Mass spectrometer calibration

Abstract

In one aspect, a system and method for calibrating a hybrid mass spectrometer is provided. The calibration may include an accurate mass calibration and, for example, a nominal mass calibration performed over multiple transmission widths and scan rates, ideally applied to each reference standard, where each reference standard represents a different m / z, to generate a matrix of correction factors corresponding to each of the transmission width and scan rate pairs at m / z values ​​for the reference standard being evaluated. Multi-parameter interpolation may be applied to identify correction factors to be used for subsequent analysis transmission width and scan rate pairs that differ from the multiple transmission widths and scan rates used to generate the correction factors.

Description

Background Art

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 63 / 250,872, filed September 30, 2021, titled "Mass Spectrometer Calibration", and U.S. Provisional Application No. 63 / 139,682, filed January 20, 2021, titled "Mass Spectrometer Calibration", each of which is incorporated herein by reference in its entirety.

[0002] A mass spectrometer is an analytical instrument used to analyze samples, identify their composition, and / or measure the quantity of analytes in a given sample.

[0003] A hybrid mass spectrometer that combines two different types of mass analyzers offers the benefits of different performance characteristics provided by each of the mass analyzers within a single instrument.

[0004] Useful types of hybrid mass spectrometers include a series of combinations of a nominal mass spectrometer and a high-precision mass spectrometer. Some manufacturers have proposed hybrid mass spectrometer products involving various different types of mass analyzers, including multipoles, orthogonal time-of-flight (ToF), electrostatic traps, linear ion traps, etc. For example, SCIEX has proposed various QTOF instruments that combine a triple quadrupole mass spectrometer with an orthogonal time-of-flight (ToF) mass spectrometer as their respective nominal mass spectrometer and high-precision mass spectrometer.

[0005] Mass spectrometer performance varies with various conditions, including environmental factors, contamination, sample composition, instrument condition, and individual instrument variability. A standard practice when operating a mass spectrometer is to perform calibration operations at regular time intervals to ensure precise instrument operation. Calibration of a hybrid mass spectrometer can be complex, as each of the two different types of mass spectrometers needs to be calibrated in relation to the other. This hybrid calibration generally involves two separate calibration operations: a nominal mass calibration and a precise mass calibration, both performed against known reference standards to ensure that each of the two mass spectrometers operates within specifications and within a defined dispersion range relative to the other.

[0006] The precision mass calibration process generally involves supplying a pure reference standard of known composition (e.g., a pure cesium reference standard) for analysis by a precision mass spectrometer. Calibration can be performed by analyzing the reference standard, comparing the analysis results to the expected values, and correcting any offset between the precision mass spectrometer analysis results and the expected values ​​with respect to the reference standard.

[0007] Depending on user requirements, the precision mass calibration operation can be performed daily, weekly, or monthly, typically requiring approximately 20-30 minutes to complete. Many users prefer to perform the precision mass calibration at least once every 24 hours to ensure that each set of daily analysis results is accurate.

[0008] Nominal mass calibration is more time-consuming and complex than precision mass calibration because the variance in the analytical results for the nominal mass instrument typically includes an m / z-dependent component. Nominal mass calibration typically requires calibration against a known reference standard and collection of analytical results at each of the different scanning speeds and transmission window widths that will be used for the analysis. For example, while supplying the reference standard, the instrument may be set with respect to a given transmission width, then stepped through to several different scanning speeds, and calibration results for that transmission width at each scanning speed may be collected. The procedure may then be repeated across multiple transmission widths to generate a matrix of calibration results containing the desired mass range. Based on this matrix of calibration results, the transmission widths may be realigned at each transmission window so that the analytical results produced by the nominal mass spectrometer match those produced by the precision mass spectrometer.

[0009] Some commercial instruments reduce the burden of performing nominal mass calibration operations by running different scan rates and transmission widths as batch operations. In this feature, the user configures the hybrid mass spectrometer with one or more standard injections and batch programs, the batch programs defining the stepwise scan rates and transmission window widths to be evaluated. Once the batch program is executed, the instrument will automatically stepwise set the scan rates and transmission window widths and build a matrix of calibration results without further user intervention. The instrument can typically complete a calibration batch run within approximately 30 minutes, depending on the number of scan rates and transmission bandwidths defined for the batch run. The user must then scrutinize the calibration data based on standard threshold levels of variance to complete the instrument calibration.

[0010] User time is freed by using batch programs, but the instrument is tied up in performing calibration operations, and reference standards and solvents are consumed. [Overview of the project] [Means for solving the problem]

[0011] The identified problems described above regarding generating calibrated mass data in hybrid mass spectrometers lead to wasted time and less precise analytical results. Additional problems exist with existing calibration methods. For example, the inventors evaluated the effectiveness of a standard calibration operation and observed that while the instrument appears calibrated during the calibration operation when performed against an implanted reference standard, the calibration can be lost when operating the instrument against an analytical sample with a full sample load. The inventors observed resolution drift when performing a full sample load, such as a large proteomics sample, on an instrument that was properly calibrated using conventional reference standard implantation. This can be attributed to contamination on the ion optics that causes a shift when the high-temperature ion beam generated from the full sample load comes into contact with the ion optics. As a result, it is considered that conventional calibration based on low implantation of the reference standard may not always be effective for large proteomics samples.

[0012] The inventors have confirmed that SCIEX's commercial hybrid mass spectrometers maintain stable nominal mass calibration over several months, but users choose to recalibrate both the nominal and precision mass spectrometers at higher intervals, such as weekly or even daily. Given the enormous investment of time, resources, and capital in research efforts, it is understandable that users would prefer to calibrate the nominal and precision mass spectrometers together to avoid the opportunity for erroneous analytical results.

[0013] Given these end-use requirements, the inventors have developed this novel approach to hybrid mass spectrometer calibration, which leverages a precision mass calibration that has proven to be stable and precise with minimal variability between calibration events, in order to calibrate the nominal mass spectrometer. Thus, it is possible to calibrate the nominal mass spectrometer while running the sample, based on the calibrated precision mass spectrometer analysis results. Furthermore, this approach enables verification and reporting to confirm the analysis results for each sample run without the need to recalibrate the nominal mass spectrometer. In other words, the embodiment can utilize data generated using a combination of a nominal mass spectrometer (i.e., a mass spectrometer not calibrated with one or more calibrators) and a precision mass spectrometer (i.e., a mass spectrometer calibrated with one or more calibrators) to derive an m / z correction for the mass signal acquired for the sample under investigation.

[0014] In some embodiments, mass data can be compiled such that the mass data is separated based on the transmission window of the nominal mass spectrometer employed to collect the data. In such embodiments, each data compilation associated with a particular transmission window of the nominal mass spectrometer may include an indicator (e.g., in the form of a file header) that provides information about the transmission window of the nominal mass spectrometer. In some such embodiments, calibration correction values ​​can be employed to adjust the header data to reposition the mass spectrometry results within each transmission window.

[0015] In some embodiments, a hybrid mass spectrometer, including a nominal mass spectrometer and a precision mass spectrometer, operates to perform a data-independent acquisition operation and calibrate the nominal mass spectrometer based on the precision mass spectrometry results. The data-independent operation may include, for example, a scanning quadrupole data-independent acquisition operation.

[0016] In some respects, a hybrid mass spectrometer may operate to calibrate the compilation of mass data by performing mass analysis on a sample to generate mass analysis results and considering the difference between one or more nominal operating settings of the nominal mass spectrometer (e.g., transmission bandwidth of the mass filter) and the actual operating settings of the nominal mass spectrometer derived from the analysis of mass data generated by a precision mass spectrometer calibrated with one or more calibrators. Calibration may include revising the nominal mass analysis results based on the calibration and generating calibrated nominal mass analysis results. In some respects, a calibration metric indicating the difference between the current calibration and previous calibrations may be evaluated.

[0017] In some embodiments, calibration may involve evaluating the mass spectrometry results and identifying one or more residual precursor ions within the m / z transmission window from the accurate mass results. The evaluation may be repeated across multiple transmission windows to include the mass range to be calibrated with respect to the sample being mass-spectrated. In some embodiments, each identified precursor ion may be paired with the mass center of the corresponding nominal mass spectrometry result within its transmission window to identify any offset or difference between the expected value, as provided by the accurate mass results, and the measured value, as provided by the nominal mass results. In some embodiments, such offsets may be used to adjust the header of the data compilation to identify the transmission bandwidth of the transmission window associated with the nominal mass spectrometer.

[0018] Conventionally, multiple precursor ions may be identified in each of the permeation windows to provide multiple corresponding offset values ​​for that permeation window. In some respects, each of the precursor ions may be evaluated relative to each other to identify any outliers that should be discarded. A correction factor for each permeation window may be generated based on the multiple offset values ​​for that permeation window. In some respects, the spread or difference of the offset values ​​may be evaluated to verify the reliability metric of the generated correction factor.

[0019] In some respects, each of the multiple correction factors may be further evaluated to confirm their consistency with one another. Consistency can be confirmed, for example, by fitting the trend line to the multiple correction factors, evaluating each correction factor, and confirming that it lies within the expected range of the trend line. In some respects, consistency can be confirmed by evaluating each correction factor and confirming that it lies within the expected range of its neighbors.

[0020] Each correction factor may be applied to shift the mass spectrometry results within the corresponding transmission window based on the offset defined by the correction factor. In some aspects, the correction factor may be applied by correcting all mass spectrometry results based on the defined offset within the corresponding transmission window. In some aspects, the correction factor may be applied by overwriting header data to reposition the mass spectrometry results within each transmission window based on the defined offset. In some aspects, the repositioning may include repositioning the mass spectrometry results within the transmission window while maintaining continuity at the boundary between that transmission window and adjacent transmission windows. In some aspects, the transmission windows may include overlaps, and the repositioning may include repositioning the mass spectrometry results within the transmission window while maintaining continuity at the boundary between that transmission window and adjacent transmission windows.

[0021] In a related aspect, a method for calibrating a hybrid mass spectrometer comprising a nominal mass spectrometer and a precision mass spectrometer is disclosed, which includes analyzing a sample using the hybrid mass spectrometer and collecting precision mass spectrometry results and nominal mass spectrometry results for the sample. With respect to at least one mass transmission window of the nominal mass spectrometer, the nominal mass spectrometry results and precision mass spectrometry results within the mass transmission window can be evaluated to identify at least one difference between the nominal mass spectrometry results and the precision mass spectrometry results. Based on the identified difference, the nominal mass spectrometry results within the mass transmission window can be corrected to align it with the precision mass spectrometry results. In some embodiments, the identification of the at least one difference includes identifying at least one precursor ion in the precision mass spectrometry results and comparing the m / z ratio of the at least one precursor ion derived from the precision mass spectrometry results with the corresponding m / z ratio corresponding to the at least one precursor ion from the nominal mass spectrometry results.

[0022] In relation to the above, a system and / or method for calibrating a hybrid mass spectrometer is provided. The calibration may include a precise mass calibration and a nominal mass calibration performed over multiple transmission widths and scan speeds applied for each reference standard, ideally each reference standard representing a different m / z ratio, to generate a matrix of correction factors corresponding to each transmission width and scan speed pair in the m / z value with respect to the reference standard being evaluated. Multiparameter interpolation may be applied to identify the correction factors to be used for different subsequent analysis transmission width and scan speed pairs than the multiple transmission widths and scan speeds used to generate the correction factors.

[0023] In relation to the present invention, a method for calibrating a hybrid mass spectrometer having a precision mass spectrometer combined with a nominal mass spectrometer is disclosed, which includes calibrating the precision mass spectrometer using one or more reference standards, analyzing a sample using the nominal mass spectrometer and the precision mass spectrometer to generate one or more corresponding nominal mass signals and calibrated precision mass signals associated with the sample, and calibrating the nominal mass spectrometer by comparing the nominal mass signals with the calibrated precision mass signals.

[0024] In some embodiments, a controller can adjust one or more operating parameters of a nominal mass spectrometer to generate multiple precursor transmission windows, which partially overlap. Precursor ions passing through each transmission window can be received by a collision cell, in which at least a portion of the precursor ions can be fragmented to generate multiple product ions. The product ions and any residual precursor ions can be received by a precision mass spectrometer (e.g., a time-of-flight (ToF) mass spectrometer). The precision mass spectrometer can generate a signal indicating the mass of the product ions and one or more residual precursor ions. In some such embodiments, the transmission windows can be scanned, and the signals generated by the precision mass spectrometer can be collected.

[0025] Operating parameters associated with scanning the transmission window of a nominal mass spectrometer can be used to determine the lower m / z limit, upper m / z limit, transmission bandwidth, and the scan rate associated with the scanned transmission window. For example, when the nominal mass spectrometer is a quadrupole mass spectrometer, the operating parameters may include one or more parameters associated with the RF and DC voltages applied to the quadrupole rods to establish the transmission bandwidth associated with the quadrupole mass spectrometer, and the adjustment of the RF and / or DC voltages for scanning the transmission window.

[0026] In some embodiments, a controller that communicates with a nominal mass analyzer establishes a transmission window for the passage of ions and can be used to adjust one or more operating parameters associated with the nominal mass analyzer, such as the RF and / or DC voltages applied to the quadrupole rods of a quadrupole mass analyzer, to scan the transmission bandwidth associated with the transmission window.

[0027] In some embodiments, mass signals generated by a high-precision mass analyzer (e.g., a ToF mass analyzer) can be stored such that mass signal data associated with each transmission window is stored in a plurality of data bins, and each data bin corresponds to a certain percentage of the transmission bandwidth of a given transmission window. As described above, each data bin is considered herein to correspond to an "experiment". In some such embodiments, an extracted ion chromatogram (XIC) can be generated for each nth experiment (e.g., for each 5th experiment), and one or more peaks can be identified as potentially corresponding to residual precursor ions (i.e., precursor ions that did not undergo fragmentation). For peaks considered to correspond to precursor ions, each peak in several experiments preceding the nth experiment and several experiments following it can be identified. In some embodiments, the total number of experiments can correspond to the transmission bandwidth of the transmission window.

[0028] An intensity profile of mass peaks across these experiments as a function of the nominal transmission bandwidth is plotted and can be used to obtain a calibration correction for each transmission window. For example, the m / z ratio associated with residual precursor ions detected via a high-precision mass analyzer is compared to the mass center of the intensity profile of the mass peaks as a function of the nominal m / z ratio to obtain a calibration correction factor.

[0029] As further discussed below, such calibration correction factors can be utilized to edit the header of a data file that includes nominal parameters associated with the transmission window, such as the starting m / z and ending m / z associated with the transmission bandwidth.

[0030] In a related aspect, a hybrid mass spectrometer is disclosed, which includes a nominal mass analyzer configured to provide a plurality of transmission windows that allow the passage of at least one precursor ion, and a collision cell positioned downstream of the nominal mass analyzer. The collision cell receives the at least one precursor ion and causes its fragmentation to generate a plurality of product ions. A high-resolution mass analyzer is positioned downstream of the collision cell to receive the ions exiting the collision cell and generate its mass spectrum. An analysis module is configured to receive one or more operating parameters of the nominal mass analyzer and the mass spectrum generated by the high-resolution mass analyzer. The analysis module is further configured to identify a mass signal associated with the precursor ion in the mass spectrum generated by the high-resolution mass analyzer and calibrate the nominal mass analyzer based on the m / z ratio of the precursor ion in the mass spectrum and the one or more operating parameters of the nominal mass analyzer.

[0031] In a related aspect, a system and / or method for calibration of mass data obtained by a hybrid mass spectrometer including a nominal mass analyzer and a high-resolution mass analyzer is provided. The calibration can include using the mass data generated by the high-resolution mass analyzer to adjust the compilation of the mass data to account for any discrepancies between the compiled data indicating the nominal parameters of the nominal mass analyzer and the values of those parameters derived through analysis of the mass data generated by the high-resolution mass analyzer.

[0032] In some embodiments, the nominal mass spectrometer may include a plurality of rods arranged in a multi-pole (e.g., quadrupole) configuration, and the nominal mass spectrometer is configured for the application of RF and DC voltages to the plurality of rods. In some such embodiments, the precision mass spectrometer may be a time-of-flight (ToF) mass spectrometer calibrated using one or more reference standards.

[0033] A further understanding of the various aspects of this instruction can be obtained by referring to the detailed explanation below and the associated diagrams briefly described below. This specification also provides, for example, the following: (Item 1) A method for calibrating a hybrid mass spectrometer equipped with a precision mass spectrometer combined with a nominal mass spectrometer, wherein the method is: Calibrating the precision mass spectrometer using one or more reference standards, Using the nominal mass spectrometer and the precision mass spectrometer, the sample is analyzed and one or more corresponding nominal mass signals and calibrated precision mass signals associated with the sample are generated. The nominal mass spectrometer is calibrated by comparing the nominal mass signal with the calibrated precision mass signal. Methods that include... (Item 2) The method according to item 1, further comprising storing one or more calibration parameters associated with the calibrated nominal mass spectrometer. (Item 3) The method according to any one of items 1-2, wherein the nominal mass spectrometer is configured to provide a plurality of ion transmission windows. (Item 4) The method according to item 3, further comprising a controller that communicates with the nominal mass spectrometer, wherein the controller adjusts at least one parameter of an ion permeable window associated with the nominal mass spectrometer in order to generate the plurality of ion permeable windows. (Item 5) The method according to item 4, wherein the nominal mass spectrometer comprises a plurality of rods arranged in a multi-pole configuration, and the nominal mass spectrometer is configured for applying RF and DC voltages to the plurality of rods. (Item 6) The method according to item 5, wherein the controller causes a change in at least one parameter associated with at least one of the RF and DC voltages in order to generate the plurality of ion permeable windows. (Item 7) The method according to any one of items 4-6, wherein the controller causes the scanning of the at least one parameter to scan the transmission bandwidth associated with the nominal mass spectrometer. (Item 8) The precision mass spectrometer is equipped with a time-of-flight (ToF) mass spectrometer, as described in any one of items 1-7. (Item 9) A method for calibrating a mass spectrometer having at least a first and a second mass spectrometer, wherein the second mass spectrometer is located downstream of the first mass spectrometer, and the method is Calibrating the second mass spectrometer using one or more reference standards, Using the first mass spectrometer, a first measurement of the mass signal of the precursor ion is generated, and using the second mass spectrometer, a second measurement of the mass signal of each of the precursor ions is generated. The first mass spectrometer is calibrated by comparing the first and second measured values ​​of the mass signal. Methods that include... (Item 10) The method according to item 9, wherein the first mass spectrometer comprises a quadrupole mass spectrometer. (Item 11) The method according to any one of items 9-10, wherein the second mass spectrometer comprises a time-of-flight (ToF) mass spectrometer. (Item 12) The method according to any one of items 9-11, further comprising a collision cell positioned between the first mass spectrometer and the second mass spectrometer, wherein the collision cell receives the precursor ion from the first mass spectrometer and generates a plurality of product ions through the fragmentation of the precursor ion. (Item 13) The method according to any one of items 9-12, wherein the first measurement of the mass signal corresponds to the m / z ratio of the precursor ion identified based on at least one nominal setting of the first mass spectrometer. (Item 14) The step of measuring the second mass signal is: Identifying the mass signal associated with the precursor ion in the mass spectrum of ions exiting the collision cell generated by the second mass spectrometer, Assigning the m / z ratio to the identified mass signal and The method described in item 13, including the method described in item 13. (Item 15) The method according to item 14, wherein the at least one nominal setting comprises either a transmission width or a speed for scanning the transmission width. (Item 16) The method according to any one of items 9-15, wherein the first mass spectrometer comprises a plurality of rods arranged in a multi-pole configuration, and RF and DC voltages can be applied to the plurality of rods. (Item 17) The method according to item 16, wherein the second mass spectrometer comprises a time-of-flight mass spectrometer. (Item 18) A hybrid mass spectrometer, wherein the hybrid mass spectrometer is A nominal mass spectrometer configured to provide multiple transmission windows for the passage of at least one precursor ion, A collision cell located downstream of the nominal mass spectrometer, the collision cell receiving the at least one precursor ion, causing its fragmentation, and generating a plurality of product ions, A precision mass spectrometer for receiving ions exiting the aforementioned collision cell and generating their mass spectra, A mass spectrometer for receiving one or more operating parameters of the nominal mass spectrometer and the mass spectrum generated by the precision mass spectrometer. Equipped with, The aforementioned mass spectrometer is To identify the mass signal associated with the precursor ion in the aforementioned precision mass spectrometer, Calibrating the nominal mass spectrometer based on the m / z ratio of the precursor ion in the mass spectrum and one or more operating parameters of the nominal mass spectrometer. A hybrid mass spectrometer configured to perform the following actions. (Item 19) The hybrid mass spectrometer according to item 18, wherein the nominal mass spectrometer comprises a plurality of rods arranged in a multi-pole configuration, and the nominal mass spectrometer is configured for the application of RF and DC voltages to the plurality of rods. (Item 20) The precision mass spectrometer is a hybrid mass spectrometer as described in any one of items 18-19, comprising a time-of-flight mass spectrometer. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 shows an example of a hybrid mass spectrometer according to one embodiment of this teaching.

[0035] [Figure 2A] Figure 2A depicts the extracted ion chromatogram (XIC) of the sample.

[0036] [Figure 2B] Figure 2B depicts the total ion chromatogram (TIC) corresponding to the XIC region around mass peak 202.

[0037] [Figure 2C] Figures 2C and 2D are spectra confirming that the reference peak corresponds to the m / z ratio within the transmission window. [Figure 2D] Figures 2C and 2D are spectra confirming that the reference peak corresponds to the m / z ratio within the transmission window.

[0038] [Figure 3] Figure 3 shows the nominal mass spectrometry results.

[0039] [Figure 4] Figure 4 shows a plot of nominal mass versus the corresponding precise mass spectrometry results.

[0040] [Figure 5A] Figures 5A and 5C show the results of precise mass spectrometry. [Figure 5B] Figures 5B and 5D show the nominal mass spectrometry results.

[0041] [Figure 5C] Figures 5A and 5C show the results of precise mass spectrometry. [Figure 5D] Figures 5B and 5D show the nominal mass spectrometry results.

[0042] [Figure 6] Figure 6 shows exemplary calibration curves plotted over the mass range of interest.

[0043] [Figure 7] Figure 7 shows a hybrid mass spectrometer according to one embodiment of this teaching.

[0044] [Figure 8] Figure 8 schematically illustrates an example of a controller / analysis module implementation suitable for practical use of the embodiments of this instruction. [Modes for carrying out the invention]

[0045] For clarity, please understand that the following discussion will detail various aspects of the embodiments of the Disclosure, while omitting certain specific details whenever it is convenient or appropriate to do so. For example, discussions of similar or analogous features in alternative embodiments may be somewhat abbreviated. Well-known concepts or ideas may also be discussed in less detail for the sake of brevity. Those skilled in the art will recognize that some embodiments of the Disclosure may not require some of the details specifically described in all implementations described herein, solely to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the embodiments described may be subject to modification or alteration in accordance with common general knowledge without departing from the scope of the Disclosure. The following detailed description of the embodiments is not intended to limit the scope of the applicant's teachings in any way.

[0046] As used herein, the terms “about” and “substantially equivalent” refer to variations in numerical quantities that may arise, for example, through real-world measurement or handling procedures, through unintentional errors in these procedures, through differences in the manufacture, source, or purity of a composition or reagent, etc. Typically, as used herein, the terms “about” and “substantially” mean 10% above or below a stated value or range of values ​​or complete condition or state. For example, a concentration value of about 30% or substantially equivalent to 30% may mean a concentration of 27% to 33%. The terms also refer to variations that would be recognized as equivalent by those skilled in the art, unless such variations encompass known values ​​practiced by the prior art.

[0047] As used herein, the term "and / or" includes all possible combinations of one or more of the listed items that are associated with each other, and may be abbreviated as " / ".

[0048] The term "nominal mass spectrometer" refers to a mass spectrometer or mass analyzer that has not been calibrated using one or more reference calibrators. The term "precision mass spectrometer" refers to a mass spectrometer or mass analyzer that has been calibrated using one or more reference calibrators.

[0049] This instruction relates in general to methods and systems for calibrating mass data generated by a hybrid mass spectrometer, and more particularly to methods and systems for calibrating a nominally calibrated mass spectrometer (e.g., one or more operating parameters of a nominally calibrated mass spectrometer) using mass signals acquired by a precisely calibrated mass spectrometer calibrated with one or more reference calibrators. More specifically, as will be discussed in more detail below, in embodiments the nominally calibrated mass spectrometer may be configured to provide a scanning transmission window that allows one or more precursor ions to pass through the mass spectrometer. A collision cell located downstream of the mass spectrometer may receive the precursor ions, cause fragmentation of at least some of them, and generate a plurality of product ions. Ions exiting the collision cell, which may contain the product ions and several residual precursor ions, are received by a precision mass spectrometer. As will be discussed herein, the mass peaks associated with one or more residual precursor ions and their respective m / z ratios determined by the precision mass spectrometer may be used to calibrate the nominal mass spectrometer.

[0050] Figure 1 presents an exemplary hybrid mass spectrometer 100 according to various embodiments of this instruction. The hybrid mass spectrometer 100 is an electromechanical instrument for separating and detecting ions of interest from a given sample. The hybrid mass spectrometer 100 includes computing resources 130 for both performing control of system components and receiving and managing data generated by the hybrid mass spectrometer 100.

[0051] In the embodiment shown in Figure 1, the computing resource 130 is illustrated to have separate components, namely a controller 135 for instructing and controlling system components, and a data handler 140 for receiving and assembling data reports of detected ions of interest. Depending on the requirements, the computing resource 130 may have more or fewer components than those depicted, may be centralized, or may be distributed across system components. Typically, the detected ion signals generated by the nominal mass spectrometer 120 and the precision mass spectrometer 125 are formatted in the form of one or more mass spectra based on control information and other process information of various system components. Subsequent data analysis using a data analyzer (not shown in Figure 1) may be performed on the data report (e.g., on the mass spectra) to interpret the results of the mass spectrometry performed by the hybrid mass spectrometer 100.

[0052] In some embodiments, the hybrid mass spectrometer 100 may include some or all of the components illustrated in Figure 1. For the purposes of this description, the hybrid mass spectrometer 100 can be considered to include all of the illustrated components, although the computing resource 130 may not have direct control over or provide data handling to the sample separation / delivery component 105.

[0053] The sample separation / delivery component 105 may comprise any known delivery component for supplying the sample to the ion source 115. For example, in some embodiments, the sample separation / delivery component 105 may comprise a liquid chromatography (LC) column for separating the sample and eluting it into the ion source 115. In some embodiments, the sample separation / delivery component 105 may comprise gas chromatography (GC) for separating the components of the sample and providing the separated sample components to the ion source 115 at different time intervals. In some embodiments, the sample separation / delivery component 105 may comprise an open port interface (OPI) for capturing a sample to be diluted without additional pretreatment, diluting it, and transporting it to the ion source 115. The OPI may be installed to receive a sample diverted from the process flow, or it may be configured to receive a metered sample from a sample delivery device. In some aspects, the sample separation / delivery component 105 may comprise a combination of the OPI and a sample delivery device in the form of an acoustic droplet ejection component for ejecting droplets of the sample into the OPI.

[0054] Conveniently, in some respects, the sample can be delivered directly to the OPI as a droplet ejected from a sample reservoir by an acoustic droplet ejector (ADE). The combination of an ADE ejecting a sample droplet into the OPI may be referred to as acoustic emission mass spectrometry (AEMS).

[0055] In the context of this application, the separation / delivery system 105 comprises a delivery system capable of delivering a measurable amount of sample, typically a combination of analyte and an accompanying solvent sampling fluid, to an ion source 115 located downstream of the separation system 105 for ionizing the delivered sample. A nominal mass spectrometer 120 may receive ions generated from the ion source 115 for mass filtering and / or fragmentation and generate a nominal mass spectrometry result indicating the detected ions to be delivered to a data handler 140. The nominal mass spectrometer 120 operates to deliver the ions of interest to a precision mass spectrometer 125, which selectively separates the ions of interest from the generated ions received from the ion source 115 based on their mass / charge ratio and / or generates them by fragmentation, generating a precision mass spectrometry result indicating the detected ions to the data handler 140. It should also be understood that the ion source 115 may have various configurations as are known in the art.

[0056] For the purposes of this invention, the components of the hybrid mass spectrometer 100 may be considered to operate as a single system. Conventionally, the combination of the mass spectrometer 120 and the ion detector 125, along with the associated components of the controller 135 and data handler 140, is typically referred to as the mass spectrometer, while the sample separation / delivery device may be considered a separate component. However, it should be understood that although some of the components, such as the separation system 105, may be considered "separate," all components of the hybrid mass spectrometer 100 work together to analyze a given sample.

[0057] When operating a hybrid mass spectrometer with orthogonal nominal mass spectrometers and precision mass spectrometers, it is standard practice to perform both precision mass calibration using one or more reference standards and nominal mass calibration using one or more reference standards to evaluate the instrument's performance with respect to multiple transmission widths and scan speeds. Precision mass correction factors may be obtained to calibrate the precision mass spectrometer's analytical results over the entire range of expected operating conditions. However, with respect to the nominal mass spectrometer, multiple correction factors are obtained, each of which corresponds to one of the transmission width and scan speed pairs evaluated against the reference standard. The nominal mass spectrometer may be operated at any of the evaluated transmission width and scan speed pairs, and the corresponding correction factor may be applied.

[0058] In one embodiment, multiparameter interpolation may be applied during nominal mass calibration to enable the calibration of the nominal mass spectrometer to operate on transmission width and scan rate pairs that have not been previously evaluated. Through experimental methods, it has been demonstrated that applying multiparameter interpolation to obtain calculated correction factors corresponding to the actual transmission width and scan rate pairs used for the reference standard and analysis provides a more accurate calibration than applying the “nearest” correction factor from multiple correction factors.

[0059] In alternative embodiments, conventional nominal mass calibration may be eliminated, and mass data may be calibrated from precision mass spectrometry results, for example, by adjusting the headers of one or more data files, where the headers indicate one or more operating parameters of the nominal mass spectrometer, such as its bandwidth. Conveniently, such embodiments can avoid the time-consuming nominal mass calibration process and provide the ability to calibrate nominal mass results in all analysis runs.

[0060] Within an analysis run, multiple transmission window width and scan rate pairs, i.e., "experiments," are performed over the mass range of interest. Each experiment may correspond to data acquired at a certain percentage of the transmission bandwidth of the sample ion transmission window associated with the nominal mass spectrometer. As mentioned above, the data acquired at each percentage can be compiled into a data bin corresponding to a single "experiment."

[0061] Without calibration of the nominal mass spectrometer, in some embodiments, the transmission bandwidth associated with the nominal mass spectrometer may be shifted by a certain amount that varies across the mass range of interest. Data generated by the precision mass spectrometer can be used to correct the compilation of data corresponding to the nominal transmission bandwidth.

[0062] During analysis, sample ions are fragmented in the nominal mass spectrometer, potentially generating a complete mass spectrum outside the transmission window in the analysis results. Referring to Figures 2A, 2B, and 2C, in this embodiment, an extracted ion chromatogram (XIC) may be performed for each nth experiment as part of the analysis. As illustrated in the complete XIC spectrum of Figure 2A, the dense region of mass peaks around m / z 589 corresponds to unfragmented precursor ions transmitted by the nominal mass spectrometer through the transmission window. In addition, masses in the m / z range of 590.5–592.5 are also transmitted through the transmission window for this experiment.

[0063] Assuming the experimental precursor transmission width is less than half the transmission width, the data can then be adjusted to a precursor width such as transmission width / X = precursor width (for example, X=5). The XIC region corresponds to the transmission width or region for that experiment. Generally, the number of XICs (N) corresponds to the number of experiments required to cover the mass range of interest. A common approach then proceeds to select the transmitted mass (e.g., based on the highest intensity), compare the precision mass spectrometry results to the nominal mass spectrometry results, and identify the shift in the nominal mass spectrometry results.

[0064] Figure 2B illustrates the mass peaks at a retention time of approximately 0.92 minutes, corresponding to the highest intensity peak 202 in Figure 2A at m / z 589.3592. Within each XIC, m peaks 205 of the highest intensity are identified. In the example in Figure 2B, m=3, however, other numbers of high-intensity peaks may also be identified. As an example, the inventors evaluated m=3 and m=5 along with other numbers of peaks. Multiple peaks, conveniently used to compare nominal mass results with precise mass results, can provide a statistically robust measure for determining appropriate correction factors to apply to nominal mass spectrometry results.

[0065] The m peaks 205 with the highest intensity can be sorted by intensity, and for each of the m peaks 205, a mass spectrum can be generated at the corresponding peak vertex.

[0066] Referring to Figures 2C and 2D, the reference peak 305 can be identified and evaluated to confirm that it corresponds to the m / z within the transmission window for that experiment. Referring to Figure 3, a precursor profile can be generated from the nominal mass result by plotting the peak intensities against adjacent spectra before and after that of the nth experiment. In general, the number of adjacent spectra before and after the nth experiment corresponds to the number of experiments within the transmission window. The highest intensity precise mass peak 305 from the precise mass spectrometry result located at mass 590.3138 can be identified from the precise mass spectrum. Referring to Figure 3, the nominal mass spectrometry result can be examined to locate the corresponding nominal mass peak in the form of a center of mass, such as represented by the maximum transmission intensity, when the transmission window is scanned across the mass range.

[0067] Referring to Figure 4, the nominal mass peak 305 identified from each of the n experiments can be used to generate a plot of the nominal mass Δm / z against the corresponding high-precision mass spectrometry results and to generate a plot of the nominal mass dispersion for each high-precision mass measurement, which provides a basis for the calibration curve. As shown in Figure 4, the calibration curve is not strictly linear, but a general trend with increasing m / z exists.

[0068] Each point on the calibration curve corresponds to a plurality of m points. Generally, the m points are clustered within a reasonable distance from each other. Outlier points that deviate considerably from the rest of the m-1 points for a given group and could affect the overall calibration curve can be excluded. Generally, there is only one extreme outlier point per cluster of points, but some embodiments can allow for multiple outliers within a group of points.

[0069] Referring to Figure 6, exemplary calibration curves are plotted over the mass range of interest. The solid line calibration curve corresponds to the calibration curve in Figure 4 with outlier exclusion. The dashed line calibration curve is calculated from the same set of analysis results with outlier inclusion. As is evident from Figure 6, a small number of outliers can significantly affect the calibration curve within a small transmission window and distort the results.

[0070] To perform outlier exclusion, each cluster of m points may be evaluated based on the spread of its group or cluster of m points to provide a reliability metric. The reliability metric may be based on various known statistical methods, including, for example, Bayesian methods, standard deviation, etc. The reliability metric can be employed to assess whether a point is an outlier from the other points in its group. In some embodiments, local variation within an m / z range, e.g., an m / z range of about 50 to about 100 Daltons, may be employed to identify and optionally exclude outliers.

[0071] As a further measure of reliability, the overall calibration curve can be evaluated to confirm the overall trend with increasing m / z.

[0072] Reliability metrics and trend analyses may be reported in relation to the corrected analysis results to provide indication that the calibration is in line with expectations.

[0073] Calibration curves can be applied in several different approaches.

[0074] In the first embodiment, the analysis results may be corrected by applying a corresponding correction factor obtained from the calibration coefficient. In this embodiment, the data file may be written using the nominal mass results corrected based on the corresponding correction factor.

[0075] In a second embodiment, the correction factor may be applied by editing each experiment to reclassify the transmission window for that experiment based on the corresponding correction factor obtained from the calibration curve. For example, each experiment may be defined by a header that identifies the start and end points of the transmission window for that experiment. In this embodiment, the header may be edited by identifying the transmission window center for that experiment, applying the corresponding correction factor from the calibration curve to generate the corrected transmission window center, modifying the header start and end locations, and reflecting the corrected transmission window center. Thus, the definition of each transmission window may be modified to shift its window based on the correction factor obtained from the calibration curve for that transmission window.

[0076] In some respects, shifts in the transparency window can lead to gaps within the data set. One way to avoid gaps would be to perform the analysis with slightly overlapping transparency windows. Provided the shift is less than the overlap, the corrected data will contain no gaps. An alternative way to avoid gaps is to correct the transparency window by forcing its start point to coincide with the end point of the preceding transparency window. The end point of the corrected transparency window is then determined by adding half the transparency width to the center of the corrected transparency window. In this method, the transparency window widths will vary slightly from one another, but the variance is too small to affect the overall analysis results.

[0077] As an illustration, Figures 5A and 5B provide examples of high-precision mass spectrometry results. Figures 5C and 5D present the corresponding nominal mass spectrometry results for a given sample. The high-precision mass spectrometry result corresponding to the residual precursor mass peak 420 is at 535.2703 m / z, while the nominal mass spectrometry result 425 is at 537.751 m / z for the same sample. Therefore, there is a difference of 1.4807 m / z between the high-precision mass spectrometer and the nominal mass spectrometer. Since the high-precision mass spectrometer was previously calibrated using a reference standard, this indicates that the nominal mass spectrometer needs to be corrected by only 1.4807 m / z.

[0078] In some embodiments, the calibration curve and / or correction factor may be monitored after each analysis run to confirm general agreement. If the calibration shows a tendency in a particular direction, for example, if the correction factor increases during a run, there may be an indication that the instrument needs inspection or cleaning.

[0079] In one embodiment, the hybrid mass spectrometer may be calibrated based on captured analytical data by calibrating the precision mass spectrometer using a reference standard and then calibrating the nominal mass spectrometer based on the captured analytical data by comparing the nominal mass spectrometer results with the calibrated precision mass spectrometer results.

[0080] In some respects, hybrid mass spectrometers can be further calibrated by evaluating mass measurements from precision and nominal mass spectrometers and eliminating outlier cases before applying the measurement pairs to a calibration curve.

[0081] In some respects, a hybrid mass spectrometer can be calibrated by calibrating a precision mass spectrometer using a reference standard, calibrating a nominal mass spectrometer using a reference standard, calibrating the nominal mass spectrometer by generating multiple correction factors for each transmission window and scanning speed pair of the nominal mass calibration and interpolating between the generated multiple correction factors, and calibrating the nominal mass spectrometer with respect to the analytical transmission window and scanning speed pair used during the analysis.

[0082] In some embodiments, a hybrid mass spectrometer may be calibrated based on captured analytical data by calibrating a precision mass spectrometer using a reference standard and then calibrating the nominal mass spectrometer based on the captured analytical data by comparing the nominal mass spectrometer results with the calibrated precision mass spectrometer results, where the statistical measure of the comparison is outside the threshold, the nominal mass spectrometer is calibrated based on the nominal mass spectrometer calibration using the reference standard. The statistical measure may include, for example, an indication that there are insufficient points of comparison between the nominal mass spectrometer and the precision mass spectrometer to generate a calibration curve for the nominal mass spectrometer. The statistical measure may also include, for example, an indication that the points of comparison between the nominal mass spectrometer and the precision mass spectrometer fluctuate too much to generate a calibration curve for the nominal mass spectrometer.

[0083] In some embodiments, the nominal mass spectrometer may be a quadrupole mass spectrometer, and the precision mass spectrometer may be a time-of-flight (ToF) mass spectrometer. As an example, Figure 7 schematically depicts a mass spectrometer 200 including an ion source 210, a quadrupole mass spectrometer 220, a collision cell 230, a downstream time-of-flight (ToF) mass spectrometer 240, and a data processing module 250. A sample delivery device 260, such as a liquid chromatography (LC) column, can deliver the sample to the ion source 210, which can ionize one or more target analytes in the sample and generate a plurality of precursor ions.

[0084] Various ion sources can be employed. Some examples of such ion sources, but not limited to, include, electrospray ionization devices, nebulizer-assisted electrospray devices, chemical ionization devices, nebulizer-assisted atomization devices, matrix-assisted laser desorption / ionization (MALDI) ion sources, and photoionization devices.

[0085] Precursor ions are received by a quadrupole mass spectrometer 220, and those precursor ions passing through the mass spectrometer 220 are received by a downstream collision cell 230. At least some of the precursor ions undergo fragmentation within the collision cell, generating multiple product ions. Ions exiting the collision cell 230, which may contain product ions and residual precursor ions (i.e., those precursor ions that did not undergo fragmentation), are then received by a downstream time-of-flight (ToF) mass spectrometer 240, which generates an ion detection signal indicating the m / z ratio of the ions.

[0086] Since the ToF mass spectrometer 240 is calibrated through the use of a reference standard, the m / z ratio determined by the ToF mass spectrometer can be considered the accurate m / z ratio for the calibration of the nominal mass spectrometer. The ion detection signal is received by the data processing module 250, which analyzes the ToF signal in the manner discussed herein for the calibration of the quadrupole mass spectrometer.

[0087] In addition to the components shown in Figure 8, the mass spectrometer 200 may include other components, such as various ion guides, that are positioned upstream of the quadrupole mass spectrometer.

[0088] In this embodiment, the time-of-flight mass spectrometer 240 can be calibrated using one or more reference calibrators in a manner known in the art. Thus, in this embodiment, the time-of-flight mass spectrometer functions as a precision mass spectrometer.

[0089] In contrast, the quadrupole mass spectrometer functions as a nominal mass spectrometer. RF voltage source 300 and DC voltage source 302 can apply RF and DC voltages to the quadrupole rods of the quadrupole mass spectrometer to establish the transmission bandwidth of the quadrupole mass spectrometer. The RF voltage source can also apply RF voltage to the rods of the collision cell (which may include, for example, four rods arranged in a quadrupole configuration) to provide radial ion confinement. A controller 303 can control the operation of the RF and DC voltage sources.

[0090] For example, the controller can scan its transmission bandwidth and cause scanning of the frequency of the RF and / or DC voltage applied to the quadrupole rods of the quadrupole mass filter so that ions with different m / z ratios can pass through to the downstream time-of-flight mass spectrometer. More specifically, in this embodiment, the ion transmission bandwidth of the quadrupole mass spectrometer can be scanned over a mass range such that consecutive ion transmission windows overlap.

[0091] Such scanning of the transmission bandwidth of a quadrupole mass spectrometer can lead to variations in intensity associated with the mass signals of generated and residual precursor ions observed within different transmission windows.

[0092] In this embodiment, the mass detection signal generated by the ToF mass spectrometer can be stored in the precursor dimension as a plurality of data bins, each data bin having an m / z width that is a certain percentage of the m / z transmission width of the quadrupole mass filter 220. For example, each data bin may have an m / z width that is 1 / 5 of the transmission bandwidth of the mass filter 220. As stated above, each data bin is considered an experiment in this specification.

[0093] Referring again to Figure 2A, as mentioned above, in some embodiments, an extracted ion chromatogram (XIC) can be generated for every nth experiment. In this case, the nth experiment corresponds to a 2-Dalton databin ranging from 590.5 Daltons to 592.5 Daltons. As illustrated in Figure 2A, the highest intensity peak in this 2-Dalton databin corresponds to an m / z ratio of 589.3592. Figure 2B shows the ion chromatogram as a function of retention time associated with this 2-Dalton databin. The peaks in the ion chromatogram depicted in Figure 2B can be sorted based on their intensity.

[0094] In this embodiment, as shown in Figures 3B and 4A, the highest intensity peak at a retention time of 0.921 minutes is selected, and the mass spectrum associated with this peak is generated. The m / z ratio of the reference peak (here, peak 305) in the mass spectrum is compared to the m / z range covered by each precursor transmission window to verify that the m / z ratio of the reference peak falls within its transmission window.

[0095] The intensity of the reference mass peak in the experiments preceding the above 2 Dalton databin, which spans from 590.5 Daltons to 592.5 Daltons, and in the experiments following this databin, can be plotted as a function of the respective m / z values ​​of the quadrupole mass spectrometer 220, and the total number of experiments may be equal to the number of experiments per quadrupole transmission window.

[0096] The plotted data can then be used to determine the m / z calibration value for the transmission window of the nominal quadrupole mass spectrometer 220. For example, the mass center of the plot can be compared to a ToF-calibrated m / z ratio associated with a reference peak to determine the m / z offset required to correct the nominal calibration of the quadrupole mass spectrometer.

[0097] As described above, the header of the data compilation associated with each experiment, which identifies the m / z range corresponding to that experiment, can then be adjusted based on the m / z calibration offset determined through the process described above.

[0098] The following examples are provided to further illustrate various aspects of this instruction and are not necessarily provided to demonstrate the best way to implement this instruction and / or the best possible results that can be obtained.

[0099] (Examples) (Example 1) (Scan SWATH settings and operation)

[0100] Scanning SWATH runs were acquired using a SCIEX triple quad 6600+ mass spectrometer operating in SWATH acquisition mode. The following settings were applied during the scanning SWATH run: (1) The precursor transmission window was set to 10 m / z, with the mass range of 400 m / z to 900 m / z included in 0.5 seconds. These settings provided a compromise between identification and quantification performance. Several precursor transmission window sizes ranging from 3 m / z to 20 m / z, including the precursor range of 400 m / z to 900 m / z, were tested for mass spectrometry of the total proteome trypsin digest of yeast (budding yeast).

[0101] Optimal results in terms of discrimination and quantitative accuracy were achieved using a window size of 10 m / z. Further reduction of the window size would result in a higher number of discriminants due to less interference, but the resulting shorter effective storage time would decrease quantitative accuracy. Raw data was binned into 2 m / z bins in the quadrupole or precursor dimension to provide resolution in the Q1 dimension (i.e., the quadrupole or precursor dimension) enabling the effective use of the Q1 score. MS1 scanning was omitted for benchmarking purposes, and data were acquired in high-sensitivity mode.

[0102] The instrument control software calculates the RF / DC ramp applied to the quadrupole mass spectrometer. The ramp can be calculated from the experimental start transmission mass, stop transmission mass, transmission width, and cycle time. The calculation uses previously obtained calibrations to calculate the ramp with respect to mass DACS and resolution DACS. The quadrupole start mass can be calculated as experimental start mass - transmission width, and the quadrupole stop mass can be calculated as experimental stop mass + transmission width. This allows obtaining the correct precursor profile for all fragments at the boundaries of the experimental mass range. The collision energy can be calculated using the +2 rolling collision energy equation, which provides a linear relationship with respect to a given charge as a function of m / z.

[0103] This results in a small spread of collision energy, depending on the width of the transmission window relative to the scanned area. In these experiments, the effect is typically a spread of about 1 eV with respect to a given precursor.

[0104] Scanning SWATH calibration was automated by running pre-built batches and directly injecting the adjustment solutions (ESI-positive calibration solutions for the SCIEX X500 system (SCIEX) with 266.16, 354.21, 422.26, 609.28, and 829.54). The quadrupole response for each standard was measured at transmission window widths of 3, 5, 10, 15, and 20 m / z, and each transmission window width was also measured while scanning the transmission window of the quadrupole mass spectrometer at 500, 1,000, 2,000, and 3,000 m / z / sec. The recorded quadrupole responses for each condition were stored in a three-dimensional matrix, the dimensions of which were width, velocity, and m / z.

[0105] The values ​​stored in the matrix were the observed m / z values ​​derived from the theoretical m / z values. The observed precursor m / z was calculated from the number of current pulses relative to the total scan pulses, which is applied as a proportion of the scanned mass range plus the starting mass. Exact calibration curves were thus derived for each of the acquired scan speeds and widths. The curves were triple-linearly interpolated for scan speeds and widths between the results from the experimental parameters.

[0106] The instrument acquisition software was configured to store the ion detection response in a calculated 2m / z precursor separation bin, assuming the current number of ToF pusher pulses relative to the start of the scan to which the scanning SWATH offset curve described above is applied. The 2m / z precursor separation bins were organized in the data file as adjacent experiments, allowing for the extraction of precursor profiles for any given fragment ion in a given cycle by tracing fragment responses across experiments and normal chromatographic profiles across cycles.

[0107] (Example 2) In another experiment, scanning SWATH calibration was acquired while processing each sample file from the sample data itself. An automated algorithm, such as the one described in this instruction, was employed to identify the maximum residual precursor for each transmission window across the entire sample. This yielded several precise mass ToF measurements, each accurate ToF measurement paired with the mass center of the quadrupole mass trace associated with each quadrupole transmission region, where typically more than 10 per 100 Da are present.

[0108] For example, if three residual potential precursor ions are identified for each transmission region, and the scanning range is 500 Da with a transmission width of 10, then there will be 500 / 10 × 3 = 150 calibration point pairs consisting of the quadrupole mass and the precise mass of ToF. Since strong mass peaks within the quadrupole transmission region may not actually correspond to residual precursor ions, a selection algorithm was employed to filter out mass peaks using an outlier exclusion algorithm that considers local variance. In particular, local variations (e.g., variations within the range of approximately 50–100 Daltons) were employed to identify and optionally exclude outlier mass peaks.

[0109] Typically, the mass peak was evaluated relative to its neighbors within the 50–100 Da region. Once multipoint calibration curves were obtained, calibration was applied to the data by updating the start and end mass regions defined in the headers from each stored experiment, so that the center was calculated from the calibration function, while maintaining the continuity of boundaries in adjacent experiments. In some cases, the calibration curves can be used to identify outlier peaks that were mistakenly assumed to correspond to precursor ions.

[0110] Controllers and / or analysis modules, such as those discussed above, that are suitable for use in the practice of this instruction can be implemented using hardware, firmware, and / or software by employing techniques known in the art, as made known by this instruction. As an example, Figure 9 schematically depicts an example of an implementation of such a controller / analysis module 500, which generally includes, among other elements known in the art, a processor 500a (e.g., a microprocessor), at least one permanent memory module 500b (e.g., ROM), at least one transient memory module (e.g., RAM) 500c, and a bus 500d.

[0111] Bus 500d enables communication between the processor and various other components of the controller. In this example, the controller 500 may further include a communication module 500e configured to enable the transmission and reception of signals.

[0112] Instructions for use by the controller 500 (for example, for adjusting the DC voltage applied to the auxiliary electrode) can be stored in the permanent memory module 500b and transferred to the transient memory module 500c during runtime for execution. The controller 500 can also be configured to control the operation of other components of the mass spectrometer, such as the ion guide and the mass spectrometer, among other things.

[0113] While some aspects are described in the context of a system and / or apparatus, these aspects also represent a description of the corresponding method, and it is evident that a block or device corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding block or item or a feature of the corresponding apparatus. Some or all of the method steps may be performed by (or using) a hardware device such as a processor, microprocessor, programmable computer, or electronic circuit. In some embodiments, one or more of the most important method steps may be performed by such a device.

[0114] Depending on certain implementation requirements, embodiments of the present invention can be implemented in hardware and / or software. Implementation can be carried out using a digital storage medium having electronically readable control signals stored thereon, which cooperates (or can cooperate) with a programmable computer system so that each method can be carried out, such as a non-transient storage medium such as a floppy disk, DVD, Blu-ray®, CD, ROM, PROM, and EPROM, EEPROM, or FLASH® memory. Thus, the digital storage medium may be computer-readable.

[0115] Those skilled in the art will understand that various modifications can be made to the above embodiments without departing from the scope of this teaching.

Claims

1. A method for calibrating a hybrid mass spectrometer equipped with a precision mass spectrometer combined with a nominal mass spectrometer, wherein the method is: Calibrating the precision mass spectrometer using one or more reference standards, Using the nominal mass spectrometer and the precision mass spectrometer, the sample is analyzed and one or more corresponding nominal mass signals and calibrated precision mass signals associated with the sample are generated. The nominal mass spectrometer is calibrated by comparing the nominal mass signal with the calibrated precision mass signal. Methods that include...

2. The method according to claim 1, further comprising storing one or more calibration parameters associated with the calibrated nominal mass spectrometer.

3. The method according to any one of claims 1-2, wherein the nominal mass spectrometer is configured to provide a plurality of ion permeable windows.

4. The method according to claim 3, further comprising a controller that communicates with the nominal mass spectrometer, the controller adjusting at least one parameter of an ion permeable window associated with the nominal mass spectrometer in order to generate the plurality of ion permeable windows.

5. The method according to claim 4, wherein the nominal mass spectrometer comprises a plurality of rods arranged in a multi-pole configuration, and the nominal mass spectrometer is configured for applying RF and DC voltages to the plurality of rods.

6. The method according to claim 5, wherein the controller causes a change in at least one parameter associated with at least one of the RF and DC voltages in order to generate the plurality of ion permeable windows.

7. The method according to any one of claims 4-6, wherein the controller causes the scanning of the at least one parameter to scan the transmission bandwidth associated with the nominal mass spectrometer.

8. The method according to any one of claims 1 to 7, wherein the precision mass spectrometer comprises a time-of-flight (ToF) mass spectrometer.

9. A method for calibrating a mass spectrometer having at least a first and a second mass spectrometer, wherein the second mass spectrometer is located downstream of the first mass spectrometer, and the method is Calibrating the second mass spectrometer using one or more reference standards, Using the first mass spectrometer, a first measurement of the mass signal of the precursor ion is generated, and using the second mass spectrometer, a second measurement of the mass signal of each of the precursor ions is generated. The first mass spectrometer is calibrated by comparing the first and second measured values ​​of the mass signal. Methods that include...

10. The method according to claim 9, wherein the first mass spectrometer comprises a quadrupole mass spectrometer.

11. The method according to any one of claims 9-10, wherein the second mass spectrometer comprises a time-of-flight (ToF) mass spectrometer.

12. The method according to any one of claims 9 to 11, further comprising a collision cell positioned between the first mass spectrometer and the second mass spectrometer, wherein the collision cell receives the precursor ion from the first mass spectrometer and generates a plurality of product ions through the fragmentation of the precursor ion.

13. The method according to any one of claims 9-12, wherein the first measurement of the mass signal corresponds to the m / z ratio of the precursor ion identified based on at least one nominal setting of the first mass spectrometer.

14. The step of measuring the second mass signal is: Identifying the mass signal associated with the precursor ion in the mass spectrum of ions exiting the collision cell generated by the second mass spectrometer, Assigning an m / z ratio to the identified mass signal and The method according to claim 13, including the method described in claim 13.

15. The method according to claim 14, wherein the at least one nominal setting comprises either a transmission width or a speed for scanning the transmission width.

16. The method according to any one of claims 9-15, wherein the first mass spectrometer comprises a plurality of rods arranged in a multi-pole configuration, and RF and DC voltages can be applied to the plurality of rods.

17. The method according to claim 16, wherein the second mass spectrometer comprises a time-of-flight mass spectrometer.

18. A hybrid mass spectrometer, wherein the hybrid mass spectrometer is A nominal mass spectrometer configured to provide multiple permeation windows for the passage of at least one precursor ion, A collision cell located downstream of the nominal mass spectrometer, the collision cell receiving the at least one precursor ion, causing its fragmentation, and generating a plurality of product ions, A precision mass spectrometer for receiving ions exiting the aforementioned collision cell and generating their mass spectra, A mass spectrometer for receiving one or more operating parameters of the nominal mass spectrometer and the mass spectrum generated by the precision mass spectrometer. Equipped with, The one or more operating parameters include the transmission width and the speed for scanning the transmission width. The aforementioned mass spectrometer is To identify the mass signal associated with the precursor ion in the aforementioned precision mass spectrometer, Calibrating the nominal mass spectrometer based on the m / z ratio of the precursor ion in the mass spectrum and one or more operating parameters of the nominal mass spectrometer. A hybrid mass spectrometer configured to perform the following actions.

19. The hybrid mass spectrometer according to claim 18, wherein the nominal mass spectrometer comprises a plurality of rods arranged in a multi-pole configuration, and the nominal mass spectrometer is configured for applying RF and DC voltages to the plurality of rods.

20. The hybrid mass spectrometer according to any one of claims 18-19, wherein the precision mass spectrometer comprises a time-of-flight mass spectrometer.

Images