or triggered from a regular sequence due to the observation of a chromatographic peak revealed by another scan, such as an AGC scan.

[0110] As mentioned above, an ion packet may be traveling through the TOF mass analyzer 150 while the next ion packet is being accumulated in the extraction trap 140. Thus, the TOF mass analyzer may be used to perform multiple MS1 and / or MS2 analyses simultaneously with the MS1 analysis performed by the orbital trap mass analyzer 110.

[0111] In the embodiment of FIG. 2, the TOF mass analyzer 150 analyzes precursor ions over an m / z range of interest using multiple MS1 analyses. As shown in FIG. 2, the m / z range of interest is 300-1100 m / z. In the MS1 analyses performed by the TOF mass analyzer 150, the m / z range of interest is subdivided into multiple m / z subranges, and an MS1 ​​analysis is performed over each m / z subrange of interest. By subdividing the m / z range of interest, the TOF mass analyzer 150 can adjust parameters of the TOF mass analyzer 150 based on the m / z subranges to improve the sensitivity of the TOF mass analyzer 150 to the m / z subranges being analyzed. Thus, subdividing the m / z range of interest for the MS1 analyses performed by the TOF mass analyzer 150 can improve detection of low abundance precursor ions in the MS1 domain.

[0112] It will be appreciated that the m / z range of interest may be subdivided according to various criteria that may be specified by a user. For example, the m / z range of interest may be subdivided into at least three, four, five, seven, or nine m / z subranges. In some embodiments, the m / z range of interest may be divided evenly among the m / z subranges of interest. In some embodiments, the user may specify one or more relatively narrow m / z subranges to target detection of precursor ions having a particular m / z. In other embodiments, each m / z subrange may have an m / z range of, for example, about 50 m / z, 100 m / z, 150 m / z, 200 m / z, or 300 m / z, with the number of m / z subranges being adjusted based on the m / z range of interest.

[0113] Each MS1 analysis performed by the TOF mass analyzer 150 in the embodiment of Figure 2 may have a duration of about 5 ms. It will therefore be appreciated that several MS1 analyses may be performed by the TOF mass analyzer 150 simultaneously with an MS1 ​​analysis performed by the orbital trap mass analyzer 110.

[0114] In addition to the method outlined with respect to Figure 2 above, a method 200 of tandem mass spectrometry is provided. Figure 3 shows a flow chart of the method 200 of tandem mass spectrometry according to an embodiment of the present disclosure. It will be understood that the blocks in the flow chart of Figure 3 correspond to similarly named blocks shown in the block diagram of Figure 2. Thus, the descriptions of the various steps of Figure 2 provided above may also be applied to the steps of method 200 further described below.

[0115] As shown in Figure 3, method 200 includes performing 201 a gain control analysis (AGC prescan), performing 202 an MS1 ​​analysis using a first mass analyzer (Orbitrap MS1 scan), and performing 203 one or more MS1 analyses using a second mass analyzer (TOF MS1 scan(s)). In the embodiment of Figure 3, these steps are performed sequentially, but in other embodiments, some of the steps may be performed in parallel. For example, steps involving the first mass analyzer may be performed in parallel with steps involving the second mass analyzer.

[0116] Following the MS1 analysis, in step 204 (Construct Unified Precursor List), the method 200 constructs a precursor ion peak combination list. The precursor ion peak combination list may include a list of m / z of precursor ion peaks identified from the MS1 analysis performed by both the first mass analyzer and the second mass analyzer. In some embodiments, the precursor ion peak combination list may be filtered as part of step 204 to remove precursor ion peaks associated with singly charged precursor ions. In some embodiments, the precursor ion peak combination list may be filtered to remove multiple charge states of precursor ions from the combination list. Thus, in some embodiments, the precursor ion peak combination list may be filtered such that only one charge state of precursor ions is included in the precursor ion peak combination list. As will be further described below, when precursor ion peaks associated with multiple different charge states are present in the combination list, the precursor ion peak having the lowest mass to charge ratio (i.e., the highest charge state) may be retained in the combination list. Alternatively, in some embodiments, the charge state of the precursor ion with the most intense precursor ion peak may be kept in the combined list (other precursor ion peaks are filtered out).

[0117] In step 205 (TOF MS2 scan), the method 200 includes performing an MS2 analysis using a second mass analyzer. The MS2 analysis fragments and analyzes precursor ions having a mass-to-charge ratio corresponding to the mass-to-charge ratio of a precursor ion peak selected from the precursor ion peak combination list. The method 200 then repeats the MS2 analysis until each precursor ion corresponding to a precursor ion peak on the combination list has been analyzed or until a predetermined time limit has been reached. Thus, the method 200 attempts to analyze each precursor ion in both the MS1 and MS2 domains within an analysis cycle.

[0118] As shown in Figure 3, in step 206 the method determines whether to continue performing the MS2 analysis or to repeat the method cycle 200 again. As shown in Figure 3, the method cycle 200 may be repeated once all precursor ions have been analyzed or once a time limit has been reached. Thus, the analysis cycle may be repeated multiple times over the duration that the sample elutes from the chromatographic device.

[0119] In some embodiments, the maximum time allowed for each cycle of the method 200 may be specified by a user. For example, the maximum time allowed may be about 1 s, 500 ms, 400 ms, 300 ms, or 250 ms or less. In some embodiments, the maximum time allowed for each cycle of the method 200 may be based on the duration of the MS1 analysis performed by the first mass analyzer (e.g., the orbital trap mass analyzer 110). That is, the maximum time allowed for each cycle 200 may be set to ensure that the first mass analyzer is used for the majority of the maximum cycle time. For example, in some embodiments, the duration of the MS1 analysis performed by the orbital trap mass analyzer 110 may be about 256 ms (at a resolution of 120,000) or about 512 ms (at a resolution of about 240,000), and the maximum cycle time may be about 270 ms or 530 ms, respectively. Preferably, the maximum cycle time may be selected to ensure that the first mass analyser is used for at least 60%, 70%, 80% or 90% of the maximum cycle duration.

[0120] One important advantage of the method 200 is that the TOF mass analyzer 150 is used to perform at least one MS1 analysis to identify precursor ion peaks for the combined list of precursor ions. Using the TOF mass analyzer 150 in this manner can result in a substantial increase in the number of precursor ion peaks identified in the analysis in the MS2 domain, as shown in FIG. 8. FIG. 8 is a bar graph showing the number of different precursor ion peaks identified using MS1 analysis of a calibration solution (Pierce® FlexMix®). As shown in FIG. 8, the MS1 analysis of the calibration solution was performed using an orbital trap analyzer 110 operating at a resolution of 60,000 (Orbi 60K), 120,000 (Orbi 120K), and 240,000 (Orbi 240K). As shown in FIG. 8, less than 1,800 precursor ion peaks were identified when the orbital trap mass analyzer was operated at a resolution of 240,000. In contrast, a single MS1 scan of the same calibration solution using a TOF mass analyzer identified approximately 3,900 precursor ion peaks.

[0121] For comparison, an MS1 ​​analysis was also performed using the orbital trap mass analyzer 110 operating in "HDR" data acquisition mode. In the HDR data acquisition mode, the mass range of interest was divided into 12 windows (i.e., 12 mass subranges) and a maximum injection time of 120 ms was given to allow a predetermined amount of precursors in each mass subrange to accumulate in the C-trap 100 before being injected into the orbital trap mass analyzer 110. Thus, when the injection time for a given mass subrange reaches 120 ms, the accumulation of ions for that mass subrange is terminated even if the predetermined amount of precursor ions has not been reached. The accumulated ions were then mass analyzed by the orbital trap mass analyzer 110. As shown in Figure 8, HDR analysis of the calibration solutions was performed using different resolutions of the orbital trap mass analyzer: 60,000 (Orbi-HDR 120ms 60K), 120,000 (Orbi-HDR 120ms 120K), and 240,000 (Orbi-HDR 120ms 240K).

[0122] As a further comparison, an HDR data acquisition mode using an injection time of 1200 ms was also performed. Although such a long injection time is not practical for normal mass spectrometry use, the relatively long injection time ensured that a given amount of ions for each mass subrange was accumulated in the C-trap 100 before mass analysis by the orbital trap mass analyzer. HDR analysis of the calibration solutions was performed using different resolutions of the orbital trap mass analyzer: 60,000 (Orbi-HDR 1200ms 60K), 120,000 (Orbi-HDR 1200ms 120K), and 240,000 (Orbi-HDR 1200ms 240K), as shown in FIG. 8.

[0123] FIG. 8 also shows data on the number of precursor ion peaks detected by the TOF mass analyzer when performing an MS1 ​​analysis of the calibration solution. FIG. 8 shows data on the number of precursor ion peaks detected from one scan and from the average mass spectrum obtained by performing ten MS1 ​​analyses of the calibration solution. As shown in FIG. 8, one scan performed by the TOF mass analyzer 150 detects more precursor ion peaks of the calibration solution than each MS1 analysis using the orbital trap mass analyzer 110, except for the HDR analysis performed at 240 K with an injection time of 1200 ms. For reference, one MS1 analysis using the TOF mass analyzer can be performed in about 5 ms, which is significantly shorter than the injection time of 1200 ms used for each of the 12 mass subranges that form part of the HDR analysis. Furthermore, the time-averaged mass spectrum obtained using the TOF mass analyzer 150 identifies more precursor ion peaks than any other analysis and can be performed on a time scale that is compatible with a typical mass analysis workflow (e.g., as shown in FIG. 2). It will therefore be appreciated from FIG. 8 that the use of a TOF mass analyzer 150 to identify precursor ion peaks can provide an effective method of identifying precursor ion peaks that would not be detected using the orbital trap mass analyzer 110.

[0124] In some embodiments, when generating the combined list of precursor ion peaks (e.g., step 206 of FIG. 3), the second set of precursor ion peaks may be thresholded. That is, the intensity of each precursor ion peak in the second set of precursor ion peaks may be compared to a first predetermined intensity level (threshold). Any precursor ion peaks below the first predetermined intensity level may be removed from the second set of precursor ion peaks (i.e., not used in generating the combined list of precursor ion peaks). By removing precursor ion peaks of relatively low intensity from the second set of precursor ion peaks, for example, single ion signals (i.e., peaks representing single precursor ions scattered during analysis) may be removed from the second set of precursor ions. Thus, the second set of precursor ion peaks better represents the precursor ions present in the sample (calibration solution) by reducing the effects of scattering of precursor ions during mass analysis.

[0125] In some embodiments, the second set of precursor ion peaks may also be thresholded to remove any precursor ion peaks above a second predetermined intensity level (threshold), the second intensity level being greater than the first intensity level. Thus, precursor ion peaks having a relatively large intensity, e.g., a signal intensity representing at least 10 precursor ions, may be removed from the second set of precursor ion peaks. Precursor ion peaks of relatively large intensity are likely to have been detected by the first mass analyzer (the orbital trap mass analyzer 110) and therefore may be removed from the second set of precursor ion peaks to reduce repetition of precursor ion peaks. That is, the second intensity level (threshold) may be set to a level corresponding to the intensity of precursor ion peaks that would be detected by the first mass analyzer as precursor ion peaks of the first set of precursor ion peaks.

[0126] 1, the TOF mass analyzer 150 outputs a voltage signal representative of precursor ions reaching the detector. Thus, the first and second intensity levels may correspond to voltage levels used to detect the presence of a precursor ion peak. In some embodiments, the controller may include a digitizer configured to apply the thresholding process described above.

[0127] FIG. 9 shows a graph illustrating the number of precursor ion peaks detected using a TOF mass analyzer when the first intensity level is varied. In the embodiment of FIG. 9, the TOF mass analyzer 150 is calibrated and the noise level is checked so that false peaks resulting from electronic noise are negligible at all threshold levels shown, and therefore all detected peaks are related to ion signals and not electronic noise. In the embodiment of FIG. 1 and as shown in FIG. 9, the first intensity level is varied from about 1 mV to 20 mV. In other embodiments, the first intensity level may depend on the nature of the signal output from the TOF mass analyzer, but in general may be configurable to allow single ion precursor ion peaks to be detected or to exclude precursor ion peaks representing one or two precursor ions.

[0128] As shown in FIG. 9, the number of precursor ion peaks detected by one scan (1x ToF 5ms in FIG. 9) using TOF mass analyzer 150 decreases as the first intensity level (digitizer HW threshold) increases from 1 mV to 20 mV. This is expected as the first intensity level increases, leading to an increase in the number of single ion peaks or well-separated multiple ion peaks that do not reach the threshold. In particular, multi-reflecting TOF mass analyzers such as TOF mass analyzer 150 often have a detector response that can be smaller than the peak width. Thus, a small detected precursor ion peak may form multiple spaced single ion events with similar heights (but much larger peak areas) as a single ion event containing multiple precursor ions.

[0129] FIG. 9 also shows a graph illustrating the variation in the number of precursor ion peaks detected using the TOF mass analyzer 150 when using the time-average acquisition method for different first intensity levels (TOF 10x 50ms 0mV SW-Th). By using the time-average acquisition method, a more complete picture can be obtained as to which of the precursor ion peaks of relatively low intensity are reproducible. It will be appreciated that the time-average graph shows that when the first intensity level is greater than 4mV, more precursor ion peaks are detected than in a single TOF mass analysis. This indicates that many of the peaks detected in the TOF mass analysis scan are associated with "real" ion events (i.e. precursor ions of different mass-to-charge ratios) and not as a result of precursor ion scattering.

[0130] It can be seen that in the time-averaged graph (TOF 50ms 0mV SW-Th), the number of precursor ion peaks begins to decrease at a lower first intensity threshold (e.g., from 6mV to 1mV). This decrease is due to the presence of scattered ion signals (i.e., spurious ion signals) appearing in the time-averaged data. This increase in peaks makes the spectrum very dense, such that closely scattered peaks merge and are difficult to distinguish as individual peaks.

[0131] To address this issue, the time-averaged data may be further thresholded to remove single ion peaks (likely to represent scattered ions). Thus, single ion peaks may be removed prior to detection of precursor ions by thresholding the time-averaged data at a third intensity level (e.g., 1 mV in FIG. 9) that is lower than the first intensity level threshold. Thus, as shown in FIG. 9, when a time-averaged graph is generated and a further 1 mV threshold is applied (TOF 50 ms 1 mV SW-Th), the number of detected precursor ion peaks increases as the first intensity level decreases, mirroring the behavior of a single TOF mass spectrometry graph.

[0132] As can be seen from FIG. 9 and the above discussion, the above described method may generate a precursor ion peak combination list that includes a significant number of peaks (e.g., FIG. 9 shows that up to 4500 precursor ion peaks may be detected by the TOF mass analyzer 150 when analyzing a calibration solution). Of these precursor ion peaks, a significant portion will have a relatively low signal intensity. Such relatively low intensity peaks may be difficult to assign a charge state to (e.g., the precursor ion peak may be associated with a singly or multiply charged precursor ion). In general, knowledge of the charge state of the precursor ion that gave rise to a given precursor ion peak may be useful for setting the fragmentation energy in a subsequent MS2 analysis of the precursor ion. Furthermore, singly charged precursor ions may be less useful in an MS2 analysis than their multiply charged precursor ions. Thus, in some embodiments, the precursor ion peak combination list may be filtered to remove singly charged precursor ions. The assignment of charge states to the precursor ion peaks may be performed by a suitable software algorithm. Known algorithms for determining charge state include Thorough High Resolution Analysis of Spectra by Horn (THRASH) (D. Horn et. al, Journal of the American Society for Mass Spectrometry, vol. 11, issue 4, April 2000), and the Advance Peak Detection (APD) algorithm available from Thermo Fisher Scientific®.

[0133] Thus, in some embodiments, the controller may be configured to determine a charge state for each precursor ion peak on the combined list of precursor ion peaks. The controller may use a charge state determination algorithm to determine the charge state associated with each precursor ion peak. In some embodiments, it may be desirable to analyze each precursor ion only once in the MS2 domain. Thus, if the same precursor ion appears in the combined list of precursor ion peaks with different charge states, the controller may filter the combined list of precursor ion peaks such that only one charge state of each precursor ion appears in the combined list of precursor ion peaks. Preferably, the controller may filter the list to remove precursor ion peaks associated with multiple charge states, in which case the precursor ion peak associated with the highest charge state of each precursor ion (i.e., the precursor ion peak with the lowest m / z of each precursor ion) is kept. Alternatively, in some embodiments, the charge state of each precursor ion having the highest intensity precursor ion peak may be kept in the combined list of precursor ion peaks (other precursor ion peaks are filtered out). In this way, the combined list of precursor ion peaks for analysis in the MS2 domain can be optimized to reduce or prevent repeated MS2 analysis of the same precursor ion.

[0134] It will be appreciated that the assignment of a charge state to each precursor ion peak in the combined list of precursor ion peaks may take some computation time. In some embodiments, the tandem mass analyzer 10 may include an ion mobility separator (not shown in FIG. 1 ) configured to filter singly charged precursor ions from the precursor ions provided to the orbital trap mass analyzer 110 and the TOF mass analyzer 150. For example, ions of different ion mobilities may be filtered by the ion mobility separator according to a compensation voltage applied to the ion mobility separator. One known ion mobility separator is the FAIMS Pro® Interface available from Thermo Fisher Scientific®.

[0135] It will be understood that the MS1 analysis performed by the second mass analyzer (TOF mass analyzer 150) may be performed at any time during the analysis cycle. For example, in the embodiment of FIG. 2, the MS1 analysis performed by the TOF mass analyzer 150 is performed near the beginning of each MS1 analysis performed by the orbital trap mass analyzer 110. For example, the MS1 analysis performed by the second mass analyzer 150 may be performed during an initial period of the MS1 analysis performed by the first mass analyzer (orbital trap mass analyzer 110). In some embodiments, the initial period may be 30% or 20% or less of the total (expected) duration of the MS1 analysis performed by the first mass analyzer. It will be understood that the initial period therefore extends from the start of the MS1 analysis performed by the first mass analyzer to the end of the initial period (i.e., when 30% of the total (expected) duration of the MS1 analysis has elapsed).

[0136] In some embodiments, it may be advantageous to distribute the MS1 analyses performed by the second mass analyzer (TOF mass analyzer 150) over the duration of the MS1 analyses performed by the first mass analyzer (orbital trap mass analyzer 110). Thus, in some embodiments in which multiple MS1 analyses are performed by the second mass analyzer, the MS1 analyses may be distributed evenly over the duration of the MS1 analyses performed by the first mass analyzer. That is, the multiple MS1 analyses performed by the second mass analyzer may be interleaved with the MS2 analyses performed by the second mass analyzer.

[0137] For example, FIG. 10 shows a block diagram of an analysis cycle performed by the tandem mass spectrometer 10 of FIG. 1. In the block diagram of FIG. 10, the MS1 analysis performed by the orbital trap mass analyzer 110 may have a duration of about 256 ms. For comparison, FIG. 10 shows a profile of a chromatographic peak eluting from a chromatographic separator over the duration of an analysis cycle. It will be understood that the MS1 analysis performed by the orbital trap ion analyzer takes samples of ions from the beginning of the elution of the chromatographic peak. Thus, for a relatively fast eluting chromatographic peak, the MS1 analysis performed by the orbital trap mass analyzer may not be able to fully characterize the chromatographic peak. For example, in FIG. 10, over the duration of a relatively fast eluting chromatographic peak, the MS1 analysis by the orbital trap mass analyzer 110 may only be performed two or three times.

[0138] To improve characterization of the chromatographic peaks, in Fig. 10, multiple MS1 analyses are performed by TOF mass analyzer 150 simultaneously with the MS1 analyses performed by orbital trap mass analyzer 110. In particular, the multiple MS1 analyses performed by TOF mass analyzer 150 are interleaved with multiple MS2 analyses performed by the TOF mass analyzer. In the embodiment of Fig. 10, at least five MS1 analyses are performed over the duration of one MS1 analysis performed by orbital trap mass analyzer 110.

[0139] In the embodiment of FIG. 10, multiple MS1 analyses may be interleaved with MS2 analyses by performing an MS1 ​​analysis followed by a predetermined number of MS2 analyses. For example, the MS1 analysis may be repeated every 5, 10, 15, or 20 MS2 analyses performed by the TOF mass analyzer 150. Thus, the analyses performed in the MS1 domain may be interleaved evenly throughout the duration of the analyses performed in the MS1 domain by the first mass analyzer. Alternatively, the MS1 analyses may be performed at predetermined time intervals during the MS1 analyses performed by the orbital trap mass analyzer 110.

[0140] It will be appreciated that in the embodiment of Figure 10, each MS1 analysis performed by the TOF mass analyzer 150 is performed over the entire m / z range of interest. In other embodiments, the MS1 analysis may be performed over an m / z subrange of interest (e.g., as described above with respect to Figure 2), with the analyses being interleaved over the duration of the MS1 analysis performed by the orbital trap mass analyzer. In some embodiments, each MS1 analysis of an m / z subrange of interest may be repeated multiple times over the duration of the MS1 analysis performed by the orbital trap mass analyzer 110.

[0141] In some embodiments, data from the MS1 analysis performed by the first mass analyzer and the second mass analyzer may be used to identify chromatographic peaks eluting from the chromatographic separation device. For example, data from the MS1 analysis performed by the TOF mass analyzer 150 may be used to identify and / or characterize the chromatographic peaks shown in FIG. 10. In some embodiments, the analysis performed in the MS2 domain by the TOF mass analyzer 150 may be selected based on the identified chromatographic peaks eluting from the mass analyzer. For example, the precursor ion peak combination list may be updated based on the identified chromatographic peaks. In particular, the order of the precursor ions analyzed in the MS2 domain in the precursor ion peak combination list may be updated based on the identified chromatographic peaks.

[0142] Thus, in accordance with the above description, there is provided a method of tandem mass spectrometry, a tandem mass spectrometer, a computer program, and a computer readable medium.

Claims

1. 1. A method of tandem mass spectrometry for analyzing precursor ions across a mass-to-charge (m / z) range of interest, comprising: analyzing some of the precursor ions across the m / z range of interest in an MS1 ​​domain using a first mass analyzer of a tandem mass spectrometer operating at a first sensitivity; analyzing some of the precursor ions across the m / z range of interest in the MS1 domain using a second mass analyzer of the tandem mass spectrometer operating at a second sensitivity, the second sensitivity being greater than the first sensitivity, and the analysis in the MS1 domain performed by the second mass analyzer being performed simultaneously with the analysis in the MS1 domain by the first mass analyzer; The method further comprises: combining data from the MS1 analyses performed by the first mass analyzer and the second mass analyzer to identify and / or quantify precursor ions; and analyzing some of the precursor ions in an MS2 domain using the second mass analyzer of the tandem mass spectrometer.

2. the first mass analyzer operates at a first sensitivity and a first mass accuracy; 2. The method of claim 1, wherein the second mass analyzer analyzes some of the precursor ions in the MS1 domain with a second sensitivity and a second mass accuracy, the second mass accuracy being lower than the first mass accuracy.

3. 2. The method of claim 1, wherein combining data from MS1 analyses performed by the first mass analyzer and the second mass analyzer includes generating a combined list of precursor ion peaks in the MS1 domain.

4. 4. The method of claim 3, wherein the combined list of precursor ion peaks includes a first set of precursor ion peaks identified from an MS1 ​​analysis performed by the first mass analyzer and a second set of precursor ion peaks identified from an MS1 ​​analysis performed by the second mass analyzer, and the combined list is filtered to remove precursor ion peaks that are repeated between the first and second sets of precursor ion peaks.

5. 5. The method of claim 4, wherein the second set of precursor ion peaks is thresholded to remove any precursor ion peaks below a predetermined first intensity level and / or any precursor ion peaks above a predetermined second intensity level.

6. 6. The method of claim 4 or 5, further comprising calibrating the tandem mass spectrometer using a first precursor ion peak produced by the second mass analyzer and a corresponding first precursor ion peak produced by the first mass analyzer, and using the calibration to identify precursor ion peaks that are repeated between the first and second sets of precursor ion peaks.

7. The method of claim 1 , wherein the analysis performed in the MS2 domain is based on the precursor ion identified by the MS1 analysis.

8. Analyzing the precursor ion in the MS2 domain comprises: fragmenting the precursor ions to generate product ions; and analyzing the product ions using the second mass analyzer.

9. The method includes performing a plurality of analytical cycles using the tandem mass spectrometer, each cycle comprising: performing a single analysis across the m / z range of interest in the MS1 domain using the first mass analyzer; performing a single analysis across the m / z range of interest in the MS1 domain using the second mass analyzer; performing an analysis of some of the precursor ions in the MS2 domain using the second mass analyzer; The method of claim 1 , wherein the analysis performed in the MS1 domain and the MS2 domain by the second mass analyzer is performed simultaneously with the one analysis in the MS1 domain performed by the first mass analyzer.

10. The method includes performing a plurality of analytical cycles using the tandem mass spectrometer, each cycle comprising: performing a single analysis across the m / z range of interest in the MS1 domain using the first mass analyzer; subdividing the m / z range of interest into a plurality of m / z subranges and performing analysis over each m / z subrange of interest in the MS1 domain using the second mass analyzer; performing an analysis of some of the precursor ions in the MS2 domain using the second mass analyzer; The method of claim 1 , wherein the analysis performed in the MS1 domain and the MS2 domain by the second mass analyzer is performed simultaneously with the one analysis in the MS1 domain performed by the first mass analyzer.

11. each cycle comprises performing multiple analyses in the MS1 domain for each m / z subrange of interest using the second mass analyzer; and averaging data from the MS1 analysis performed by the second mass analyzer for each m / z subrange of interest.

12. The method includes performing a plurality of analytical cycles using the tandem mass spectrometer, each cycle comprising: performing a single analysis across the m / z range of interest in the MS1 domain using the first mass analyzer; performing multiple analyses across the m / z range of interest in the MS1 domain using the second mass analyzer; and performing an analysis of some of the precursor ions in the MS2 domain using the second mass analyzer.

13. For each analysis cycle, The method of claim 12 , further comprising averaging data from the analysis performed in the MS1 domain by the second mass analyzer.

14. The method of claim 12 , wherein averaging the data from the analysis performed in the MS1 domain comprises thresholding the data to remove precursor ion peaks below a third intensity level.

15. 15. The method of any one of claims 12 to 14, wherein the analyses performed in the MS1 domain by the second mass analyzer are interleaved with the analyses performed in the MS2 domain by the second mass analyzer.

16. 16. The method of claim 15, wherein for each analysis cycle, at least 3, 5, or 7 analyses are performed in the MS1 domain by the second mass analyzer.

17. 16. The method of claim 15, wherein the analyses performed in the MS1 domain are evenly interleaved throughout the duration of the analyses performed in the MS1 domain by the first mass analyzer.

18. 9. The method of claim 8, wherein each analysis cycle further comprises performing a gain control analysis using the first mass analyzer or the second mass analyzer, and an injection time for each of the MS1 analyses performed by the first mass analyzer and / or the second mass analyzer is adjusted based on the gain control analysis.

19. The method of claim 1 , wherein the precursor ions analyzed are filtered to remove or reduce the number of singly charged precursor ions.

20. The method of claim 1 , wherein the precursor ions to be analyzed are provided by an ion source configured to ionize molecules provided from a chromatographic separation device.

21. 21. The method of claim 20, wherein data from the MS1 analysis performed by the first mass analyzer and the second mass analyzer is used to identify chromatographic peaks eluting from the chromatographic separation device.

22. 22. The method of claim 21, wherein the analysis performed in the MS2 domain by the second mass analyzer is selected based on the identified chromatographic peaks eluting from the mass analyzer.

23. the first mass analyzer is a mass analyzer selected from the group including an orbital trap mass analyzer, a Fourier transform ion cyclotron resonance (FTICR) mass analyzer, and a time-of-flight (TOF) mass analyzer; and / or The method of claim 1 , wherein the second mass analyzer is a TOF mass analyzer.

24. the first mass analyzer operates in a first dynamic range to perform each of the MS1 analyses; 2. The method of claim 1, wherein the second mass analyzer operates at a second dynamic range greater than the first dynamic range for performing each of the MS1 analyses.

25. 1. A tandem mass spectrometer for analyzing precursor ions over a mass-to-charge (m / z) range of interest, comprising: a first mass analyzer configured to analyze precursor ions in an MS1 ​​domain; a second mass analyzer configured to analyze the precursor ions in the MS1 domain and the MS2 domain; and a controller, the controller comprising: causing the first mass analyzer to analyze some of the precursor ions across the m / z range of interest in the MS1 domain at a first sensitivity; and causing the second mass analyzer to analyze some of the precursor ions across the m / z range of interest in the MS1 domain at a second sensitivity, the second sensitivity being greater than the first sensitivity, the second mass analyzer analyzing the precursor ions in the MS1 domain at the same time that the first mass analyzer analyzes the precursor ions in the MS1 domain, the controller further configured to: combining data from the MS1 analyses performed by the first mass analyzer and the second mass analyzer to identify and / or quantify precursor ions; causing the second mass analyzer to analyze some of the precursor ions in the MS2 domain.

26. 26. A computer program comprising instructions for causing a tandem mass spectrometer according to claim 25 to carry out the steps of the method according to claim 1.

27. 27. A computer readable medium storing a computer program according to claim 26.

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