CN116897410A - Mass spectrometer calibration - Google Patents

Abstract

In one aspect, systems and methods for calibrating a hybrid mass spectrometer are provided. Calibration may include, for example, accurate and nominal quality calibration performed across multiple transmission width and scan speeds applied to each reference standard, each reference standard ideally representing a different m/z, to produce a matrix of correction factors corresponding to each transmission width and scan speed pair at the m/z value of the reference standard evaluated. A multiparameter interpolation may be applied to identify correction factors to be used for subsequent analysis of pairs of transmission widths and scan speeds that are different from the plurality of transmission widths and scan speeds used to generate the correction factors.

Description

Mass spectrometer calibration

RELATED APPLICATIONS

The present application claims priority from U.S. provisional application No.63/250,872 entitled "Mass Spectrometer Calibration" filed on 9/30 of 2021 and U.S. provisional application No.63/139,682 entitled "Mass Spectrometer Calibration" filed on 20 of 2021, each of which is incorporated herein by reference in its entirety.

Background

A mass spectrometer is an analytical instrument used to analyze a sample to identify its constituents and/or to measure the amount of analyte in a given sample.

A hybrid mass spectrometer incorporating two different types of mass analyzers provides the benefit of providing different performance characteristics from each of the mass analyzers within a single instrument.

One useful type of hybrid mass spectrometer includes a tandem combination of a nominal mass spectrometer and an accurate mass spectrometer. A number of manufacturers offer hybrid mass spectrometer products with a variety of different mass analyzer types, including multipoles, orthogonal time of flight (ToF), electrostatic traps, linear ion traps, etc. SCIEX, for example, provides a series of QTOF instruments that combine triple quadrupole mass spectrometers with orthogonal time of flight (ToF) mass spectrometers as respective nominal and accurate mass spectrometers.

Mass spectrometer performance varies with a variety of conditions including environment, contamination, sample composition, instrument status, variability of individual instruments, etc. Standard practice in operating mass spectrometers is to perform calibration operations at regular intervals to ensure accurate operation of the instrument. The calibration of a hybrid mass spectrometer can be complex because each of the two different types of mass spectrometers need to be calibrated with respect to the other. Such hybrid calibration generally involves two separate calibration operations, namely a nominal mass calibration operation and an accurate mass calibration, both performed in accordance with known reference standards to ensure that each of the two mass analyzers is operating within specifications and within a defined range of variation of the other.

Accurate mass calibration operations generally involve providing a pure reference standard (e.g., pure cesium reference standard) of known composition for analysis by an accurate mass spectrometer. Calibration may be performed by analyzing the reference standard, comparing the analysis results against expected values, and correcting for any offset between the accurate mass spectrometer analysis results and the expected values of the reference standard.

Accurate mass calibration operations may be performed daily, weekly or monthly, depending on the user requirements, and typically require about 20-30 minutes to complete. Many users prefer to perform at least one accurate quality calibration every 24 hours to ensure that the daily analysis result set is accurate.

The nominal mass calibration operation is more time consuming and complex than the accurate mass calibration operation because variations in the analysis results of the nominal mass instrument typically include m/z dependent components. The nominal mass calibration operation typically requires calibration according to known reference standards and collecting the analysis results at each different scan speed and transmission window width to be used for analysis. For example, while providing a reference standard, the instrument may be set to a given transport width and stepped through a plurality of different scan speeds to collect calibration results for the transport width at each scan speed. This process can then be repeated over multiple transmission widths to generate a matrix of calibration results covering the desired mass range. Based on this matrix of calibration results, the transmission widths can be realigned at each transmission window such that the analysis results produced by the nominal mass analyzer match the results produced by the accurate mass analyzer.

Some commercial instruments alleviate the burden of running nominal mass calibration operations by performing different scan speeds and transmission widths as batch operations. With this feature, the user sets the hybrid mass spectrometer to inject a batch process defining the step-wise scan speed and transmission window width to be evaluated, along with one or more criteria. Once the batch process is performed, the instrument will automatically step through the scan speed and transmission window width to build up a matrix of calibration results without further user intervention. The instrument can typically complete a calibrated batch run in about 30 minutes, depending on the number of scan speeds and transmission bandwidths defined for the batch run. The user must then check the calibration data based on the varying standard threshold level in order to complete the calibration of the instrument.

While the user time is released by using a batch procedure, the instrument is busy running calibration operations and consumes reference standards and solvents.

Disclosure of Invention

The problems described above associated with generating calibrated mass data in a hybrid mass spectrometer can lead to wasted time and inaccurate analysis results. Additional problems exist with existing calibration methods. For example, the inventors evaluated the effectiveness of standard calibration operations and observed that while the instrument appeared to be calibrated when run on an injected reference standard during the calibration operation, the calibration may be shut down when the instrument was operated on an analysis sample at full sample load. The inventors observed that resolution drifts when a full sample load (such as a large proteome sample) was run on an instrument that was successfully calibrated using conventional reference standard injection. This may be due to contamination on the ion optics causing drift when the thermionic beam generated by the full sample load contacts the ion optics. Thus, conventional calibration of low injection based on reference standards does not seem to be necessarily effective for large proteome samples.

While the inventors have confirmed that SCIEX commercial hybrid mass spectrometers remain stable nominal mass calibration for months, the user chooses to recalibrate the nominal mass analyzer and accurate mass analyzer at a higher pace, such as weekly and even daily. In view of the significant time, resources and capital invested in research effort, it is appreciated that users prefer to calibrate a nominal mass spectrometer and an accurate mass spectrometer together to avoid the opportunity to produce erroneous analysis results.

In view of these end use requirements, the inventors developed a new method of hybrid mass spectrometer calibration of the present invention that leverages accurate mass calibration to calibrate a nominal mass spectrometer, which proved to be stable and accurate and with little variation between calibration events. Thus, the nominal mass spectrometer can be calibrated while running the sample based on the calibrated accurate mass spectrometer analysis results. Furthermore, this method enables verification and reporting to confirm the analysis results of each sample run without the need to recalibrate the nominal mass spectrometer. In other words, embodiments may utilize data generated using a combination of a nominal mass analyzer (i.e., a mass analyzer that has not been calibrated via one or more calibrators) and an accurate mass analyzer (i.e., a mass analyzer that has been calibrated using one or more calibrators) to derive an m/z correction of a mass signal obtained for a sample under study.

In some embodiments, the quality data may be compiled such that the quality data is separated based on a transmission window of a nominal quality analyzer used to collect the data. In such embodiments, each data compilation associated with a particular transmission window of the nominal mass analyzer may include an indicator (e.g., in the form of a file header) that provides information about the transmission window of the nominal mass analyzer. In some such embodiments, the calibration correction values may be used to adjust the head data to reposition the mass analysis results in each transmission window.

In some embodiments, a hybrid mass spectrometer comprising a nominal mass spectrometer and an accurate mass spectrometer is operable to perform a data independent acquisition operation and calibrate the nominal mass spectrometer based on accurate mass analysis results. The data independent operation may include, for example, a scanning quadrupole data independent acquisition operation.

In some aspects, the hybrid mass spectrometer is operable to perform mass analysis on a sample to generate mass analysis results, and calibrate compilation of mass data by taking into account differences between one or more nominal operational settings of the nominal mass spectrometer (e.g., transmission bandwidth of a mass filter) and actual operational settings of the nominal mass spectrometer resulting from analysis of mass data generated via an accurate mass analyzer calibrated by one or more calibrators. Calibration may include revising the nominal mass analysis results based on the calibration to generate calibrated nominal mass analysis results. In some aspects, a calibration metric may be evaluated that indicates a change between a current calibration and a previous calibration.

In an embodiment, the calibration may include evaluating the mass analysis results to identify 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 cover a mass range to be calibrated for the sample being mass analyzed. In some embodiments, each identified precursor ion may be paired with a centroid of a corresponding nominal mass analysis result within the transmission window to identify any offset or change between an expected value provided by the accurate mass result and a measured value provided by the nominal mass result. In some embodiments, such an offset may be used to adjust the header of the data compilation identifying the transmission bandwidth of the transmission window associated with the nominal mass spectrometer.

Conveniently, a plurality of precursor ions may be identified in each transmission window to provide a plurality of corresponding offset values for that transmission window. In some aspects, each of the precursor ions may be evaluated relative to each other to identify any outliers to discard. The correction factor for each transmission window may be generated based on a plurality of offset values for the transmission window. In some aspects, the spread or change in offset values may be evaluated to confirm a confidence measure of the generated correction factor.

In some aspects, each of the plurality of correction factors may also be evaluated to confirm each other's consistency. For example, consistency may be confirmed by fitting a trend line to a plurality of correction factors and evaluating each correction factor to confirm that it is within the expected range of the trend line. In some aspects, consistency may be confirmed by evaluating each correction factor to confirm that it is within the expected range of adjacent correction factors.

Each correction factor may be applied to move the quality analysis results within a corresponding transmission window based on an offset defined by the correction factor. In some aspects, the correction factor may be applied by correcting all quality analysis results based on an offset defined within the corresponding transmission window. In some aspects, the correction factor may be applied by rewriting the header data to reposition the quality analysis results in each transmission window based on the defined offset. In some aspects, repositioning may include repositioning quality analysis results within the transmission window while maintaining continuity at the boundary of the transmission window and an adjacent transmission window. In some aspects, the transmission windows may include an overlap, and repositioning may include repositioning quality analysis results within the transmission window while maintaining continuity at the boundary of the transmission window and an adjacent transmission window.

In a related aspect, a method for calibrating a hybrid mass spectrometer including a nominal mass spectrometer and an accurate mass spectrometer is disclosed, the method comprising analyzing a sample using the hybrid mass spectrometer and collecting accurate mass analysis results and nominal mass analysis results of the sample. For at least one mass transfer window of the nominal mass spectrometer, nominal mass analysis results and accurate mass analysis results within the mass transfer window may be evaluated to identify at least one difference between the nominal mass analysis results and the accurate mass analysis results. Based on the identified differences, nominal mass analysis results within the mass transfer window may be corrected to align them with accurate mass analysis results. In some embodiments, the identifying of the at least one difference includes identifying at least one precursor ion in the accurate mass analysis result and comparing an m/z ratio of the at least one precursor ion derived from the accurate mass analysis result to a corresponding m/z ratio corresponding to the at least one precursor ion derived from the nominal mass analysis result.

In related aspects, systems and/or methods for calibrating a hybrid mass spectrometer are provided. Calibration may include, for example, nominal and accurate quality calibration performed across multiple transmission width and scan speeds applied for each reference standard, each reference standard ideally representing a different m/z ratio, to produce a matrix of correction factors corresponding to each transmission width and scan speed pair at the m/z value of the reference standard evaluated. A multiparameter interpolation may be applied to identify correction factors to be used for subsequent analysis of transmission width and scan speed pairs, which are different from the plurality of transmission widths and scan speeds used to generate the correction factors.

In a related aspect, a method for calibrating a hybrid mass spectrometer having an accurate mass analyzer in combination with a nominal mass analyzer is disclosed, the method comprising: the method includes calibrating an accurate mass analyzer using one or more reference standards, analyzing the sample using the nominal mass analyzer and the accurate mass analyzer to generate one or more corresponding nominal mass signals and calibrated accurate mass signals associated with the sample, and calibrating the nominal mass analyzer by comparing the nominal mass signals to the calibrated accurate mass signals.

In some embodiments, the controller may adjust one or more operating parameters of the nominal mass analyzer to generate a plurality of precursor transmission windows, wherein the transmission windows partially overlap. The precursor ions passing through each of the transmission windows may be received by a collision cell in which at least a portion of the precursor ions may be fragmented to generate a plurality of product ions. The product ions and any residual precursor ions may be received by an accurate mass analyzer (e.g., a time of flight (ToF) mass analyzer). The accurate mass analyser may generate a signal indicative of the mass of the product ions and the one or more residual precursor ions. In some such embodiments, the transmission window may be scanned and the signals generated by the accurate mass analyzer may be collected.

The operating parameters associated with scanning of the transmission window of the nominal mass analyzer may be used to determine a lower m/z end, a higher m/z end, a transmission bandwidth, and a scanning rate associated with scanning the transmission window. For example, when the nominal mass analyzer is a quadrupole mass analyzer, the operating parameters may include RF and DC voltages applied to the quadrupole rods to establish a transmission bandwidth associated with the quadrupole mass analyzer and one or more parameters associated with the adjustment of the RF and DC voltages for scanning the transmission window.

In some embodiments, a controller in communication with the nominal mass analyzer may be used to adjust one or more operating parameters associated with the nominal mass analyzer, such as RF and/or DC voltages applied to quadrupole rods of the quadrupole mass analyzer, for establishing a transmission window for ion passage and for scanning a transmission bandwidth associated with the transmission window.

In some embodiments, the quality signal generated by the accurate quality analyzer (e.g., toF quality analyzer) may be stored such that the quality signal data associated with each transmission window is stored in a plurality of data intervals, where each data interval corresponds to a portion of the transmission bandwidth of a given transmission window. As described above, each data interval is considered herein to correspond to an "experiment". In some such embodiments, for each nth experiment (e.g., for each 5 th experiment), an extracted ion chromatogram (XIC) may be generated and one or more peaks may be identified as likely corresponding to residual precursor ions (i.e., precursor ions that have not been fragmented). For peaks considered to correspond to precursor ions, corresponding peaks in multiple experiments before and after the nth experiment can be identified. In some embodiments, the total number of experiments may correspond to the transmission bandwidth of the transmission window.

The intensity profile of the mass peaks across these experiments as a function of nominal transmission bandwidth can be plotted and used to obtain calibration corrections for the corresponding transmission windows. For example, the m/z ratio associated with residual precursor ions detected via an accurate mass analyzer may be compared to the centroid of the intensity profile of the mass peak as a function of the nominal m/z ratio to arrive at a calibration correction factor.

As discussed further below, such calibration correction factors may be used to edit the header of a data file containing nominal parameters associated with the transmission window, such as a start m/z and an end m/z associated with the transmission bandwidth.

In a related aspect, a hybrid mass spectrometer is disclosed that includes a nominal mass analyzer configured to provide a plurality of transmission windows that allow passage of at least one precursor ion, and a collision cell positioned downstream of the nominal mass analyzer for receiving the at least one precursor ion and causing fragmentation thereof to generate a plurality of product ions. An accurate mass analyser is positioned downstream of the collision cell for receiving ions exiting the collision cell and generating a mass spectrum thereof. The analysis module is configured to receive one or more operating parameters of the nominal mass analyzer and a mass spectrum generated by the accurate mass analyzer. The analysis module is further configured to identify a mass signal associated with a precursor ion in a mass spectrum generated by an accurate mass analyzer and calibrate a nominal mass analyzer based on an m/z ratio of precursor ions in the mass spectrum and the one or more operating parameters of the nominal mass analyzer.

In related aspects, systems and/or methods are provided for calibrating mass data acquired by a hybrid mass spectrometer that includes a nominal mass analyzer and an accurate mass analyzer. Calibration may include adjusting compilation of quality data with quality data generated by an accurate quality analyzer to account for any differences between compiled data indicative of nominal parameters of a nominal quality analyzer and values of those parameters derived via analysis of the quality data generated by the accurate quality analyzer.

In some embodiments, the nominal mass analyzer may include a plurality of rods arranged in a multipole (e.g., quadrupole) configuration and configured for applying RF and DC voltages thereto. In some such embodiments, the accurate mass analyzer may be a time of flight (ToF) mass analyzer calibrated using one or more reference standards.

A further understanding of the various aspects of the present teachings can be obtained by reference to the following detailed description and related drawings, which are briefly described below.

Drawings

Fig. 1 is an example of a hybrid mass spectrometer according to an embodiment of the present teachings.

Fig. 2A depicts an extracted ion chromatogram (XIC) of a sample.

Fig. 2B depicts a Total Ion Chromatogram (TIC) corresponding to the region around the mass peak 202 of XIC.

Fig. 2C and 2D are spectra confirming that the fundamental peak corresponds to m/z in the transmission window.

Fig. 3 shows nominal mass analysis results.

Fig. 4 shows a graph of nominal mass versus corresponding accurate mass analysis.

Fig. 5A and 5C show accurate mass analysis results.

Fig. 5B and 5D show nominal mass analysis results.

FIG. 6 presents an exemplary calibration curve plotted across a mass range of interest.

Fig. 7 shows a hybrid mass spectrometer according to an embodiment of the present teachings.

Fig. 8 schematically depicts an example of an implementation of a controller/analysis module suitable for use in the practice of embodiments of the present teachings.

Detailed Description

It should be appreciated that for clarity, the following discussion will set forth various aspects of embodiments of the disclosure, while omitting certain specific details where convenient or appropriate. For example, discussion of the same or similar features in alternative embodiments may be somewhat simplified. Well-known ideas or concepts may not be discussed in detail for brevity. One of ordinary skill will recognize that some embodiments of the present disclosure may not require certain specific details in each implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it is apparent that modifications or variations of the described embodiments may be readily made in accordance with well known general knowledge without departing from the scope of the present disclosure. The following detailed description of the embodiments should not be taken as limiting the scope of the applicant's teachings in any way.

As used herein, the terms "about" and "substantially equal" relate to a variation in the number of values, which may occur, for example, by: measurement or treatment processes in the real world; unintentional errors in these processes; differences in the manufacture, source, or purity of the compositions or reagents; etc. Generally, the terms "about" and "substantially" as used herein mean greater or less than 10% of the stated value or range of values or complete condition or state. For example, a concentration value of about 30% or substantially equal to 30% may refer to a concentration between 27% and 33%. These terms also refer to equivalent variations that will be recognized by those skilled in the art, so long as the variations do not encompass known values practiced in the art.

As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".

The term "nominal mass spectrometer" or "nominal mass analyzer" refers to a mass spectrometer or mass analyzer that is not calibrated via one or more reference calibrators. The term "accurate mass spectrometer" or "accurate mass analyzer" refers to a mass spectrometer or mass analyzer that has been calibrated by one or more reference calibrators.

The present teachings relate generally to methods and systems for calibrating mass data generated by a hybrid mass spectrometer, and in particular, for calibrating a nominally-calibrated mass analyzer (e.g., one or more operating parameters of the nominally-calibrated mass analyzer) via mass signals obtained by an accurately-calibrated mass analyzer that is calibrated via one or more reference calibrators. More specifically, as discussed in more detail below, in an embodiment, the nominally calibrated mass analyzer may be configured to provide a scanning transmission window that allows one or more precursor ions to pass through the mass analyzer. A collision cell positioned downstream of the mass analyzer may receive the precursor ions and cause at least a portion thereof to fragment to generate a plurality of product ions. Ions exiting the collision cell (which may include product ions as well as some residual precursor ions) are received by an accurate mass analyzer. As discussed herein, the mass peaks associated with one or more residual precursor ions and their respective m/z ratios determined by the accurate mass analyzer may be used to calibrate the nominal mass analyzer.

Fig. 1 presents an exemplary hybrid mass spectrometer 100 according to various embodiments of the present teachings. 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 to perform control of system components and to receive and manage data generated by the hybrid mass spectrometer 100.

In the embodiment of fig. 1, computing resources 130 are shown as having separate components: a controller 135 for directing and controlling the system components, and a data handler 140 for receiving and assembling data reports of the detected ions of interest. Depending on the requirements, the computing resources 130 may include more or fewer components than those depicted, may be centralized, or may be distributed across system components. In general, the detected ion signals generated by the nominal mass analyzer 120 and the accurate mass analyzer 125 are formatted into the form of one or more mass spectra based on control information as well as other process information for various system components. Subsequent data analysis using a data analyzer (not shown in fig. 1) may be performed on the data report (e.g., on the mass spectrum) in order to interpret the results of the mass analysis performed by the hybrid mass spectrometer 100.

In some embodiments, the hybrid mass spectrometer 100 can include some or all of the components as shown in fig. 1. For purposes of this explanation, the hybrid mass spectrometer 100 may be considered to include all of the components shown, but the computing resources 130 may not directly control the sample separation/delivery component 105 or provide data handling to the sample separation/delivery component 105.

The sample separation/delivery assembly 105 may include any known delivery assembly for supplying a sample to the ion source 115. For example, in some embodiments, the sample separation/delivery assembly 105 may include a Liquid Chromatography (LC) column for separating the sample and eluting the sample to the ion source 115. In some embodiments, the sample separation/delivery assembly 105 may include Gas Chromatography (GC) for separating components of a sample and providing the separated sample components to the ion source 115 at different time intervals. In some embodiments, the sample separation/delivery assembly 105 may include an Open Port Interface (OPI) for capturing, diluting and transporting the diluted sample to the ion source 115 without additional pre-processing. The OPI may be positioned to receive diverted samples from the process stream or may be arranged to receive metered samples from a sample delivery device. In some aspects, sample separation/delivery assembly 105 may include a combination of an OPI and a sample delivery device in the form of an acoustic drop ejection assembly for ejecting sample drops into the OPI.

Conveniently, in some aspects, the sample may be delivered directly to the OPI as a droplet ejected from the sample reservoir by an Acoustic Droplet Ejector (ADE). The combination of ADE that ejects sample droplets into an OPI may be referred to as Acoustic Ejection Mass Spectrometry (AEMS).

In the context of the present application, the separation/delivery system 105 comprises a delivery system capable of delivering a measurable amount of sample (typically a combination of analyte and accompanying solvent sampling fluid) to an ion source 115 disposed downstream of the separation system 105 for ionizing the delivered sample. The nominal mass spectrometer 120 receives ions generated from the ion source 115 for mass filtering and/or fragmentation, and may generate nominal mass analysis results indicative of the detected ions for delivery to the data handler 140. The nominal mass analyzer 120 is operable to selectively separate and/or generate ions of interest from generated ions received from the ion source 115 based on the mass-to-charge ratio of the generated ions received from the ion source 115 and deliver the ions of interest to the accurate mass spectrometer 125, which accurate mass spectrometer 125 generates accurate mass analysis results indicative of the detected ions to the data handler 140. It should also be appreciated that the ion source 115 may have a variety of configurations known in the art.

For purposes of the present application, the components of the hybrid mass spectrometer 100 may be considered to operate as a single system. Conventionally, the combination of the mass analyzer 120 and ion detector 125, and the associated components of the controller 135 and data handler 140 are commonly referred to as a mass spectrometer, and the sample separation/delivery apparatus may be considered as a separate component. However, it should be appreciated that while some of the components may be considered "separate," such as separation system 105, all components of the hybrid mass spectrometer 100 operate cooperatively to analyze a given sample.

When operating a hybrid mass spectrometer with orthogonal nominal and accurate mass spectrometers, standard practice is to perform both accurate mass calibration using one or more reference standards and nominal mass calibration using one or more reference standards and evaluating instrument performance for a variety of transmission widths and scan speeds. An accurate mass correction factor can be obtained for an accurate mass spectrometer to calibrate its analysis results across the entire range of expected operating conditions. However, for a nominal mass spectrometer, a plurality of correction factors are obtained, each correction factor corresponding to one of the transmission width and scan speed pairs evaluated against the reference standard(s). The nominal mass spectrometer may operate at any one of the estimated transmission width and scan speed pairs and a corresponding correction factor may be applied.

In an embodiment, multiparameter interpolation may be applied to allow calibration of a nominal mass spectrometer to operate at transmission width and scan speed pairs that have not been previously evaluated during nominal mass calibration. By experimentation, it has been demonstrated that applying multiparameter interpolation to obtain a calculated correction factor corresponding to the reference standard(s) and the actual transmission width and scan speed pair for analysis provides a more accurate calibration than applying the "closest" correction factor of the plurality of correction factors.

In alternative embodiments, conventional nominal mass calibration may be eliminated, and mass data may be calibrated from accurate mass analysis results, for example, via adjusting the header of one or more data files, wherein the header indicates one or more operating parameters of the nominal mass spectrometer, such as its bandwidth. Conveniently, such an embodiment may provide the ability to avoid time consuming nominal mass calibration procedures and provide calibration of nominal mass results in each analysis run.

In the analysis run, a plurality of transmission window width and scanning speed pairs ("experiments") are performed over the mass range of interest. Each experiment may correspond to data obtained, for example, in a portion of the transmission bandwidth of a sample ion transmission window associated with a nominal mass spectrometer. As described above, the data obtained in each section may be compiled into a data section corresponding to one "experiment".

In an embodiment, without calibration of the nominal mass spectrometer, the transmission bandwidth associated with the nominal mass spectrometer may be shifted by an amount that varies across the mass range of interest. The data generated by the accurate mass spectrometer can be used to correct the compilation of data corresponding to the nominal transmission bandwidth.

During analysis, sample ions may be fragmented in a nominal mass spectrometer, thereby generating a potentially full mass spectrum in the analysis results outside the transmission window. Referring to fig. 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 shown in the full XIC spectrum of fig. 2A, dense areas of mass peaks around m/z 589 correspond to unfractionated precursor ions transmitted by a nominal mass spectrometer through the transmission window. In addition, for this experiment, masses in the m/z range from 590.5 to 592.5 will also pass through the transmission window.

Assuming that the experimental precursor transport width is less than or equal to half the transport width, the data may be organized into precursor widths, such as transport width/x=precursor width (x=5 for example). The XIC region corresponds to the transmission width or region of the experiment. In general, the number of XICs (N) corresponds to the number of experiments required to span the mass range of interest. The transmitted quality is then selected using a general method (e.g., based on highest intensity) and the accurate quality analysis results are compared against the nominal quality analysis results to identify shifts in the nominal quality analysis results.

FIG. 2B illustrates a mass peak with a retention time of about 0.92 minutes, corresponding to the highest intensity peak 202 at m/z 589.3592 of FIG. 2A. Within each XIC, the m peaks 205 of highest intensity are identified. In the example of fig. 2B, m=3, but other numbers of high intensity peaks can be identified. For example, the inventors evaluated m=3, m=5, and other numbers of peaks. Conveniently, the plurality of peaks used to compare the nominal mass result to the accurate mass result may provide a statistically robust measure for determining an appropriate correction factor to be applied to the nominal mass analysis result.

The m peaks 205 of highest intensity may be ordered by intensity, and for each of the m peaks 205, a mass spectrum may be generated at the corresponding peak top point.

Referring to fig. 2C and 2D, the base peak 305 can be identified and evaluated to confirm that it corresponds to m/z within the transmission window of the experiment. Referring to fig. 3, a precursor profile can be generated from nominal mass results by plotting the peak intensities of adjacent spectra before and after 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 accurate mass peak 305 (located at mass 590.3138) from the accurate mass analysis results can be identified from the accurate mass spectrum. Referring to fig. 3, nominal mass analysis results may be examined to locate a corresponding nominal mass peak, for example, in the form of a centroid, as represented by a maximum transmission intensity when scanning a transmission window across a mass range.

Referring to fig. 4, the nominal mass peaks 305 identified from each of the n experiments can be used to generate a plot of nominal mass Δm/z against the corresponding accurate mass analysis results to generate a plot of nominal mass change for each accurate mass measurement, which provides a basis for a calibration curve. As indicated in fig. 4, the calibration curve is not strictly linear, but has an overall trend as m/z increases.

Each point of the calibration curve corresponds to m (m being a plurality of) points. In general, m points are clustered within a reasonable distribution range of each other. In some cases outliers that are very different from the remaining m-1 points of the group and that may affect the overall calibration curve may be culled. In general, there is only one extreme outlier point per point cluster, but some embodiments may allow multiple outliers to exist within a group of points.

Referring to FIG. 6, an exemplary calibration curve is plotted across a mass range of interest. The calibration curve of the solid line corresponds to the calibration curve of fig. 4 with outliers removed. The calibration curve of the dashed line is calculated from the same analysis results in which the outlier points are included. As is apparent from fig. 6, a small number of outlier points can greatly affect the calibration curve within a small number of transmission windows, skewing the results.

For outlier rejection, each cluster of m points may be evaluated to provide a confidence metric based on the set of m points or the spread of clusters. The confidence measure may be based on a variety of known statistical methods including, for example, bayesian, standard deviation, and the like. Confidence measures may be used to evaluate whether a point is an outlier to other points in the set. In some embodiments, local variations in the m/z range, such as an m/z range of about 50 to about 100 daltons, may be used to identify outliers and optionally eliminate outliers.

As a further measure of confidence, the overall calibration curve may be evaluated to confirm the overall trend as m/z increases.

Confidence measures and trend analysis may be reported in association with the corrected analysis results to provide an indication that the calibration is expected.

The calibration curve can be applied in a number of different ways.

In the first embodiment, the analysis result can be corrected by applying the corresponding correction factor obtained from the calibration factor. In this embodiment, the data file may be written with nominal quality results corrected based on the corresponding correction factors.

In a second embodiment, the correction factors may be applied by editing each experiment to reclassify the transmission window of the 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 the experiment. In this embodiment, the header may be edited by identifying the transmission window center of the experiment, applying the corresponding correction factor from the calibration curve to generate a corrected transmission window center, and modifying the header start and end positions to reflect the corrected transmission window center. Thus, the definition of each transmission window may be modified to shift the window based on the correction factor for that transmission window obtained from the calibration curve.

In some aspects, shifting of the transmission window may result in gaps in the data set. One way to avoid gaps is to use slightly overlapping transmission windows to perform the analysis. If the shift is less than the overlap, the corrected data will not include any gaps. An alternative way to avoid gaps is to correct the transmission window by forcing the start of the transmission window to coincide with the end of the previous transmission window. The end of the corrected transmission window is then determined by adding half the transmission width to the corrected transmission window center. In this way, the transmission window widths will be slightly different from each other, but the variation is too small to affect the overall analysis result.

By way of illustration, fig. 5A and 5B provide examples of accurate quality analysis results. Fig. 5C and 5D present the corresponding nominal mass analysis results for a given sample. The exact mass analysis corresponding to the residual precursor mass peak 420 is at 535.2703m/z, while for the same sample, the nominal mass analysis result 425 is at 537.751m/z. Thus, the variation of the accurate mass spectrometer from the nominal mass spectrometer is 1.4807m/z. Since the accurate mass spectrometer was previously calibrated using the reference standard, this indicates that the nominal mass spectrometer needs to be calibrated up to 1.4807m/z.

In some embodiments, the calibration curve and/or correction factor may be monitored between analysis runs to confirm overall consistency. In the case of a calibration that tends in a particular direction, for example, the correction factor between runs becomes large, it may indicate that the instrument needs maintenance or cleaning.

In an embodiment, a hybrid mass spectrometer may be calibrated by: the accurate mass spectrometer is calibrated using a reference standard, and the nominal mass spectrometer is calibrated based on the captured analysis data by comparing the nominal mass analysis results to the calibrated accurate mass analysis results.

In one aspect, the hybrid mass spectrometer can also be calibrated by evaluating mass measurements from the accurate mass spectrometer and the nominal mass spectrometer and rejecting outlier cases before applying the measurements to the calibration curve.

In one aspect, a hybrid mass spectrometer may be calibrated by: calibrating an accurate mass spectrometer using a reference standard, calibrating a nominal mass spectrometer using the reference standard to generate a plurality of correction factors for each transmission window and scan speed pair for nominal mass calibration, and calibrating the nominal mass spectrometer by interpolating between the generated plurality of correction factors to calibrate the nominal mass spectrometer for each analysis transmission window and scan speed pair used during an analysis run.

In some embodiments, the hybrid mass spectrometer may be calibrated by: calibrating the accurate mass spectrometer using a reference standard, and calibrating the nominal mass spectrometer based on analysis data captured by comparing the nominal mass analysis result to the calibrated accurate mass analysis result, wherein if the statistical measure of the comparison exceeds a threshold, calibrating the nominal mass spectrometer based on the nominal mass spectrometer calibration using the reference standard. The statistical measurement may include, for example, an indication that there are not enough points of comparison between the nominal mass spectrometer and the accurate mass spectrometer to generate a calibration curve for the nominal mass spectrometer. The statistical measurement may include, for example, an indication that the point of comparison between the nominal mass spectrometer and the accurate mass spectrometer is too varied to generate a calibration curve for the nominal mass spectrometer.

In some embodiments, the nominal mass analyzer may be a quadrupole mass analyzer and the accurate mass analyzer may be a time of flight (ToF) mass analyzer. For example, fig. 7 schematically depicts a mass spectrometer 200 that includes an ion source 210, a quadrupole mass analyzer 220, a collision cell 230, a downstream time-of-flight (ToF) mass analyzer 240, and a data processing module 250. A sample delivery apparatus 260 (e.g., a Liquid Chromatography (LC) column) may deliver a sample to the ion source 210, and the ion source 210 may ionize one or more target analytes within the sample to generate a plurality of precursor ions.

A variety of ion sources may be employed. Some examples of such ion sources include, but are not limited to, electrospray ionization devices, nebulizer-assisted electrospray devices, chemical ionization devices, nebulizer-assisted atomization devices, matrix-assisted laser desorption/ionization (MALDI) ion sources, photoionization devices, and the like.

Precursor ions are received by the quadrupole mass analyzer 220, and those precursor ions that pass through the mass analyzer 220 are received by the downstream collision cell 230. At least a portion of the precursor ions undergo fragmentation within the collision cell to generate a plurality of product ions. Ions exiting the collision cell 230 (which may include product ions as well as residual precursor ions, i.e., those precursor ions that have not undergone fragmentation) are then received by a downstream time-of-flight (ToF) mass analyzer 240, which ToF mass analyzer 240 generates an ion detection signal indicative of the ion m/z ratio.

Since the ToF mass analyzer 240 is calibrated via use of a reference standard, the m/z ratio determined by the ToF mass analyzer can be considered as the exact m/z ratio for calibration of the nominal mass analyzer. The ion detection signal is received by the data processing module 250, which data processing module 250 analyzes the ToF signal in the manner discussed herein for calibrating a quadrupole mass analyzer.

In addition to the components shown in fig. 8, mass spectrometer 200 can also include other components, such as various ion guides positioned upstream of the quadrupole mass analyzer.

In this embodiment, the time-of-flight mass analyzer 240 may be calibrated using one or more reference calibrators in a manner known in the art. Thus, in this embodiment, the time-of-flight mass analyzer is used as an accurate mass analyzer.

As a control, a quadrupole mass analyzer was used as the nominal mass analyzer. The RF voltage source 300 and the DC voltage source 302 may apply RF and DC voltages to quadrupole rods of a quadrupole mass analyzer in order to establish a transmission bandwidth of the quadrupole mass analyzer. The RF voltage source may also apply RF voltages to rods of the collision cell (which may comprise, for example, four rods arranged in a quadrupole configuration) to provide radial confinement of ions. The controller 303 may control the operation of the RF and DC voltage sources.

For example, the controller may cause a sweep of the frequency of the RF and/or DC voltages applied to the quadrupole rods of the quadrupole mass filter to sweep its transmission bandwidth to allow ions having different m/z ratios to pass to the downstream time-of-flight mass analyzer. More specifically, in this embodiment, the ion transmission bandwidth of a quadrupole mass analyzer can be scanned across the mass range such that successive ion transmission windows overlap.

Such scanning of the transmission bandwidth of a quadrupole mass analyzer can result in variations in intensity associated with the mass signal of product ions and residual precursor ions observed in different transmission windows.

In this embodiment, the mass detection signal generated by the ToF mass analyzer may be stored in the precursor dimension as a plurality of data intervals, wherein each data interval has an m/z width that is part of the m/z transmission width of the quadrupole mass filter 220. For example, each data interval may have an m/z width of 1/5 of the transmission bandwidth as the quality filter 220. As described above, each data interval is considered herein as one experiment.

Referring again to fig. 2A and as described above, in some embodiments, an extracted ion chromatogram (XIC) may be generated for each nth experiment. In this case, the nth experiment corresponds to a 2 dalton data interval extending from 590.5 dalton to 592.5 dalton. As shown in fig. 2A, the highest intensity peak within this 2-dalton data interval corresponds to the m/z ratio of 589.3592. Fig. 2B shows an ion chromatogram as a function of retention time associated with this 2-daltons data interval. Peaks in the ion chromatogram depicted in fig. 2B may be ordered based on their intensities.

In this example, the highest intensity peak at a retention time of 0.921 minutes was selected and a mass spectrum associated with this peak was generated, as shown in fig. 3B and 4A. The m/z ratio of the fundamental peak (here peak 305) in the mass spectrum is compared to the m/z range spanned by the corresponding precursor transmission window to verify that the m/z ratio of the fundamental peak is within that transmission window.

The base mass peak intensities in the experiments before the 2-dalton data interval extending between 590.5 dalton and 592.5 dalton as described above and after this data interval may be plotted as a function of the corresponding m/z values of the quadrupole mass analyzer 220, wherein the total number of experiments may be equal to the number of experiments per quadrupole transmission window.

The plotted data may then be used to determine m/z calibration values for the transmission window of the nominal quadrupole mass analyzer 220. For example, the centroid of the plot can be compared to the m/z ratio of the ToF calibration associated with the base peak to determine the m/z offset required to correct the nominal calibration of the quadrupole mass analyzer.

As described above, the header of the data compilation associated with each experiment, which identifies the m/z range corresponding to the experiment, may then be adjusted based on the m/z calibration offset determined via the above-described process.

The following examples are provided to further illustrate various aspects of the present teachings, but are not necessarily indicative of the best mode of practicing the present teachings and/or of the best results that may be obtained.

Example

Example 1

Scanning SWATH settings and operations

Scanning SWATH runs were acquired using a SCIEX triple quadrupole 6600+ mass spectrometer operating in SWATH acquisition mode. The following settings apply in the scan SWATH run: (1) The precursor delivery window was set to 10m/z and covered a mass range from 400m/z to 900m/z in 0.5 seconds. These settings provide a compromise between identification and quantitative performance. Several precursor delivery window sizes ranging from 3m/z to 20m/z (covering precursor ranges from 400m/z to 900 m/z) were tested for mass analysis of yeast (Saccharomyces cerevisiae) whole proteome trypsin digests.

In the case of a window size of 10m/z, optimal results are obtained in terms of identification and quantification accuracy. Further decreasing the window size will result in an even higher number of identifications due to less interference, but the resulting shorter effective accumulation time will decrease the accuracy of the quantification. The raw data is binned into 2m/z intervals in the quadrupole or precursor dimension, providing a resolution in the Q1 dimension (i.e., the quadrupole or precursor dimension), which allows for efficient use of the Q1 fraction. MS1 scanning was omitted in benchmark testing (benchmark) and data was acquired in high sensitivity mode.

The instrument control software calculates the RF/DC ramp applied to the quadrupole mass analyzer. The ramp may be calculated from the experimental start transmission quality, stop transmission quality, transmission width and cycle time. The calculation uses the previously acquired calibration to calculate the slope of the mass DACS and the resolution DACS. The quadrupole start mass can be calculated as the experimental start mass minus the transmission width and the quadrupole stop mass can be calculated as the experimental stop mass plus the transmission width. This allows to obtain the correct precursor profile of all fragments at the boundary of the experimental mass range. The collision energy may be calculated using a +2 rolling collision energy equation that provides a linear relationship for a given charge as a function of m/z.

This results in a small collision energy spread, which depends on the width of the transmission window relative to the scanned range. In these experiments, the effect is typically about 1eV spread for a given precursor.

When running a pre-built batch and injecting tuning solution directly (ESI positive calibration solution for SCIEX X500 System (SCIEX)), with 266.16;354.21;422.26;609.28;829.54 Scanning SWATH calibration is performed automatically. The quadrupole response of each standard was measured at transmission window widths 3, 5, 10, 15 and 20m/z, with each transmission window width additionally measured while scanning the transmission window of the quadrupole mass analyzer at 500, 1000, 2000 and 3000 m/z/sec. The recorded quadrupole responses for each condition are stored in a three-dimensional matrix, where the dimensions of the matrix are width, speed and m/z.

The values stored in the matrix are the m/z observed from the theoretical m/z values. The observed precursor m/z is calculated from the current number of pulses relative to the total scan pulse applied as a fraction of the scanned mass range plus the starting mass. Thus, an exact calibration curve is derived for each acquired scan speed and width. For scan speeds and widths between those generated by experimental parameters, the curve is interpolated trilinear.

The instrument acquisition software is configured to store ion detection responses into the calculated 2m/z precursor isolation interval given the current ToF thruster pulse number relative to the scan start applying the scan SWATH offset curve described above. The 2m/z precursor isolation intervals are organized as adjacent experiments in the data file, allowing the extraction of the precursor profile of any given fragment ion in a given cycle by tracking the fragment response across the experiments and the nominal chromatogram profile across the cycles.

Example 2

In another experiment, a scanning SWATH calibration was obtained as each sample file was processed from the sample data itself. An automated algorithm as described in the present teachings is used to identify the largest residual precursor across each transmission window of the entire sample. This results in several accurate mass ToF measurements, each paired with the centroid of a quadrupole mass trace associated with a respective quadrupole transmission region, typically 10 or more per 100 Da.

For example, if 3 residual potential precursor ions are identified per transport region, with a scan range of 500Da and a transport width of 10, there will be 500/10×3=150 calibration point pairs consisting of quadrupole mass and ToF exact mass. Since strong mass peaks in the quadrupole transmission region may not actually correspond to residual precursor ions, a selection algorithm is employed to filter out mass peaks using an outlier rejection algorithm that takes local variations into account. In particular, local variations (e.g., variations in the range of about 50-100 daltons) are employed to identify and optionally eliminate outlier mass peaks.

Typically, the mass peaks are evaluated against their neighbors in the 50-100Da region. Once the multi-point calibration curve is obtained, the calibration is applied to the data by: the start and end quality areas defined in the header of each experiment for which data is stored are updated to calculate the center from the calibration function while maintaining continuity of boundaries in adjacent experiments. In some cases, the calibration curve may be used to identify outlier peaks that are mistakenly considered to correspond to precursor ions.

The controller and/or analysis module (such as those discussed above as being suitable for use in the practice of the present teachings) may be implemented using hardware, firmware, and/or software using techniques known in the art as taught by the present teachings. For example, fig. 9 schematically depicts an example of an implementation of such a controller/analysis module 500, which includes a processor 500a (e.g., a microprocessor), at least one persistent memory module 500b (e.g., a ROM), at least one transient memory module (e.g., a RAM) 500c, and a bus 500d, among other elements well known in the art.

Bus 500d allows communication among the various other components of the processor and controller. In this example, the controller 500 may further include a communication module 500e configured to allow transmission and reception of signals.

Instructions used by the controller 500, for example instructions for adjusting the DC voltage applied to the auxiliary electrode, may be stored in the permanent memory module 500b and may be transferred to the transient memory module 500c for execution during run time. The controller 500 may also be configured to control the operation of other components of the mass spectrometer, such as the ion guide and mass analyzer.

Although some aspects have been described in the context of systems and/or apparatus, it will be apparent that these aspects also represent descriptions of corresponding methods in which a block or apparatus corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of method steps also represent descriptions of corresponding blocks or items or features of corresponding apparatus. Some or all of the method steps may be performed by (or using) hardware devices (e.g., processors, microprocessors, programmable computers, or electronic circuits). In some embodiments, one or more of the most important method steps may be performed by such an apparatus.

Embodiments of the present invention may be implemented in hardware and/or software, depending on the requirements of some implementations. The embodiments may be performed using a non-transitory storage medium such as a digital storage medium, e.g., floppy disk, DVD, blu-ray, CD, ROM, PROM, and EPROM, EEPROM, or FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system, such that the corresponding method is performed. Thus, the digital storage medium may be computer readable.

Those of ordinary skill in the art will appreciate that various modifications may be made to the above-described embodiments without departing from the scope of the present teachings.

Claims (20)

1. A method for calibrating a hybrid mass spectrometer, the hybrid mass spectrometer comprising an accurate mass analyser in combination with a nominal mass analyser, the method comprising:

one or more reference standards are used to calibrate the accurate mass analyzer,

analyzing the sample using the nominal mass analyzer and the accurate mass analyzer to generate one or more corresponding nominal mass signals and calibrated accurate mass signals associated with the sample, and

The nominal mass analyzer is calibrated by comparing the nominal mass signal to the calibrated accurate mass signal.

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

3. The method of any of claims 1-2, wherein the nominal mass analyzer is configured to provide a plurality of ion transmission windows.

4. The method of claim 3, further comprising a controller in communication with the nominal mass analyzer for adjusting at least one parameter of an ion transmission window associated with the nominal mass analyzer to generate the plurality of ion transmission windows.

5. The method of claim 4, wherein the nominal mass analyzer comprises a plurality of rods arranged in a multipole configuration and configured for applying RF and DC voltages thereto.

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

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

8. The method of any one of the preceding claims, wherein the accurate mass analyser comprises a time of flight (ToF) mass analyser.

9. A method for calibrating a mass spectrometer having at least a first mass analyzer and a second mass analyzer, wherein the second mass analyzer is positioned downstream of the first mass analyzer, the method comprising:

calibrating the second mass analyzer using one or more reference standards,

generating a first measurement of a mass signal of a precursor ion using a first mass analyzer and a second measurement of a corresponding mass signal of the precursor ion using a second mass analyzer, and

the first mass analyzer is calibrated via comparing the first measurement of the mass signal with the second measurement.

10. The method of claim 9, wherein the first mass analyzer comprises a quadrupole mass analyzer.

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

12. The method of any of claims 9-11, further comprising a collision cell positioned between the first mass analyzer and the second mass analyzer for receiving the precursor ions from the first mass analyzer and generating a plurality of product ions via fragmentation of the precursor ions.

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

14. The method of claim 13, wherein the step of measuring a second mass signal comprises identifying a mass signal associated with the precursor ion in a mass spectrum of ions exiting the collision cell generated by the second mass analyzer, and assigning an m/z ratio to the identified mass signal.

15. The method of claim 14, wherein the at least one nominal setting includes any one of a transport width and a speed for scanning the transport width.

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

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

18. A hybrid mass spectrometer, comprising:

a nominal mass analyzer configured to provide a plurality of transmission windows for passage of at least one precursor ion,

A collision cell positioned downstream of the nominal mass analyzer for receiving and causing fragmentation of the at least one precursor ion to thereby generate a plurality of product ions,

an accurate mass analyser for receiving ions exiting the collision cell and generating a mass spectrum thereof,

a mass analyzer for receiving one or more operating parameters of the nominal mass analyzer and the mass spectrum generated by the accurate mass analyzer,

wherein the mass analyzer is configured to identify a mass signal associated with the precursor ions in the accurate mass analyzer and to calibrate the nominal mass analyzer based on an m/z ratio of the precursor ions in the mass spectrum and the one or more operating parameters of the nominal mass analyzer.

19. The hybrid mass spectrometer of claim 18, wherein the nominal mass analyzer comprises a plurality of rods arranged in a multipole configuration, the plurality of rods configured for applying RF and DC voltages thereto.

20. The hybrid mass spectrometer of any of claims 18-19, wherein the accurate-mass analyzer comprises a time-of-flight mass analyzer.