Quantifying substances present in sample

Abstract

The sample is separated into constituent analytes at a time parameter by a first separator. The compositional analytes are analyzed by a mass spectrometer that provides intensity measurements for the mass-to-charge ratio of each compositional analyte. For each compositional analyte, the relationship of the measured intensity at the mass-to-charge ratio to the time parameter defines a respective peak. A peak quality factor may be determined for each of the peaks and / or a common peak position with respect to a time parameter may be determined for at least some of the peaks. A particular range of time parameters used to quantify a substance may be based on one peak quality factor (or peak quality factors). Alternatively, a subset of peaks is selected for quantifying the substance based on the determined peak quality factor and / or the determined common peak location.

Description

Technical Field

[0001] The present disclosure relates to quantifying substances present in a sample, separating the sample into constituent analytes by a first separator (e.g., a chromatographic separator or an ion mobility separator) over a time parameter, and then further analyzing the constituent analytes by a mass spectrometer. This can be embodied in a method, a computer program, a controller for a mass spectrometry system, and / or a mass spectrometry system. Background Art

[0002] Software analysis of mass spectrometry data is increasingly useful in identifying and quantifying the substances present in a sample. Analysis of MS1 data is generally considered to be more accurate than MS n Analysis of the data is more straightforward, and analysis of data independent acquisition (DIA) data can be even more complex. Mass analysis can be performed after the initial separation stage, for example using a chromatographic separator, including a gas chromatograph-mass spectrometer (GC-MS) or a liquid chromatograph-mass spectrometer (LC-MS), or an ion mobility separator, commonly referred to as an ion mobility spectrometer-mass spectrometer (IMS-MS). This only adds to the complexity of the analytical workflow.

[0003] In general, there are two main methods for DIA data analysis. The first method uses a database-based search engine, which is commonly used to analyze data-dependent acquisition (DDA) data. The second option is directed analysis, also known as SWATH-MS (Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectra). These data analysis algorithms focus primarily on sample identification, but also take quantification into account.

[0004] WO-2009 / 146345 discusses matching a precursor ion with one or more related product ions. A data set including information about the precursor ions is obtained from multiple injections and then normalized according to a single retention time. By determining which product ions are within a predetermined retention time window relative to the single retention time, the presence of such product ions allows their relationship to the precursor ion to be established.

[0005] WO-2012 / 035412 relates to the use of multiple product ions to characterize unknown sample compounds. Chromatographic peak retention times can be correlated between product ions to improve identification.

[0006] Typical existing methods for quantification use a single raw data sample (or single event) basis. For example, Barkovits, Katalin et al. "Reproducibility, specificity and accuracy of relative quantification using spectral library-based data-independent acquisition" (Molecular Cellular Proteomics 19.1 (2020): 181-197) discusses the selection of suitable spectral libraries for peptide identification and quantification. Searle, Brian C. et al. "Generating high quality libraries for DIA MS with empirically corrected peptide prediction" (Nature Communications 11.1 (2020): 1-10) describes library generation using empirical data, such as fragmentation and retention time prediction. Demichev, Vadim et al., “DIA-NN: neural networks and interference correction enabled deep proteome coverage in high throughput” (Nature Methods, 17.1 (2020): 41-44) describe the removal of interfering product ions by selecting chromatographic product ion peaks that best correlate with their corresponding product ion partners.

[0007] It is therefore desirable to improve the analysis of mass spectrometry data in order to provide a more accurate quantification of substances within a sample. This is particularly true where a sample is first separated into constituent analytes and then mass analyzed for each of the constituent analytes. Summary of the invention

[0008] Against this background, there are provided a method of quantifying a substance present in a sample according to claim 1 and according to claim 9, a computer program according to claim 23, a controller for a mass spectrometry system as defined by claim 24 and a mass spectrometry system according to claim 25. Preferred and / or optional features are disclosed in the dependent claims.

[0009] The present disclosure relates to quantifying substances present in a sample. The sample is separated into constituent analytes on a time parameter by a first separator. For example, the first separator can be a chromatographic separator (e.g., GC or LC). In this case, the sample is typically a liquid or gas phase sample. Then, the time parameter relates to retention time, and the constituent analytes are components separated from the chromatographic separator at different retention times. In another example, the first separator is an ion migration separator. Then, the sample is a mixture of sample ions. In this case, the time parameter relates to drift time or retention time, and the constituent analytes are constituent ions of the sample ion mixture.

[0010] Aspects according to the present disclosure may be embodied in a controller for a mass spectrometry system and / or software in a mass spectrometry system (as a computer program stored on a computer readable medium, optionally non-transitory). Additionally or alternatively, these aspects may be implemented as a process.

[0011] In any case, the constituent analytes are further analyzed by a mass spectrometer (where the first separator is a chromatographic separator that initially ionizes each constituent analyte). For example, analysis of the constituent analytes in a mass spectrometer can include: processing the ions; and mass analyzing the processed ions. In some cases, the processing does not involve fragmentation (although it may involve mass filtering or selection and / or cooling), and the mass analysis may be of the unfragmented ions. Alternatively, the ions may be precursor ions, and the processing of the ions includes fragmenting the precursor ions to produce fragment ions, such that the fragment ions are mass analyzed. The mass analysis of the fragment ions can then include data dependent acquisition (DIA) MS n analyze.

[0012] For each of the one or more component analytes, a measurement result of the intensity for the mass-to-charge ratio is obtained from a mass spectrometer. Such measurements are performed multiple times on a time parameter. For each component analyte, the relationship between the measured intensity of the selected mass-to-charge ratio (or mass-to-charge ratio range) and the time parameter defines a peak value. Preferably, each peak value includes at least a minimum number of intensity measurements, each intensity measurement result for different values ​​of the time parameter (e.g., at least 5 measurements).

[0013] In some cases, the ions of the substance being quantified have one of the selected mass-to-charge ratios, which can allow the disclosed techniques to be applied to the analysis of MS1 full scans, typically analyzing multiple ions within a single scan (rather than quantifying a single peak). More commonly, each of the selected mass-to-charge ratios relates to a corresponding fragment of the ions of the substance being quantified. Quantification can involve identifying the substance and / or identifying the chemical constituent corresponding to one or more peaks (such that the selected mass-to-charge ratio or range of the one or more peaks corresponds to the identified chemical constituent).

[0014] In the first aspect, there may be a single peak (i.e., providing data for only a single mass-to-charge ratio or range over a time parameter), but more typically multiple peaks are obtained. A peak quality is established for each peak, and this is used to determine a specific range of the time parameter. The determined specific range of the time parameter can then be used to quantify the substance. It is particularly noted that the peak quality factor depends on the range of the time parameter used to establish the peak quality factor.

[0015] A flatness detection algorithm may additionally be used (eg, as an algorithm or one of multiple algorithms used to determine a peak quality factor) to determine a particular range of a temporal parameter.

[0016] The specific range of the time parameter can be determined iteratively. For example, a first range of the time parameter can be used to establish a first peak quality factor for each peak. Then, a second range of the time parameter is identified, which can be narrower or wider than the first range of the time parameter. For example, the second range can be based on a predetermined increase or decrease of the time parameter from the first range (at either end or both ends of the range). Optionally, the second range can be based on a first peak quality factor (or multiple first peak quality factors), for example by comparing them to a criterion (or multiple criteria, such as one or more thresholds). The identified second range of the time parameter can be used to establish a second peak quality factor for each peak. The specific range of the time parameter can therefore be based on a first peak quality factor (or multiple first peak quality factors) and a second peak quality factor (or multiple second peak quality factors). The process can continue until a peak quality factor (or multiple peak quality factors) meets a criterion (or multiple criteria). For example, the range of the time parameter can be gradually widened until a criterion (or multiple criteria) is met.

[0017] The initial evaluation measurement result for the intensity of the mass-to-charge ratio can be used for the first range of the time parameter. If the second range of the time parameter is wider than the first range, additional measurements for the mass-to-charge ratio can be evaluated for a component analyte (or multiple component analytes). In this case, the initial intensity measurement result and the additional intensity measurement result can together cover the second range of the time parameter. A second peak quality factor (or multiple second peak quality factors) can therefore be based on the initial intensity measurement result and the additional intensity measurement result.

[0018] The substance is advantageously quantified based on the intensity measurement results of each peak value in one or more peak values ​​falling within the determined specific range. The determined specific range of the time parameter can be used not only to quantify the measured sample, but also to quantify the same substance from different samples. Alternatively, a parallel evaluation of a specific range of the time parameter can be performed for each sample in multiple samples. In this case, a specific range of the time parameter can be established for the sample being evaluated.

[0019] In a second aspect, a plurality of peaks of intensity measurements over a time parameter are obtained (each peak being associated with a respective mass-to-charge ratio or range). A respective peak quality factor may be established for each peak (the peak quality factor being dependent on the range of the time parameter) and / or a common peak position may be determined for at least some of the peaks with respect to the time parameter. A subset of the peaks is then selected for quantifying the substance based on the determined peak quality factors and / or the determined common peak positions.

[0020] In one approach, a subset of peaks may be selected by evaluating the peak position of each of the peaks against the determined common peak position. For example, only peaks having peak positions within a predetermined amount away from the determined common peak position may be selected for the subset.

[0021] In another method (which can be used with the method discussed above), a subset of peaks can be selected by evaluating the respective peak quality factor of each of the peaks according to a criterion (e.g., greater than, at least equal to, not greater than, or less than a threshold). The subset of peaks can then be selected based on whether the criterion is met for each peak (i.e., the selected peaks can be those peaks that meet the criterion). Optionally, there can be multiple criteria.

[0022] Each peak in the selected subset of peaks may then be quantified.The substance may then be quantified based on the quantification of the selected subset of peaks.

[0023] The process according to the invention does not require the use of correlation of peak intensities between product ions, nor does it require the use of correlation with precursor masses.

[0024] It should be noted that various aspects of the present disclosure may be combined.According to any aspect, the mass spectrometer may be controlled to perform mass analysis of each of the constituent analytes and provide intensity measurements of the mass-to-charge ratio for each of the plurality of components. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present disclosure may be put into practice in many ways and a preferred embodiment will now be described by way of example only and with reference to the accompanying drawings, in which:

[0026] Figure 1 schematically depicts a mass spectrometry system for use in accordance with the present disclosure;

[0027] Figure 2 The measured intensity is plotted against the time peak for three exemplary mass-to-charge ratio ranges;

[0028] Figure 3 A block diagram of a first process according to the present disclosure is shown;

[0029] Figure 4 shows a block diagram of a second process according to the present disclosure; and

[0030] Figure 5a to Figure 5d Each illustrates a graph of measured intensity versus time peak, showing the effect of a process according to the present disclosure. DETAILED DESCRIPTION

[0031] First reference Figure 1 , schematically depicts a mass spectrometry system used in accordance with the present disclosure. The mass spectrometry system 10 receives a sample 5 and includes: a first separator 20; a mass spectrometer 30; and a controller 40. The mass spectrometer 30 includes: at least one ion optical device 32 (e.g., an ion guide, an ion trap, and / or an ion selector or filter); a collision cell 34; a mass analyzer 36; and a detector 38. Optionally, the mass analyzer 36 and the detector 38 can be combined into a single device.

[0032] The first separator 20 separates the sample 5 into its constituent analytes. The separation occurs over time, with different constituent analytes being output at different times from the first separator 20. Thus, there is a time parameter associated with the first separator 20, with each constituent analyte having a different corresponding value (or range) for the time parameter.

[0033] Preferably, the first separator 20 is a chromatographic separator, such as a gas chromatography (GC) device or a liquid chromatography (LC) device, so that the time parameter can be a retention time (RT). Then, the mass spectrometry system 10 can be a GC-MS or LC-MS system. In such cases (and optionally with other types of separators), the mass spectrometer 30 also includes an ion source 31, which is advantageously configured to receive the separated analyte (effluent or eluate) from the first separator 20 and produce ions from the received separated analyte.

[0034] Alternatively, the first separator 20 may be an ion mobility spectrometer (IMS), such that the time parameter may be a drift time (e.g., in the case of drift tube type or traveling wave type ion mobility separation) or a retention time (e.g., in the case of trapped ion mobility separation (TIMS)). The mass spectrometry system 10 may then be an IMS-MS system. The first separator 20 may then include an ion source (not shown), and in this case the output of the first separator 20 includes ions. Therefore, the mass spectrometer 30 does not need to include an ion source 31, and the ion source 31 is therefore Figure 1 is shown as optional.

[0035] Controller 40 is configured to control the operation of first separator 20 and mass spectrometer 30. In addition, controller 40 receives mass spectrum data from detector 38. Optionally, controller 40 can also receive data from first separator 20. Therefore, controller can make mass spectrometry system 10 run experiment, and then receive the result of experiment for analysis. This analysis can be performed by controller or external processor or processing system (not shown). Controller 40 is usually a single device, but it can include multiple parts that usually operate together.

[0036] As discussed above, the methods according to the present disclosure are useful for analyzing n (usually MS 2 ) experiment is particularly advantageous. In this case (and controlled by controller 40), mass spectrometry system 10 is operated to separate sample 5 into one or more constituent analytes, each constituent analyte having a relevant range of time parameters. Therefore, a single constituent analyte can be output from first separator 20 within the range of time parameters, thus resulting in multiple outputs of constituent analytes from first separator 20. Each output of constituent analytes with associated time parameters is then provided to mass spectrometer 30, in which the constituent analytes are fragmented in collision chamber 34, analyzed by mass analyzer 36, and mass spectral data are detected by detector 38. Mass spectral data is then communicated to controller 40. Each mass analysis typically indicates multiple fragments, each fragment being at a different mass-to-charge ratio (m / z) and having a corresponding detection intensity. This is referred to as MSn As a result, the controller 40 receives a plurality of MS n Mass spectrometry, each MS n The mass spectrum is associated with one or more output component analytes from the first separator 20 (and therefore has an associated time parameter). Typically, multiple MS spectra are acquired for each individual output component analyte. n In other words, multiple MS spectra are acquired during the output of each constituent analyte. n Mass spectrometry.

[0037] Alternatively, multiple MS1 mass spectra may also be obtained for each individual output constituent analyte, ie, multiple MS1 mass spectra may be obtained during the output of each constituent analyte.

[0038] Initial identification of one or more constituent analyte compounds is usually performed by first quantifying using a DIA identification tool on a mass spectrometer. Each compound identification provides a point in the time parameter (e.g., a retention time point) at which the compound has been detected and a list of detected product (i.e., fragment) ions. The list can be exhaustive or simply a selection of the strongest or most accurately detected product (fragment) ions.

[0039] Then, by targeting specific m / z and time parameter values ​​for this set of product (fragment) ions, the MS n It is possible to recover peaks in the analysis. For example, for a point in the time parameter determined by the DIA identification tool, the intensity of the set of product ions can be obtained at a specific m / z value of the product (fragment) ion (optionally subject to a user-defined or automatically estimated tolerance). Then, for spectra at adjacent values ​​of the time parameter, the intensity of the same m / z value can be retrieved (again, optionally subject to a user-defined or automatically estimated tolerance). The retrieval process can then be repeated until a minimum number of spectra min_s is reached (typically, min_s=5). In doing so, the intensity measurements of the same fragment (based on m / z value or range) can be associated or matched on the time parameter.

[0040] This data can be presented graphically. Figure 2, plotting the peak values ​​of measured intensity relative to time for three exemplary fragments (each corresponding to a respective m / z range) analyzed from DIA. The first peak 61 corresponding to the first fragment is relatively low (intensity of approximately 3500), with a peak position of approximately 31.58 on the time axis. The first peak 62 corresponding to the second fragment is relatively high (intensity of approximately 32000), with a peak position of approximately 31.57 on the time axis. The third peak 63 corresponding to the third fragment has an intermediate intensity (approximately 18000), with a peak position of approximately 31.64 on the time axis. All three peaks are present in approximately the same retention time range.

[0041] It is desirable to use the information from these three peaks to quantify the substances from which these three fragments appear to originate. Quantification can be performed by summing the peak intensities of the identified product ions over a specific range of time parameters. It is beneficial to use the same range for all peaks. Identification points to relatively arbitrary points on the range of time parameters (e.g., a specific time at which a compound elutes), which may not be a peak retention or even an indication of a suitable range.

[0042] Return to reference Figure 2 , showing a selected range 65 of a time parameter from about 31.53 to about 31.62. Data points from the first peak 61, the second peak 62, and the third peak 63 that fall within the selected range 65 are highlighted. It is therefore desirable to improve the selection of the range 65. The second peak 62 starts slightly earlier than the first peak 61. The third peak 63 starts at a significantly later time than the first peak 61 and the second peak 62. It can also be seen that the peak position of the third peak 63 is slightly different from the peak positions of the first peak 61 and the second peak 62 (in other words, the third peak 63 is offset from the other two peaks). Therefore, it is unclear whether the fragment corresponding to the third peak 63 originates from the same substance (parent or precursor ion) as the corresponding fragment corresponding to the first peak 61 and the second peak 62.

[0043] It has been determined that mathematical methods can be used to solve these problems. Specifically, a peak quality factor (PQF) algorithm can be used to objectively and quantitatively evaluate the quality of each peak value on the selected range of the time parameter. More typically, multiple PQF algorithms can be used, such as in "MetaClean: A machine learning-based classifier for reduced false positive peak detection in untargeted LC-MS metabolomics data", Chetnik et al., Metabolomics 16 (2020): discussed in 1-13. Therefore, the range of the time parameter that maximizes the result of the PQF algorithm can allow for improved quantification of peaks and substances.

[0044] Initially only data from a limited range of the time parameter may be provided to the quantification algorithm. Thus, the algorithm may ideally retrieve ion product intensities from mass spectra at adjacent time parameter points. The PQF algorithm may also inform the direction in which to retrieve additional data (upward and / or downward with respect to the time parameter) and when to stop the search. The process may be run simultaneously on all identifications of the same compound in all samples of an experiment, which may ensure that the same set of product ions is used in all samples. This allows product ions to be discarded from all samples when the output of the PQF algorithm determines that a peak of a product ion does not have sufficient (user defined) quality on any of the samples. This also allows a fixed time parameter range (or at least width) to be defined for all samples if the user so desires (in other words, multiple samples use the same width or range).

[0045] Reference now Figure 3 , a block diagram of a first process according to the present disclosure is shown. The process is specifically used to select a range of time parameters for the purpose of quantifying a substance. In an initial step 100, a mass spectrometer provides (or a controller retrieves from a mass spectrometer) data including intensity measurements of the mass-to-charge ratio for each of one or more constituent analytes. As described above, the relationship between the measured intensity of each constituent analyte at each selected mass-to-charge ratio and the time parameter defines a corresponding peak (e.g., Figure 2 Typically, data are obtained from DIA experiments where multiple MS have been acquired as a function of a time parameter. 2Spectra, such as retention time (RT) for GC-MS or LC-MS. As a result, there are usually multiple peaks (each corresponding to a different m / z value or range), each peak corresponding to a specific fragment of one of the constituent analytes.

[0046] Advantageously, an optional identification step 105 is run first. This uses a (known) identification algorithm to process the received data in order to determine the identity of the compounds in the data. Each identification may preferably include a list of m / z ratios of a plurality of (n) product or fragment ions of the identified parent ion. Known algorithms typically allow for the identification of compounds in the MS 2 Any (but usually only one) MS 2 Each identification was made within the spectrum. The MS used 2 The spectrum is not necessarily the MS corresponding to the vertex of the intensity peak over time. 2 Spectra. Usually, in MS 2 Identifications are made in the spectrum at an arbitrarily chosen value of a time parameter, which may be referred to as a reference time (t0). Thus, the identified parent ion (and its corresponding starting compound) is assumed to include all fragments identified at that value of the time parameter.

[0047] In the PQF step 110, one or more PQF algorithms are performed on the retrieved data aggregated within a selected range of the time parameter to determine a PQF result. The selected range desirably includes a minimum number of data points, typically at least 5 data points. To achieve this, the selected range is typically set to include: a reference time (t0); two data points before the reference time (t0), i.e., t -1 and t -2 ; and two data points after the reference time (t0), namely t1 and t2. Any peak whose intensity is below a predetermined threshold (e.g., 5) of these first minimum number of data points is preferably discarded, as a minimum number of data points is generally required to assess the peak characteristics of the fragment.

[0048] The PQF results are then compared to at least one PQF criterion in a validation step 120. One or more PQF algorithms may thus determine whether each peak shape is good enough or whether the product ions do not produce good peaks for quantification. A flatness detector algorithm may form part of a PQF algorithm to determine when a peak boundary has been reached in either or both directions. Thus, at least one PQF criterion may include determining whether a threshold minimum number of peaks (min_peak) of sufficient quality (according to the PQF results) may be used for quantification. The threshold may be user defined or automatically defined. Another criterion may involve optimization (e.g., maximization) of the PQF results.

[0049] If at least one PQF criterion is not met, then the range change path 124 is taken to reach the range change step 130. In the range change step 130, it is determined how to change the selected range of the time parameter. For example, the selected range of the time parameter may extend at one or both ends of the range (e.g., including t -3 The fragment is then updated with a minimum number of data points. ...

[0050] Based on the updated selected range of the time parameter, additional data may be retrieved. This is done in a retrieval step 135. It will be appreciated that this is not always required and therefore this step is optional (indicated by the dashed outline).

[0051] The process then continues by repeating the PQF step 110, but now with an updated selected range for the time parameter. It will be appreciated that this cycle may be repeated multiple times if at least one of the PQF criteria considered in the validation step 120 is not met. It is contemplated that the selected range may be adjusted both forward and backward in time with reference to the reference time (t0).

[0052] If at least one of the PQF criteria considered in the validation step 120 is met (e.g., the PQF result is optimized and / or any one or more other criteria are met), the quantification path 126 is taken. In the quantification step 140, the material is quantified based on the selected range of the time parameter (if appropriate, as updated immediately in the range change step 130). This can be achieved by summing the intensities of the remaining fragments between the boundaries defined by the updated selected range.

[0053] The above method focuses on determining the range of time parameters for use in quantization. The fragments selected for inclusion in the quantization are also considered. However, this is not the main focus of the method. In another method, which is preferably combined with the above method, the main focus is on which fragments are included in the quantization.

[0054] In general, a method for quantifying the substance present in a sample can be considered. The sample is separated into constituent analytes by a first separator on a time parameter. The constituent analytes are then further analyzed by a mass spectrometer. In practice, a constituent analyte can be provided (output) from the first separator within the scope of the time parameter (within the time range). Therefore, a mass spectrometer can provide multiple mass spectra about each constituent analyte.

[0055] In one example, the sample comprises (or is) a liquid or gas phase sample. Then, the first separator may be a chromatographic separator and the time parameter relates to a retention time. In this case, the constituent analytes are components separated from the chromatographic separator at different retention times, and the constituent analytes are further analyzed by a mass spectrometer by ionizing each analyte to provide ions. The constituent analytes ionized to provide ions are then analyzed in a mass spectrometer by: processing the ions, in particular by fragmentation; and mass analysis of the processed (fragmented) ions.

[0056] Alternatively, the sample comprises (or is) a mixture of sample ions, and the first separator is an ion mobility separator. Then, the time parameter relates to drift time or retention time, and the constituent analytes are constituent ions of the mixture of sample ions. These sample ions may also be processed (e.g., by fragmentation), and then the processed (fragmented) is subjected to mass analysis.

[0057] In any case, each of the peaks may generally correspond to a DIA MS peak from a constituent analyte. n (For example, MS 2 ) corresponding fragment ions analyzed.

[0058] In the method according to the first aspect, intensity measurements of the mass-to-charge ratio of each of the one or more constituent analytes are received from a mass spectrometer. For each constituent analyte, the relationship between the measured intensity and the time parameter at each selected mass-to-charge ratio in one or more selected mass-to-charge ratios defines the corresponding peak. In an embodiment, the selected mass-to-charge ratio (which may be a mass-to-charge ratio range) can be determined based on fragmentation. Then, a specific range of the time parameter for quantifying the substance is determined based on the corresponding peak quality factor of each peak in the one or more peaks, and the peak quality factor depends on the range of the time parameter. Typically, multiple peak quality factors can be used, each of which can provide a statistical measure of the peak, such as in terms of peak clarity, symmetry, or other shape features.

[0059] Preferably, a substance and / or chemical composition corresponding to at least one peak (and typically each peak) is identified (after receiving the measurement data). In the latter case, one or more selected mass-to-charge ratios of at least one peak may correspond to the identified chemical composition. The identification of the peak and / or substance may help determine the starting point and / or initial range of the time parameter of each peak.

[0060] Advantageously, each peak comprises at least a predetermined number of intensity measurements, typically at least 5 intensity measurements (but optionally 7, 9 or 11). Peaks having less than the predetermined number of intensity measurements may be ignored.

[0061] Optionally, a flatness detection algorithm may be used to further determine a specific range of the time parameter. This may allow a determination to be made whether the full range of each peak is captured by the selected range of the time parameter.

[0062] In some embodiments, determining a specific range of a time parameter includes: establishing a corresponding first peak quality factor for each of the one or more peaks relative to a first range of the time parameter; and establishing a corresponding second peak quality factor for each of the one or more peaks relative to a second range of the time parameter, the second range of the time parameter being narrower or wider than the first range of the time parameter. Then, a specific range of the time parameter for quantifying a substance can be determined based on the one or more first peak quality factors and the one or more second peak quality factors. For example, a change between the one or more second peak quality factors and the one or more first peak quality factors can indicate whether the specific range of the time parameter should be the first range of the time parameter, the second range of the time parameter, below the first range of the time parameter, above the second range of the time parameter, or between the first range of the time parameter and the second range of the time parameter.

[0063] Advantageously, the second range of the time parameter may be selected based on one or more first peak quality factors (e.g., if the one or more first peak quality factors are too low, then increasing the second range of the time parameter compared to the first range of the time parameter) and / or based on a predetermined differential factor (e.g., a step increase or decrease in the second range of the time parameter compared to the first range of the time parameter).

[0064] Not all data required to determine one or more second peak quality factors may be initially evaluated. For example, the received intensity measurements for the mass-to-charge ratio for each of the one or more constituent analytes may be initial intensity measurements for a first range of time parameters. Then, additional intensity measurements for the mass-to-charge ratios in the one or more constituent analytes may be evaluated in the case where the second range of the time parameter is wider than the first range of the time parameter. In this case, the initial intensity measurements and the additional intensity measurements may together cover the second range of the time parameter. Then, a corresponding second peak quality factor for each of the one or more peaks may be established based on the initial intensity measurements and the additional intensity measurements.

[0065] In a preferred embodiment, a particular range of the time parameter is iteratively determined by repeating the step of establishing a corresponding second peak quality factor for each of the one or more peaks associated with a second range of the time parameter. Preferably, each second range of the time parameter is progressively wider (so that the range of the time parameter is iteratively increased until a peak quality factor is found that satisfies the set one or more criteria).

[0066] The specific range of the time parameter may be determined by evaluating the respective peak quality factor of each of the one or more peaks according to a criterion (or criteria).The specific range of the time parameter may be determined based on the criteria that are satisfied.

[0067] Preferably, the substance is quantified based on (summing) the intensity measurements of each of the one or more peaks falling within the determined specific range.

[0068] Alternatively, the determined specific range of the time parameter may be used to quantify the same substance from different samples.

[0069] Reference now Figure 4 , along these lines a block diagram of the second process is shown. For the purpose of quantifying a substance, the process is specifically used to determine the fragments involved. In an initial step 200, the mass spectrometer provides (or the controller retrieves from the mass spectrometer) data including intensity measurements of the mass-to-charge ratio for each of one or more constituent analytes. In this case, there must be multiple peaks (each for a different m / z value or range). This typically occurs when considering a fragment of one of the constituent analytes (e.g., from a DIA MS 2 ), but there may be other cases where multiple peaks of a substance are considered for quantification.

[0070] Advantageously, the optional identification step 205 is performed first. This is described above with reference to Figure 3 Based on the initial step 200 or based on the optional identification step 205, an initial set of peaks are identified for quantification.

[0071] In the evaluation step 210, the peak values ​​identified for quantification are evaluated. There are two options for this evaluation. The first option is to perform one or more PQF algorithms on the search data gathered within the time parameter range. The second option is to determine the common peak position. This can be established by statistical measures, such as determining the average value, which can include the average value, median or mode of the peak position. These two options can be used together.

[0072] The evaluation results are then compared to at least one criterion in a verification step 220. For example, the results of a PQF algorithm (or multiple PQF algorithms) for each peak value can be compared to a threshold value. Additionally or alternatively, the peak position of each peak in the peak value can be compared to a determined common peak position. The threshold value can be predetermined for the spacing between the peak position and the determined common peak position so that the criterion can include a spacing that exceeds the threshold value. For example, one of the peak values ​​can be an interfering peak originating from another parent ion. Separation in the time parameter domain can be sufficient to identify peaks that should be excluded from quantification.

[0073] If at least one criterion is not met, then a peak reselection path 224 is taken to a peak removal step 230. In the peak removal step 230, at least one peak is selected to be ignored from those peaks previously identified for quantification. This is typically determined based on the evaluation step 210. For example, if a peak does not meet a PQF-based criterion (threshold) and / or has a peak position that is at least (or greater than) a threshold away from a determined common peak position, then the peak is selected to be ignored from a set of peaks identified for quantification.

[0074] The process then continues by repeating the evaluation step 210, but now with an updated set of peaks identified for quantification. It will be appreciated that this cycle may be repeated multiple times if at least one of the criteria considered in the verification step 220 is not met.

[0075] If at least one of the criteria considered in the validation step 220 is met (e.g., all peaks have sufficient PQFs and / or the peak positions of all peaks do not exceed (or are less than) a threshold from a determined common peak position), the quantification path 226 is taken. In the quantification step 240, the substance is quantified based on the set of peaks identified for quantification as updated during the process. This can be achieved by summing the intensities of the fragments in the set of peaks identified for quantification, particularly between the boundaries defined by the selected time range.

[0076] Advantageously, these two processes can be performed simultaneously or sequentially. As a result, for quantification purposes, a selected time range and a selected set of peak values ​​can be determined.

[0077] In either or both methods, the results (selected time range and / or selected set of peaks) used to quantify one sample are beneficially used to quantify the same substance in another sample. This may apply, for example, if multiple sample replicates are processed in multiple experimental runs. This can ensure consistent quantification between runs.

[0078] Returning to the general terms discussed above, another aspect can be considered (which can be combined with any other aspect disclosed herein). In this aspect, a method of quantifying a substance present in a sample can also be considered. The same considerations as discussed above can apply to this aspect.

[0079] In this method, intensity measurements of the mass-to-charge ratio of each of the one or more constituent analytes are received from a mass spectrometer. For each constituent analyte, the relationship between the measured intensity and the time parameter at each of the multiple selected mass-to-charge ratios defines a corresponding peak. In other words, peaks for multiple selected mass-to-charge ratios are received. For example, these can each represent fragments from the same substance (e.g., in a DIA MS n (under experiment).

[0080] A respective peak quality factor may be determined for each of the peaks. The peak quality factor depends on the range of the time parameter. Preferably, a plurality of peak quality factors may be determined for each of the peaks. Further details regarding optional aspects and / or specific implementations of the peak quality factor determination have been discussed above and are equally applicable here. Additionally or alternatively, a common peak position with respect to the time parameter is determined for at least some of the peaks. A subset of the peaks is then selected for quantifying the substance based on the determined peak quality factors and / or the determined common peak positions.

[0081] In this way, the peaks selected for quantifying the substance are determined based on their characteristics, in particular the quality of the peaks and / or whether the peaks are aligned by a common (maximum) position. The common peak position can be determined statistically, for example, by averaging, weighted averaging, or a heuristic algorithm. Peaks of poor quality and / or offset peaks can indicate interference, and ignoring such peaks in the quantification of the substance can improve accuracy.

[0082] Optionally, selecting the subset of peaks may include evaluating the peak position of each peak with respect to the determined common peak position (e.g., determining an offset). The subset of peaks is then advantageously selected based on the evaluation (e.g., by ignoring any peak having a peak position that is at least or more than a threshold away from the determined common peak position).

[0083] Advantageously, selecting the subset of peaks comprises evaluating a respective peak quality factor (or peak quality factors) of each of the peaks according to a criterion (e.g., whether one or more thresholds are met or exceeded, e.g., each threshold may be applied to a respective peak quality factor). The subset of peaks may then be selected based on whether the criterion is met for each peak. There may be a plurality of criteria, wherein each criterion may be applied to a respective peak quality factor.

[0084] Each peak in the selected subset of peaks is preferably quantified (by summing the measured intensities over the selected range of the time parameter).The substance may then be quantified based on the quantification of the selected subset of peaks (eg, summing them).

[0085] Preferably, the substance and / or chemical composition corresponding to at least one peak (and typically each peak) is identified (after receiving the measurement data). This may further assist in determining a set of peaks from which to select a subset.

[0086] As mentioned above, each peak advantageously comprises at least a predetermined number of intensity measurements, typically at least 5 intensity measurements (but optionally 7, 9 or 11). Peaks having fewer than the predetermined number of intensity measurements may be ignored.

[0087] Reference now Figure 5a to Figure 5d , each of which illustrates a graph of measured intensity versus time peak, showing the effect of different PQF algorithms when their time ranges are changed. As discussed above, these use Figure 2 The same data is shown, with three peaks. Three exemplary PQF algorithms are used: ZigZagId (smaller numbers are considered better); Symmetry (-1 is best, and the closer to this value the better); and trigPeakSim (1 is best, and the closer to this value the better).

[0088] For completeness, a brief description of these exemplary PQF algorithms is now provided (more complete details can be found in "MetaClean: a machine learning-based classifier for reduced false positive peak detection in untargeted LC-MS metabolomics data" by Chetnik et al., detailed above). ZigZagId is short for zigzag index. The zigzag index (ZZ) captures shape quality by measuring the normalized variance between a point on either side and its immediate neighbors. The normalized average of all zigzag index values ​​(a zigzag index value can be calculated for each point of a peak except for the two extreme values) is the value used as the quality factor for the peak. Where ZZ is the zigzag index of the peak and I n is the nth intensity measurement (there are N measurements in the peak), which can be expressed mathematically as:

[0089] ZZ=ZZ1 / ZZ2;

[0090] ZZ1=sum((2I n -I n-1 -I n+1 ) 2 ), from n=2 to n=N-1;

[0091] ZZ2=N*EPI 2 ;as well as

[0092] EPI=I A -avg(I1+I2+I N-1 +I N ), where I A is the value of maximum intensity.

[0093] Symmetry (SY) measures the correlation between the left and right halves of the peak. This can be expressed mathematically as:

[0094] SY=cor([I1,...,IN / 2],[IN / 2,...,IN]), range: [-1,1].

[0095] TrigPeakSim (TPASR) is short for Triangle Peak Area Symmetry or Similarity Ratio. This peak quality factor estimates the shape quality by comparing the peak area to the area of ​​the triangle formed by the vertices and the boundary. This can be expressed mathematically as:

[0096] TPASR = abs(triangle_area - AUC) / max(triangle_area, AUC), where AUC is the area under the peak curve (or simply peak area).

[0097] exist Figure 5a , select five data points as highlighted, and Figure 5b , the time range is extended to seven data points, Figure 5c Using nine data points, and Figure 5d Eleven data points are selected. For the first peak (with the lowest peak density) and the second peak (with the highest peak density), it can be seen that the ZigZagId value and the trigPeakSim value decrease as the number of data points increases. The symmetry values ​​of the first peak and the second peak change, but not significantly. For the third peak (with an intermediate peak density and a slight shift in the peak position compared to the other two peaks), the ZigZagId value decreases as the number of data points increases. However, the symmetry value is uncertain until the number of data points reaches 11. In contrast, the trigPeakSim value performs best when the number of data points is 5, 7, or 9, but is lower when the number of data points reaches 11.

[0098] It will therefore be appreciated that a combination of PQF algorithms is beneficially used to evaluate peak values ​​and determine the number of data points (for a selected range of a time parameter).

[0099] Returning to the general terms discussed above (in any respect), it will be appreciated that a mass spectrometer can be controlled to perform mass analysis of each of the constituent analytes and provide intensity measurements of the mass-to-charge ratio for each of the plurality of components.

[0100] Aspects according to the invention may be embodied as a computer program (optionally on a computer readable medium, which may be non-transitory), a controller for a mass spectrometry system (in hardware and / or software form), or a mass spectrometry system including such a controller. It should be understood that such a mass spectrometry system may also include: a first separator configured to separate the sample into constituent analytes on a time parameter; and a mass spectrometer configured to receive and analyze the constituent analytes.

[0101] Because a minimum number of intensities (which may be necessary or desirable) are retrieved in order to have reliable quantification, processes, methods or specific implementations according to the present disclosure can consume minimal RAM memory. Such processes can be run on multiple samples simultaneously. This can allow control of the consistency of the product ion set used for quantification and similar peak boundaries for all samples.

[0102] The separation of DIA identification and DIA quantification may allow a second validation step, thereby improving the quantification and even discarding the quantification of unreliable compounds (in the case where all product ions from the common product ion set in the sample fail to produce any reliable peak). Moreover, a (more) consistent quantification may be provided, since the same set of product ions may be used on all samples. Furthermore, if any product ion fails to produce a reliable peak, the product ion may be removed from the common product ion set for all samples. Separating identification from quantification may in particular give the opportunity to evaluate the best and most common product ion set for each identified compound on all samples involved.

[0103] Although embodiments according to the present disclosure have been described with reference to specific types of equipment and applications (specifically, mass spectrometry) and these embodiments have specific advantages in this case, as discussed herein, methods according to the present disclosure can be applied to other types of equipment and / or applications. Specifically, devices according to the present disclosure can be used for other applications. Although the specific structure, arrangement and operating details (e.g., parameters) of the processes described herein may be advantageous (especially in view of known configurations and capabilities), there may be great changes to achieve an operating mode with similar or identical performance. Other types of separations can be considered from those disclosed herein. Certain features may be omitted or replaced, for example, as shown herein. Unless otherwise stated, each feature disclosed in this specification may be replaced with an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise stated, each feature disclosed is only an example of a series of equivalent or similar attribute features.

[0104] It has been suggested above that the retention time range (or equivalently, the drift or other suitable time range) can be determined based on the measurement result of one sample and then used for another sample, especially if multiple sample replicas are processed in multiple experimental runs. Alternative methods are possible. For example, a parallel evaluation of the retention time range can be performed for all (or at least multiple) samples. This can allow the consensus (which can be common or statistically determined, such as average or weighted average) retention time range of the sample being evaluated to be determined. When using the parallel analysis of multiple samples, the peak value with less than a predetermined number of intensity measurements does not need to be ignored, but can be integrated into the quantitative measurement result. Then, the determined time range can be used for the quantification of all samples (possibly including other samples, not used for determining the range).

[0105] A variation of the method described above is to apply the same algorithm to a full MS1 scan, thereby searching only for the expected (precursor) mass of each identified compound. In this case, the term "fragment" used above can be replaced by the identified compound, and the method can be used in the same manner.

[0106] In the general terms discussed above, it may be optionally considered that the ions of the species being quantified have one of the selected mass-to-charge ratios. The quantification of the peaks may then correspond to the quantification of the species.

[0107] In the detailed description of various embodiments, for the purpose of explanation, many specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced with or without these specific details. In addition, those skilled in the art may readily appreciate that the specific order in which the methods are presented and executed is illustrative, and it is contemplated that the order may be changed and still remain within the scope of the various embodiments disclosed herein.

[0108] As used herein, including in the claims, unless the context indicates otherwise, the singular form of the terms herein should be interpreted as including the plural form, and vice versa. For example, unless the context indicates otherwise, a singular reference herein (including in the claims), such as "one / kind" (such as an ion multipole device) means "one or more" (for example, one or more ion multipole devices). In the specification and claims of the present disclosure, the words "include", "comprising", "having" and "containing" and variations of these words, for example, "including" or similar words mean "including but not limited to", and are not intended to (and do not) exclude other components. In addition, the use of "or" is inclusive, so that when "A" is true, "B is true", or "A" and "B" are both true, the phrase "A or B" is true.

[0109] The use of any and all examples or exemplary language ("for example," "such as," "for example," and similar language) provided herein is intended to merely better illustrate the present disclosure and does not indicate a limitation on the scope of the present disclosure unless otherwise required. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0110] The terms "first" and "second" may be reversed without changing the scope of the present disclosure. That is, an element referred to as a "first" element may instead be referred to as a "second" element, and an element referred to as a "second" element may instead be considered a "first" element.

[0111] Unless otherwise specified or the context requires otherwise, any steps described in this specification may be performed in any order or simultaneously. In addition, where a step is described as being performed after a step, this does not exclude intermediate steps being performed.

[0112] It should also be understood that, unless otherwise implicitly or explicitly understood or stated, for any given component or embodiment described herein, any possible candidate or alternative listed for that component may generally be used alone or in combination with each other. It should be understood that any list of such candidates or alternatives is merely illustrative and not restrictive, unless otherwise implicitly or explicitly understood or stated.

[0113] All literature and similar materials cited in this disclosure (including but not limited to patents, patent applications, articles, books, treaties, and internet web pages) are expressly incorporated by reference for any purpose. Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong.

Claims

1. A method for quantifying a substance present in a sample, wherein the sample is separated into constituent analytes by a first separator in terms of a time parameter, and the constituent analytes are then further analyzed by a mass spectrometer, the method comprising: receiving from the mass spectrometer an intensity measurement for a mass-to-charge ratio of each of the one or more constituent analytes, for each constituent analyte, a relationship of the measured intensity at each of the one or more selected mass-to-charge ratios to the time parameter defining a corresponding peak; as well as A particular range of the time parameter for quantifying the substance is determined based on a corresponding peak quality factor for each of the one or more peaks, the peak quality factor being dependent on the range of the time parameter. 2 . The method of claim 1 , wherein a flatness detection algorithm is used to further determine the specific range of the time parameter.

3. The method according to claim 1 or claim 2, wherein determining the specific range of the time parameter comprises: establishing a respective first peak quality factor for each of the one or more peaks relative to the first range of the time parameter; establishing a respective second peak quality factor for each of the one or more peaks relative to a second range of the time parameter, the second range of the time parameter being narrower or wider than the first range of the time parameter; and The specific range of the time parameter for quantifying the substance is determined based on one or more first peak quality factors and one or more second peak quality factors. 4 . The method of claim 3 , wherein the second range of the time parameter is selected based on the one or more first peak quality factors and / or a predetermined differential factor.

5. The method of claim 3 or claim 4, wherein the received intensity measurements for the mass-to-charge ratio of each of the one or more constituent analytes are initial intensity measurements for the first range of the time parameter, and the second range of the time parameter is wider than the first range of the time parameter, the method further comprising: receiving additional intensity measurements for mass-to-charge ratios of the one or more constituent analytes, the initial intensity measurements and the additional intensity measurements together covering the second range of the time parameter; and Wherein the respective second peak quality factor of each of the one or more peaks is established based on the initial intensity measurement and the additional intensity measurement.

6. A method according to any one of claims 3 to 5, wherein the specific range of the time parameter is iteratively determined by repeatedly establishing a corresponding second peak quality factor for each of the one or more peaks associated with a second range of the time parameter, each second range of the time parameter becoming gradually wider.

7. The method according to any preceding claim, further comprising: using the determined specific range of the time parameter to quantify the same substance from different samples; and / or The substance is quantified based on the intensity measurement of each of the one or more peaks that fall within the determined particular range.

8. A method according to any preceding claim, wherein the step of determining the specific range of the time parameter comprises: The respective peak quality factor of each of the one or more peaks is evaluated according to a criterion, and the particular range of the time parameter is determined based on satisfying the criterion.

9. A method for quantifying a substance present in a sample, wherein the sample is separated into constituent analytes by a first separator in terms of a time parameter, and the constituent analytes are then further analyzed by a mass spectrometer, the method comprising: receiving from the mass spectrometer an intensity measurement for a mass-to-charge ratio of each of the one or more constituent analytes, for each constituent analyte, a relationship of the measured intensity to the time parameter at each selected mass-to-charge ratio of a plurality of selected mass-to-charge ratios defines a corresponding peak; determining for each of the peaks a respective peak quality factor, the peak quality factor being dependent on a range of the time parameter and / or determining for at least some of the peaks a common peak position with respect to the time parameter; as well as A subset of the peaks is selected for quantifying the substance based on the determined peak quality factors and / or the determined common peak positions.

10. The method of claim 9, wherein selecting the subset of the peaks comprises: The peak position of each of the peaks is evaluated according to the determined common peak position, and the subset of the peaks is selected based on the evaluation.

11. The method of claim 9 or claim 10, wherein selecting the subset of the peaks comprises: The respective peak quality factor of each of the peaks is evaluated according to a criterion, and the subset of the peaks is selected based on whether each peak satisfies the criterion.

12. The method according to any one of claims 9 to 11, further comprising: quantizing each peak in the selected subset of the peaks; as well as The substance is quantified based on the quantification of the selected subset of the peaks.

13. The method according to any one of claims 9 to 12, further comprising the method according to any one of claims 1 to 8.

14. A method according to any one of claims 1 to 11 or 13, wherein the ions of the species being quantified have one of the selected mass-to-charge ratios.

15. A method according to any preceding claim, wherein the constituent analytes are analysed in the mass spectrometer in the form of ions by: processing the ions; and mass analysing the processed ions.

16. A method according to claim 15, wherein the sample is a liquid or gas phase sample, the first separator is a chromatographic separator, the time parameter relates to retention time, the constituent analytes are components separated from the chromatographic separator at different retention times, and the constituent analytes are further analyzed by the mass spectrometer by ionizing each analyte to provide the ions.

17. The method of claim 15, wherein the sample is a mixture of sample ions, the first separator is an ion mobility separator, the time parameter relates to a drift time or a retention time, and the constituent analytes are constituent ions of the mixture of sample ions.

18. A method according to any one of claims 15 to 17, wherein the ions are precursor ions and the processing of the ions comprises fragmenting the precursor ions to produce fragment ions such that the fragment ions are mass analysed.

19. The method of claim 18, wherein each of the peaks corresponds to a data independent acquisition (DIA) MS from the constituent analytes. n The corresponding fragment ions analyzed.

20. The method according to any preceding claim, further comprising: Identify the substance; and / or A chemical constituent corresponding to the at least one peak is identified, the one or more selected mass-to-charge ratios of the at least one peak corresponding to the identified chemical constituent.

21. A method according to any preceding claim, wherein each peak comprises at least a predetermined number of intensity measurements.

22. The method according to any preceding claim, further comprising: The mass spectrometer is controlled to perform mass analysis of each of the constituent analytes and provide the intensity measurement results for the mass-to-charge ratio of each of the plurality of components.

23. A computer program configured, when operated by a processor, to perform the method according to any preceding claim.

24. A controller for a mass spectrometry system, the controller being configured to control the mass spectrometry system to perform the method according to any one of claims 1 to 22.

25. A mass spectrometry analysis system, comprising: a first separator configured to separate the sample into component analytes over a time parameter; a mass spectrometer configured to receive and analyze the constituent analytes; and A controller according to claim 24.

Images