Methods and systems for compound identification in untargeted mass spectrometry

Abstract

In one aspect, a method for compound identification using mass spectrometry is disclosed, which includes passing a plurality of different precursor ions through an ion dissociation device to cause fragmentation thereof to generate a plurality of fragment ions, acquiring one or more fragment ion signals associated with said plurality of fragment ions, and assigning to at least one of the fragment ion signals an uncertainty corresponding to at least one ion attribute such that the assigned uncertainty is independent of a respective uncertainty assigned to any other one of said fragment ion signals.

Description

[0001] P2023-3015-WO (PLG Ref. 4277-0407W001)

[0002] METHODS AND SYSTEMS FOR COMPOUND IDENTIFICATION IN UNTARGETED MASS SPECTROMETRY

[0003] Related Applications

[0004] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 706,513, filed on October 11, 2024, the content of which is incorporated by reference herein in its entirety.

[0005] Technical Field

[0006] The present disclosure is generally related to methods and systems for performing mass spectrometry, and in particular to such methods and systems that can be used for compound identification in complex MS / MS spectra.

[0007] Background

[0008] Mass spectrometry (MS) can be useful for identifying unknown compounds, determining the composition of atomic elements in a molecule, determining the structure of a compound by observing its fragmentation, and quantifying the amount of a particular chemical compound in a mixed sample.

[0009] Tandem mass spectrometry, can employ multiple stages of mass analysis for providing detailed information about molecules. In one example, in a two-stage mass spectrometry, one or more analytes within a sample under analysis are ionized to generate a plurality of precursor ions (MSI) and a mass filter is utilized to select those precursor ions having mass-to-charge ratios within a bandpass of the mass filter. Subsequently, the selected precursor ions are fragmented to generate fragment ions and a mass spectrum of the fragment ions is acquired and analyzed (MS2) to obtain information about the analytes.

[0010] Untargeted mass spectrometry can be used for identification and relative quantification of compounds in a sample. Untargeted mass spectrometry is increasingly used for compound identification in complex samples. For example, untargeted metabolomic analysis can involve the identification of thousands of metabolites in a complex biological sample.

[0011] High resolution mass spectrometers can typically measure the exact mass of a compound. However, interferences and low statistics for less abundant ions can adversely affect mass P2023-3015-WO (PLG Ref. 4277-0407W001) accuracy determination, resulting in the reduction of relevant compound identifications. Further, the efficiency of the generation of search results, e.g., from either proteomics or metabolomics, can be reduced and the size of a possible search space in both cases can be increased, thus reducing the likelihood of accurate matches.

[0012] Unambiguous spectrum identification is still a challenge, for low signal / noise (S / N) and DIA (data independent acquisition) deconvoluted MS / MS spectra.

[0013] Summary

[0014] In one aspect, a method for compound identification using mass spectrometry is disclosed, which comprises passing a plurality of different precursor ions through an ion dissociation device to cause fragmentation thereof to generate a plurality of fragment ions, acquiring one or more fragment ion signals associated with said plurality of fragment ions, assigning to at least one of the fragment ion signals a corresponding experimentally-derived uncertainty associated with at least one ion attribute such that the assigned uncertainty is independent of a respective uncertainty assigned to any of the other of said fragment ion signals. The method further includes generating at least one candidate for the identity of a compound corresponding to at least one of the precursor ions by utilizing the at least one ion attribute and its corresponding experimentally-derived uncertainty.

[0015] In various embodiments, the experimentally-derived uncertainty can include an uncertainty interval and one or more uncertainty values over that interval. In some such embodiments, the uncertainty values are distributed uniformly over that interval, and in some other embodiments, the uncertainty values are distributed non-uniformly over that interval. For example, the uncertainty can include an m / z range and uncertainty values over that range, e.g., in the form of a continuous function of uncertainty values.

[0016] In various embodiments, the at least one candidate can include a plurality of candidates for the identity of said compound. In some such embodiments, the plurality of candidates can be ranked and the ranking can then be employed to identify the most probable candidate among the plurality of candidates.

[0017] The experimentally-derived uncertainties can be determined by evaluating one or more of the fragment ion signals. The signals can be one-dimensional or multi-dimensional and can be P2023-3015-WO (PLG Ref. 4277-0407W001) analyzed in a single or multiple dimensions. Further, when analyzing the signals in multiple dimensions, they can be analyzed in each dimension independently of the other dimensions or in combination with one or more of the other dimensions.

[0018] By way of example, the step of evaluating the one or more of the fragment ion signals can include evaluating at least one fragment ion signal shape relative to an expected signal shape.

[0019] By way of example, the ion attributes can be any of m / z ratio of a precursor or a fragment ion, an association between a fragment ion and an m / z of a respective precursor ion, an ion mobility of a precursor ion, an electrophoretic mobility of a precursor ion, a retention time associated with passage of an analyte corresponding to a precursor ion through an LC column.

[0020] In various embodiments, the method can further include associating at least one of the fragment ion signals with an attribute of at least one of a plurality of different co-isolated precursor ions. By way of example, such an attribute can be any of an m / z ratio and an ion mobility.

[0021] In various embodiments, an intensity variation of the at least one of the fragment ion signals is determined as a function of a change in a parameter of a mass filter through which the precursor ions pass to reach said dissociation device and the intensity variation is utilized to determine an association between the fragment ion signal and a precursor m / z as well as an uncertainty corresponding to that association.

[0022] By way of example, the parameter of the mass filter can be a transmission m / z range associated with the mass filter.

[0023] In various embodiments, the intensity variation function can correspond to a variation of the intensity of the fragment ion signal as a function of a change of said transmission m / z range of the mass filter. In some such embodiments, the intensity variation function can have a substantially triangular shape and the m / z associated with the at least one of the precursor ions can correspond to an apex of the substantially triangular shape. By way of example, the corresponding experimentally-derived uncertainty can correspond to an uncertainty in the m / z position of the apex. By way of example, and without limitation, in some embodiments, the transmission m / z range of the mass filter can be varied by scanning the mass filter m / z transmission range along the m / z dimension. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0024] The intensity variation functional profile is not limited to a triangular shape. By way of example, in various embodiments, such a function can have a Gaussian profile and the m / z associated with the precursor ion and its corresponding experimentally-derived uncertainty can relate to a particular feature of the Gaussian profile. For example, in various embodiments, the maximum of the Gaussian profile can correspond to the m / z associated with the precursor ion and the width (e.g., full width at half maximum) can correspond to an uncertainty associated with the m / z.

[0025] In various embodiments, the use of the ion attributes and their respective uncertainties can lead to multiple candidates for the identity of the precursor ion giving rise to at least one of the fragment ion signals. In such embodiments, the candidates can be ranked using one or more attributes and their associated uncertainties, such as mass accuracy, the match scores for fragment ion signals and the intensities of the fragment ion signals. Such ranking can then be utilized to identify the most likely compound corresponding to the precursor ion. The identity of the compound, or the most likely compound, can be presented to a user.

[0026] In various embodiments, the precursor level attributes can be determined based on a combination of information derived from both MSI and MS2 spectra. By way of example, the MS2 data can be utilized, e.g., in a manner discussed above, to determine an estimate of m / z range for a precursor ion. The MS 1 spectrum can then be used to identify one or more mass peaks are present in that m / z range. If a single mass peak is identified in that range, the m / z associated with that mass peak can be used as a candidate precursor m / z. On the other hand, if two or more mass peaks are found in that m / z range, the m / z values corresponding to those peaks can be identified as the candidates for the precursor m / z. Each candidate can be ranked to identify the most probable candidate. In various embodiments, the ranking of the candidates can be based on the experimentally-derived uncertainties.

[0027] In various embodiments, as one or more compounds listed in a library database can be identified as one or more candidates based on at least one ion attribute and its corresponding experimentally-derived uncertainty.

[0028] In various embodiments, the fragment ion signals can be generated and analyzed via a data independent acquisition (DIA) mass spectrometric method, such as scanning SWATH. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0029] In various embodiments, the identity of the compound can be presented to a user.

[0030] In various embodiments, a programmed computer can be used to perform various steps of the methods according to the present teachings, such as the step of assigning to one or more of the fragment ions a respective experimentally-derived uncertainty associated with at least one ion attribute and the step of generating the at least one candidate for the identity of a compound corresponding to at least one of the precursor ions. In various embodiments, the above method can include passing the plurality of different precursor ions or a plurality of compounds giving rise to said precursor ions through a separation device prior to introduction of the precursor ions into the ion dissociation device, wherein at least one operating parameter of the separation device causes selection of one or more precursor ions for passage through the separation device. In general, the separation device can separate analytes and / or ions derived from those analytes based on time and / or one or more physiochemical properties of the analytes and / or the ions. By way of example, the separation device can be an ion mass filter that can separate ions based on their m / z ratios. By way of another example, the separation device can be an ion mobility spectrometer that can separate ions based on their mobility. Alternatively, the separation device can be a liquid chromatography column that can separate analytes based on their retention times. By way of another example, the separation device can be a capillary electrophoresis device that can separate ions based on their electrophoretic mobility.

[0031] By way of example, in some embodiments, an ion mobility spectrometer (IMS) can be utilized to separate ions generated via ionization of one or more analytes in a sample along the ion mobility dimension. The ions exiting the IMS can then be introduced into a mass spectrometer, which can employ a scanning transmission window to provide ion separation along the m / z dimension.

[0032] In various embodiments in which the separation device is a mass filter, the method can include scanning an m / z transmission window of the mass filter so as to generate a plurality of overlapping m / z transmission windows. In such embodiments, the association of any of the fragment ion signals with an m / z ratio of a precursor ion can be performed via the analysis of the variation of an intensity of that fragment ion signal as a function of the scanning of the m / z transmission window of the mass filter. By way of example, the peak of such variation can be identified as the m / z ratio of the precursor ion. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0033] In various embodiments, the fragment ion signals can be processed, e.g., by a digital microprocessor, to determine the ion attributes and the corresponding experimentally-derived uncertainties. Further, the digital microprocessor can be employed to generate one or more candidates for the identity of one or more of the precursor ions and to rank multiple candidates to arrive at the most probable one.

[0034] In a related aspect, a method for compound identification using mass spectrometry is disclosed, which includes passing a sample through a chromatographic device to generate an eluate containing one or more compounds, ionizing the one or more compounds to generate a plurality of precursor ions, passing the plurality of precursor ions through a mass filter as an m / z transmission window of the mass filter is scanned so as to generate a plurality of overlapping m / z transmission windows, generating one or more fragment ions via dissociation of the precursor ions passing through the mass filter, and acquiring one or more fragment ion signals associated with said one or more fragment ions. For at least one of the fragment ions, the respective fragment ion signal can be associated with a precursor ion m / z to generate a plurality of associations each corresponding to a pair including one of said fragment ion signals and a precursor ion m / z. An independent uncertainty is assigned to each of said associations. The method further includes generating at least one candidate for an identity of a compound corresponding to at least one of the precursor ions based on the associations and their respective uncertainties.

[0035] In various embodiments, the step of associating the at least one of the fragment ion signals with a precursor m / z is performed by determining a variation of an intensity of that fragment ion signal as a function of variation of a parameter of a mass filter positioned upstream of an ion dissociation device. For example, the parameter can correspond to a transmission m / z range of the mass filter, e.g., an m / z position of the transmission window of the mass filter as the transmission window is scanned over the m / z dimension. By way of example, the intensity variation can have a substantially triangular shape and the step of associating the fragment ion signal with the precursor m / z can include associating that fragment ion signal with an m / z ratio corresponding to an apex of the substantially triangular shape. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0036] The method can also include assigning an uncertainty to the association between the fragment ion signal and the respective precursor m / z based on the degree of sharpness of the apex of the substantially triangular shape.

[0037] Alternatively, the intensity variation can have other shapes, such as a Gaussian shape, where a parameter associated with the intensity variation can be used to associate a fragment ion signal with at least one precursor m / z.

[0038] In various embodiments, the ion attributes and their associated uncertainties can be utilized to generate at least one candidate for an identity of at least one compound corresponding to at least one of the precursor ions. By way of example, such a candidate can be generated via assigning probabilities to one or more compounds in a library database based on the ion attributes and their associated uncertainties.

[0039] The above methods can be implemented by using a computer having a digital processor, and at least one memory module in which instructions for carrying out the steps of the above methods can be stored and executed during runtime by the processor.

[0040] In a related aspect, a computer-implemented method for identifying a compound in a sample using mass spectrometry is disclosed, which includes using a microprocessor to process a plurality of fragment ion signals corresponding to fragment ions generated via dissociation of one or more precursor ions, using the microprocessor to assign to at least one of said fragment ion signals a corresponding experimentally- derived uncertainty associated with at least one ion attribute, where the uncertainty assigned to the at least one of said fragment ion signals is independent from a respective uncertainty assigned to any of the other ones of the plurality of fragment ion signals, and using the microprocessor to generate at least one candidate for identity of a compound corresponding to at least one of the precursor ions by utilizing the at least one ion attribute and the corresponding experimentally-derived uncertainty.

[0041] The methods according to various embodiments can be employed to analyze data generated by a variety of mass spectrometers. By way of example, in a related aspect, a mass spectrometer is disclosed, which includes an ion source for ionizing a sample to generate a plurality of different precursor ions, a mass filter configured to allow passage of at least one subset of the plurality of precursor ions having one or more m / z ratios within an m / z P2023-3015-WO (PLG Ref. 4277-0407W001) transmission window of the mass filter therethrough, a controller in communication with the mass filter to change a parameter thereof, e.g., to cause scanning of the m / z transmission window of the mass filter over an m / z range so as to generate a plurality of overlapping m / z transmission windows, and an ion dissociation device for receiving ions exiting the mass filter and causing dissociation thereof to generate a plurality of fragment ions. The mass spectrometer further includes a mass analyzer configured to receive the fragment ions and generate one or more fragment ion signals. Moreover, the mass spectrometer includes a data analysis module having a microprocessor, where the data analysis module is configured: to process the fragment ion signals to derive a plurality of ion attributes, assign to at least one of the fragment ion signals a corresponding experimentally-derived uncertainty associated with at least one of the ion attributes, where the experimentally-derived uncertainty assigned to the at least one of the fragment ion signals is independent from a respective uncertainty assigned to any of the other ones of the plurality of fragment ion signals, and generate at least one candidate for the identity of a compound corresponding to at least one of the precursor ions by utilizing the at least one ion attribute and its corresponding experimentally-derived uncertainty.

[0042] In various embodiments, one of the ion attributes can correspond to an association between a fragment ion signal and an m / z ratio of one of the precursor ions. By way example, an intensity variation of the fragment ion signal as a function of a change in an m / z associated with a scanning m / z transmission window of a mass filter through which the precursor ions pass prior to reaching said dissociation device can be used to determine the association. For example, in various embodiments, the variation can have a substantially triangular shape, where the m / z associated with the apex of the triangular shape can be assigned as the precursor ion m / z corresponding to the respective fragment ion.

[0043] Further understanding of various aspects of the present teachings can be obtained with reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.

[0044] Brief Description of the Drawings

[0045] FIG. 1 is a flow chart depicting various steps of a method according to an embodiment of the present teachings, P2023-3015-WO (PLG Ref. 4277-0407W001)

[0046] FIG. 2A is an example of a spectrum of a plurality of fragment ions generated via dissociation of a plurality of precursor ions,

[0047] FIG. 2B in an enlarged view of several low-intensity mass peaks in the mass spectrum illustrated in FIG. 2A,

[0048] FIG. 3 is an exemplary plot that shows the variation of the mass peak intensity of a fragment ion mass peak as a function of the m / z of the leading edge of a scanning ion transmission window of a mass filter through which the precursor ions, which give rise to the fragment ions, pass,

[0049] FIG. 4A shows a plot similar to that depicted in FIG. 3 but with an apex that is not as sharp as the one in the plot of FIG. 3,

[0050] FIG. 4B shows a plot similar to that depicted in FIG. 4A but with the quadrupole shape of a fragment originating from two precursor ions with distinct m / z ratios, but co-isolated by the mass filter,

[0051] FIG. 5 schematically depicts a computer system according to various embodiments that can be used to implement methods according to various embodiments of the present teachings,

[0052] FIG. 6 schematically depicts a mass spectrometer according to an embodiment of the present teachings,

[0053] FIGS. 7A shows an example of a mass spectrum containing ion mass signals (mass peaks) corresponding to a plurality of precursor ions,

[0054] FIG. 7B shows a mass spectrum containing ion mass signals (mass peaks) corresponding to a plurality of fragment ions generated via dissociation of the plurality of precursor ions as shown in FIG. 7A,

[0055] FIGS. 7C-1 - 7C-7 present plots exhibiting the variation of the mass peak intensity of select fragment ion mass peaks in the fragment ions spectrum depicted in FIG. 7B as a function of the m / z of the leading edge of a scanning ion transmission window of a mass filter through which the precursor ions, which give rise to the fragment ions, pass, P2023-3015-WO (PLG Ref. 4277-0407W001)

[0056] FIG. 8A shows fit scores of analytes in a given cycle before and after weighting various probabilities regarding a compound identification and after deconvolution, and

[0057] FIG. 8B shows purity scores of analytes at a given cycle before and after weighting various probabilities regarding a compound identification and after deconvolution (filtered).

[0058] P2023-3015-WO (PLG Ref. 4277-0407W001)

[0059] Detailed Description

[0060] It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of the specifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

[0061] As used herein, the terms "about" and "substantially equal" refer to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the terms "about" and "substantially" as used herein mean 10% greater or less than the value or range of values stated or the complete condition or state. For instance, a concentration value of about 30% or substantially equal to 30% can mean a concentration between 27% and 33%. The terms also refer to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art.

[0062] 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 " / ".

[0063] The term “dissociation device,” as used herein, refers to a component or an apparatus used in a mass spectrometry system that is configured to induce dissociation of ions through various physical and / or chemical mechanisms. Some examples of such mechanisms include, without limitation, collision-induced dissociation (CID), electron capture dissociation (EDC), P2023-3015-WO (PLG Ref. 4277-0407W001) electron transfer dissociation (ETD), dissociation via application of radiation, such as ultraviolet (UV) and infrared radiation (e.g., infrared multiphoton dissociation (IRMPD).

[0064] The term “uncertainty,” as used herein, refers to a parameter that characterizes the probability distribution of values that can be attributed to a measurement or an ion attribute derived from a measurement. As discussed in more detail below, and by way of example, in the context of the present application, a measurement can be directed to the operating parameters associated with a transmission window of a mass filter, the ion detection signals generated by an ion detector, elution profile of eluates exiting an LC column, etc. The measurements can be used to derive the ion attributes, such as the centroid m / z of a precursor or a fragment mass peak, an association between a fragment ion signal and a precursor m / z, the LC peak apex and / or shape, ion mobility, etc. An independent uncertainty can be assigned to each ion attribute. For example, the uncertainty associated with an ion attribute can provide an interval of values within which the true value of the ion attribute can he with a stated probability, e.g., 95%. In various embodiments, an uncertainty associated with an attribute can include an uncertainty range as well as uncertainty values over that range, which can be uniformly or non-uniformly distributed.

[0065] The term “data-independent acquisition (DIA)” refers to a mass spectrometry technique characterized by the systematic and unbiased fragmentation of all precursor ions within predetermined mass-to-charge (m / z) windows, regardless of their abundance or intensity. DIA is a particularly useful technique for analyzing complex samples, proteomics, metabolomics and lipidomics. An example of a DIA technique is scanning SWATH in which precursor ions passing through a wide ion transmission window of a mass filter are subjected to dissociation and mass analysis as the ion transmission window is scanned along the m / z dimension.

[0066] The present disclosure generally provides methods and systems for mass spectrometry and in particular, such methods and systems that can be utilized in untargeted identification of compounds in samples, including complex biological samples. As discussed in more detail below, in various embodiments, the methods and systems of the present teachings can be used in combination with DIA to assign optimal precursor and fragment tolerances associated with precursor and fragment ions. The assignment of optimal independent fragment tolerances can in turn reduce the number of candidates for the identification of compounds. Further, in various P2023-3015-WO (PLG Ref. 4277-0407W001) embodiments, the use of feature-weighting strategies for fragment scoring can reduce the chance of incorrect compound identification associated with MS / MS spectra, which can lead to an increase in the discriminatory power of the multiple identification candidates. In various embodiments, the application of compound identification methods according to the present teachings can lead to lower rates of incorrect identification.

[0067] Automated searching tools employed in peptide identification compare experimental data against either in-silico predictions or data from previous identifications. In both approaches, search tools generally utilize a single mass tolerance at MSI and MS2 levels to generate a list of peptide-spectrum matches, which are then scored / ranked to determine the most likely identification. In such conventional methods, the precursor tolerance parameter is optimized for the corresponding instrument.

[0068] As noted above and discussed in more detail below, in various embodiments, multidimensional features corresponding to each individual precursor / fragment pair can be employed to derive optimal precursor and fragment tolerances. As discussed in more detail below, in various embodiments, such tolerances can be determined for each precursor / fragment pair independent of the other precursor / fragment pairs. In various embodiments, utilizing feature-weighting for fragment scoring can additionally reduce the chances of incorrect identification of precursor ions based on MS / MS spectra, particularly when multiple candidates exist for the identification. As such, various embodiments of the present teachings for compound identification based on MS / MS spectra can improve identification of compounds.

[0069] In various embodiments, experimentally-derived uncertainties for one or more attributes of fragment ions generated via dissociation of one or more precursor ions are assigned to the fragment ions, where the uncertainties are independently obtained for each fragment ion. This is in contrast to conventionally used uncertainties (tolerances) with respect to attributes of fragment ions in which the same uncertainty is employed for all fragment ions, e.g., typically m / z is used as an attribute with the same uncertainty for all fragment ions.

[0070] In conventional methods of compound identification, an assigned uncertainty is uniformly distributed over a fixed interval and the same uncertainty is employed for different fragment ions. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0071] In contrast, in various embodiments, the uncertainty is experimentally derived for each fragment ion independently and can have the following properties: the uncertainty interval and the uncertainty values over that interval are a function of the attribute type and can be uniformly, or non-uniformly distributed over the interval. For example, the m / z of a fragment ion is one type of attribute and the relation of a fragment ion to a respective precursor ion is another type of attribute. By way example, the association between a fragment ion and a respective precursor can be determined using measurements in one or more of these dimensions; LC, ion mobility, and / or mass filter.

[0072] By way of example, a fragment-precursor attribute and its associated uncertainty can be determined by analyzing a fragment ion signal as a function of a change in at least one parameter of a mass filter through which precursor ions pass prior to reaching an ion dissociation device in which the precursor ions are dissociated to generate the fragment ion. Further, in some embodiments, the fragment-precursor association and its corresponding uncertainty can be determined as a two dimensional function of LC retention time (RT) and a mass filter parameter or as separate analysis of one-dimensional functions of RT and a mass filter parameter.

[0073] Without any loss of generality, various embodiments of the present teachings are described below by reference to fragment ions generated via dissociation of a plurality of precursor ions. The principles of the present teachings apply equally to other types of product ions, e.g., adduct ions, that may be generated from precursor ions.

[0074] FIG. 1 is a flow chart depicting various steps of a method according to an embodiment of the present teachings for compound identification using mass spectrometry. The illustrated method includes passing a plurality of different precursor ions through an ion dissociation device to cause fragmentation thereof to generate a plurality of fragment ions and acquiring one or more fragment ion signals associated with the plurality of the fragment ions. The method further includes assigning to at least one of the fragment ion signals a corresponding experimentally- derived uncertainty corresponding to at least one fragment and / or precursor ion attribute (herein also referred to for brevity as “ion attribute”) such that the uncertainty corresponding to the assigned ion attribute is independent of a respective uncertainty assigned to any of the other fragment ion signals. The ion attributes and the associated uncertainties can then be utilized to generate candidates for the identity of one or more compounds within a sample under analysis. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0075] Without any loss of generality and for illustration of various aspects of the present teachings, an example of a method according to an embodiment of present teachings is discussed below in connection with mass data obtained using a DIA technique, known as scanning SWATH. In scanning SWATH, a sample undergoes ionization to generate a plurality of precursor ions. The precursor ions are transmitted to a mass filter having an ion transmission window that is scanned in m / z dimension as the precursor ions are received by the mass filter to provide a plurality of overlapping ion transmission windows. The ions passing through the mass filter are dissociated to generate a plurality of fragment ions, which can then be analyzed.

[0076] By way of illustration, FIG. 2A shows a fragment ion spectrum obtained using scanning SWATH data acquisition method, wherein the fragment ions mass peaks provide an example of fragment ion signals that can be utilized in various embodiments of the present teachings. The illustrated spectrum in FIG. 2A includes both high-intensity mass peaks and low-intensity mass peaks. For low-intensity mass peaks, the accuracy of the derived m / z ratio may be adversely affected by low ion statistics. By way example, FIG. 2B shows one of such low-intensity mass peaks. In this example, one ion attribute associated with each of the fragment ion mass peak is the m / z ratio. In various embodiments, at least one of the fragment ion mass peaks, an independent uncertainty is assigned to the m / z value associated with that mass peak. The m / z uncertainty can be computed in a variety of different ways. By way of example, the m / z uncertainty corresponding to a fragment ion mass peak can be determined based on the maximum intensity of that ion mass peak. For example, as the maximum intensity of a mass peak decreases, an uncertainty assigned to the m / z of that mass peak increases. By way of example, in the fragment ions mass spectrum illustrated in FIG. 2A, in various embodiments, a lower uncertainty will be assigned to the m / z of the fragment ion mass peak at m / z of 217.1335 relative to a respective m / z uncertainty assigned to the mass peak at m / z of 691.3775.

[0077] In various embodiments, the m / z associated with a mass peak corresponds to the centroid of the mass peak and the confidence interval for Poisson statistics can be used to estimate ion spectral peak centroid uncertainty interval. By way of further illustration, if the centroid of a spectral peak is identified as x, the confidence interval can be obtained using the following relation: x + z-^=, where z is defined as: z = , where x denotes m / z, .s denotes the standard / n s deviation, and n denotes the number of ion events. In such embodiments, the 95% confidence P2023-3015-WO (PLG Ref. 4277-0407W001) interval for peak centroid is obtained by the following relation:=

[0078] 0.95, where a denotes the variance, which can be known from instrument resolution and can also be estimated from the spectral peak as well, and n is defined as the number of ion events over the spectral peak. The m / z associated with a peak can then be x ± - .

[0079] Another ion attribute corresponds to the association between a fragment ion mass peak and an m / z value of a precursor ion giving rise to that fragment ion mass peak. By way of example, the techniques described in U.S. Patent No. 10,068,753 (herein also referred to as “the ‘753 Patent”) titled “Systems and methods for identifying precursor ions from product ions using arbitrary transmission windowing,” which is herein incorporated by reference in its entirety, can be utilized to associate a fragment ion mass peak with an m / z ratio corresponding to a respective precursor ion.

[0080] More specifically, based on the teachings of the ‘753 Patent, the variation of the intensity of a fragment ion mass peak as a function of a parameter associated with the ion transmission m / z window (e.g., the m / z range associated with the transmission window, e.g., a scanning transmission window) of the mass filter (herein the mass filter is also referred to as QI filter) as the transmission window is scanned can be plotted and the fragment ion signal can be associated with a precursor ion having an m / z corresponding to the apex of the plot. For example, FIG. 4 of the ‘753 Patent shows the variation of the mass peak intensity of a fragment ion mass peak as a function of the m / z of the leading edge of a scanned ion transmission window of a mass filter through which the precursor ions, which give rise to the fragment ions, pass. The apex of the plot corresponds to the m / z of a precursor ion corresponding to that fragment ion signal.

[0081] More specifically, the plot illustrated in FIG. 4 of the ‘753 Patent is generated by summing the intensity of a fragment ion mass peak from successive groups of overlapping rectangular precursor ion transmission windows to produce a triangular function that describes fragment ion intensity as a function of precursor ion m / z.

[0082] While in some cases such a plot can exhibit a sharp peak, in other cases the plot may not exhibit a sharp peak and hence may introduce some degree of uncertainty regarding the precursor m / z that would correspond to the fragment ion mass peak. By way of illustration, FIG. 3 is an exemplary plot that shows the variation of the mass peak intensity of a fragment ion mass peak P2023-3015-WO (PLG Ref. 4277-0407W001)

[0083] (such as a fragment ion mass peak depicted in FIG. 2A) as a function of the scanned leading edge of a scanning ion transmission window of a mass filter through which precursor ions that give rise to the fragment ions pass. By way of example, the vertical lines in FIG. 3 define an uncertainty in the m / z of the precursor ion (A(m / z)) associated with the fragment ion. By way of reference, the solid grey triangle shows an ideal intensity variation of the fragment ion signal as a function of the scanned leading edge of the mass filter.

[0084] FIG. 4A shows a similar plot for another fragment ion signal but with an apex that is not as sharp as the plot illustrated in FIG. 3, thereby leading to a higher uncertainty of the precursor ion m / z. By way of further illustration, FIG. 4B shows a plot similar to that depicted in FIG. 4A but with the quadrupole shape of a fragment ion originating from two precursor ions with distinct m / z ratios, but co-isolated by the mass filter. In this example, the uncertainty associated with m / z inference of the precursor ion is higher than that in FIG. 3. Further, in general, the uncertainty associated with m / z inference of the precursor ion is higher for lower intensity fragment signals.

[0085] Another ion attribute that can be utilized in the identification of a compound using various embodiments of the present teachings is an estimate of the electric charge of the precursor and / or fragment ions.

[0086] The measurement parameters and their associated uncertainties, which are assigned for each fragment ion mass peak independent of other fragment ion mass peaks, can then be utilized to generate a candidate for the identity of one or more precursor ions in a sample under analysis. By way of example, a database, such as a metabolomic or a proteomic database, can be searched for precursor ions having an m / z within the range of uncertainty computed for the precursor m / z. In some cases, such a search may result in identification of only one compound as a precursor candidate while in other cases, the search may result in the identification of multiple candidates for the precursor compound.

[0087] In cases in which the search of a database results in multiple candidates for the identity of a precursor ion (i.e., it results in the identification of a plurality of compounds as candidates corresponding to the precursor ion), several precursor level attributes, such as mass accuracy, the number of matched fragment ion signals and the intensities of the matched fragment ion signals, P2023-3015-WO (PLG Ref. 4277-0407W001) can be employed to rank the different candidates, e.g., to arrive at the most likely candidate, e.g., as described in more detail in the examples provided below.

[0088] The ion attributes that can be utilized in various embodiments of the present teachings are not limited to those discussed above. By way of example, in some embodiments, a plurality of precursor ions can be introduced into an ion mobility device, which can separate the ions based on their mobility, prior to the introduction of the ions into a mass spectrometer. In some embodiments, the ion mobility device can be a differential ion mobility spectrometer (DMS) in which ions are separated by the application of a high-voltage asymmetric waveform at radio frequency combined with a static DC waveform (a compensation voltage (CV)) applied between two electrodes. As the compensation voltage is scanned, ions with different mobilities pass through the DMS. The ions passing through the DMS can be received by a downstream mass spectrometer in which the ions can undergo dissociation to generate a plurality of fragment ions. A mass spectrum of the fragment ions can be generated (e.g., such as the hypothetical mass spectrum illustrated in FIG. 2A), which can be used to generate a candidate for the identity of one or more precursor ions within a sample.

[0089] In various embodiments, machine learning techniques can be utilized to assign uncertainties to various ion attributes. By way of example, the present teachings can be employed to train a machine learning algorithm to assign experimentally-derived uncertainties to a plurality of ion attributes, develop one or more candidates for the identity of compound, and rank those candidates to arrive at the most likely candidate.

[0090] By way of example, FIG. 5 is a block diagram that illustrates such a computer system 100, upon which embodiments of the present teachings may be implemented. Computer system 100 includes a bus 102 or other communication mechanism for communicating information, and a processor 104 coupled with bus 102 for processing information. Computer system 100 also includes a memory 106, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus 102 for determining base calls, and instructions to be executed by processor 104. Memory 106 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 104. Computer system 100 further includes a read only memory (ROM) 108 or other static storage device coupled to bus 102 for storing static information and instructions P2023-3015-WO (PLG Ref. 4277-0407W001) for processor 104. A storage device 110, such as a magnetic disk or an optical disk, is provided and coupled to bus 102 for storing information and instructions.

[0091] Computer system 100 may be coupled via bus 102 to a display 112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 114, including alphanumeric and other keys, is coupled to bus 102 for communicating information and command selections to processor 104. Another type of user input device is cursor control 116, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 104 and for controlling cursor movement on display 112. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane. The computer system 100 further includes a communication module 118 that allows the computer system to communicate with other devices and systems, and in particular, a database 120.

[0092] A computer system 100 can perform the present teachings. Consistent with certain implementations of the present teachings, results are provided by computer system 100 in response to processor 104 executing one or more sequences of one or more instructions contained in memory 106. Such instructions may be read into memory 106 from another computer-readable medium, such as storage device 110. Execution of the sequences of instructions contained in memory 106 causes processor 104 to perform the process described herein. Alternatively hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0093] By way of example, in various embodiments, the computer system 100 can receive fragment ion signals and process those signals according to the present teachings to derive various ion attributes associated with the fragment ion signals and further assign uncertainties to those ion attributes, e.g., in a manner discussed above. By way of example, the computer system 100 can be configured to derive the m / z values of various fragment ion mass peaks present in an MS2 spectrum and assign uncertainties to those derived m / z values according to the present teachings. In addition, in various embodiments, the computer system 100 can associate each of the plurality of fragment ion signals with a precursor m / z and, for each such association, assign P2023-3015-WO (PLG Ref. 4277-0407W001) an uncertainty according to the present teachings. As noted above, the ion attributes that can be utilized in a method according to the present teachings are not limited to m / z values of fragment ion signals and the associations between the fragment ion signals and precursor m / z values. For example, in some cases, the computer system 100 can associate fragment ion signals with precursor ion mobilities, and further assign uncertainties to such associations.

[0094] In various embodiments, the computer system can communicate the ion attributes and their respective uncertainties to the database 120, such as a metabolomic or proteomic database, to search for candidates for the precursor ions. For example, the database can be searched for one or more precursor ions having m / z values corresponding to the m / z values derived by the computer system 100 with the uncertainty computed as discussed above.

[0095] The term “computer-readable medium” as used herein refers to any media that can participate in providing instructions to processor 104 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 110. Volatile media can include dynamic memory, such as memory 106. Transmission media can include coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 102.

[0096] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, papertape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0097] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 104 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 100 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 102 can receive the data carried in the infra-red signal and place P2023-3015-WO (PLG Ref. 4277-0407W001) the data on bus 102. Bus 102 carries the data to memory 106, from which processor 104 retrieves and executes the instructions. The instructions received by memory 106 may optionally be stored on storage device 110 either before or after execution by processor 104.

[0098] In accordance with various embodiments, instructions configured to be executed by a processor to perform a method according to various embodiments of the present teachings are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer- readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0099] The present teachings for analysis of mass spectral data can be utilized in a variety of different mass spectrometers as well as in connection with a variety of data acquisition methods employed in mass spectrometry. In particular, the present methods can be utilized in mass spectrometer systems that employ the scanning SWATH data acquisition method. By way of example, FIG. 6 schematically depicts a mass spectrometer 600 according to an embodiment that includes an LC column 602 that can receive a sample and separate a plurality of analytes in the sample based on their elution time from the LC column. The mass spectrometer 600 further includes an ion source 604 that receives an eluate exiting the LC column and ionizes one or more analytes contained in the eluate to generate a plurality of precursor ions. In many implementations, one or more ion guides 606 receive the precursor ions and provide focusing of the ions to generate an ion beam that is received by a mass filter 608. By way of example, the ion guide(s) can include a plurality of rods arranged in a quadrupole configuration to which RF and DC voltages generated by an RF voltage source 610 and a DC voltage source 612 can be applied in a manner known in the art to provide radial confinement of the received ions.

[0100] The mass filter 608 provides an ion transmission window that allows transmission of ions having m / z values within an m / z range through the mass filter. By way of example, the mass filter 608 can include a plurality of rods arranged in a quadrupole configuration to which RF voltages as well as a resolving DC voltage can be applied via the RF and the DC voltage sources, respectively, to generate an ion transmission window. In this implementation, the ions passing P2023-3015-WO (PLG Ref. 4277-0407W001) through the mass filter 608 are received by an ion fragmentation device 614 that causes fragmentation of the precursor ions to generate a plurality of product ions. The product ions are received by a mass analyzer 616, which generates mass signal data associated with the product ions.

[0101] When operating in the scanning SWATH data acquisition mode, a controller 618 can control the operation of the RF and the DC voltage sources to scan the ion transmission window over an m / z range so as to generate a plurality of overlapping transmission windows.

[0102] In use, the mass spectrometer can be employed to generate a mass spectrum of the fragment ions, and optionally a survey scan of the precursor ions.

[0103] An analysis module 620 (herein also referred to as an analysis unit) that is in communication with the mass analyzer 616 and the controller 618 can receive the fragment ions signals and process them in a manner disclosed herein to generate a plurality of ion attributes and their respective uncertainties. Further the analysis module can communicate with a database to identify one or more compounds within the sample based on the ion attributes and their associated uncertainties. By way of example, the analysis module 620 can be implemented as the computer 100 discussed above.

[0104] As noted above, various embodiments of the present teachings provide advantages relative to conventional systems and methods for compound identification, especially in complex samples. For example, the optimal assignment of uncertainties to ion attributes associated with product ion signals, such as fragment ion signals, can reduce the number of candidates for the identification of the precursor ions and can further reduce FDR in identification of compounds.

[0105] The following examples are provided for further elucidation of various aspects of the present teachings and are not necessarily provided to indicate optimal ways of practicing the present teachings and / or optimal results that may be achieved.

[0106] Examples

[0107] Example 1 P2023-3015-WO (PLG Ref. 4277-0407W001)

[0108] In many cases, one or more of the observed precursor ions can be confidently associated with a respective compound, e.g., via the search of a database. For example, it may be feasible to identify a compound corresponding to the most abundant precursor ion with a high degree of confidence. However, in some cases, the search of a database for the identification of some precursor ions may lead to multiple candidates. As illustrated in the example below, in various embodiments, several precursor level attributes, such as mass accuracy, the number of matched fragment ion signals and the intensities of the matched fragment ion signals, can be employed to rank the different candidates, e.g., to arrive at the most likely candidate.

[0109] By way of illustration, FIGS. 7A shows an example of a mass spectrum containing ion mass signals (mass peaks) corresponding to a plurality of precursor ions. FIG. 7B in turn depicts a mass spectrum containing ion mass signals (mass peaks) corresponding to a plurality of fragment ions generated via dissociation of the plurality of precursor ions as shown in FIG. 7A.

[0110] FIGS. 7C-1, 7C-2, 7C-3, 7C-4, 7C-5, 7C-6, and 7C-7 present plots exhibiting the variation of the mass peak intensity of select fragment ion mass peaks in the fragment ions spectrum depicted in FIG. 7B as a function of the m / z of the leading edge of a scanning ion transmission window of a mass filter through which the precursor ions, which give rise to the fragment ions, pass. In particular, FIG. 7C-1 is a plot corresponding to a fragment ion with an m / z in the range of 630.6 to 630.37, FIG. 7C-2 is a plot corresponding to a fragment ion with an m / z in the range of 175.11 to 175.12, FIG. 7C-3 is a plot corresponding to a fragment ion with an m / z in the range of 727.46 to 727.47, FIG. 7C-4 is a plot corresponding to a fragment ion with an m / z in the range of 288.20, FIG. 7C-5 is a plot corresponding to a fragment ion with an m / z in the range of 492.25 to 492.27, FIG. 7C-6 is a plot corresponding to a fragment ion with an m / z in the range of 516.28 to 51630, and FIG. 7C-7 is a plot corresponding to a fragment ion with an m / z in the range of 938.51 to 983.52.

[0111] Table 2 below provides three potential compounds that may correspond to the precursor ion associated with the mass peak at the m / z ratio of 565.7976 in the mass spectrum depicted in FIG. 7 A. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0112] Table 2

[0113] Table 3 below provides the intensities of the fragment ion signals that can be associated with the first compound (i.e., FPGQNADLR) and a degree of confidence (certainty) for each such association. In this example, the QI m / z for each fragment ion signal is computed in a manner discussed above as the maximum of the variation of the intensity of each fragment ion mass peak as a function of an m / z of the ion transmission window (e.g., the m / z associated with the leading edge of the scanning transmission window) of the mass filter (QI) as the transmission window is scanned. The dark grey bars identify those fragment ions that were matched to a single compound and the light grey bars identify those fragment ions that were matched to more than one compound. The degree of confidence for the association of each fragment ion with the respective compound is shown via the length of the respective shaded bar. By way of example, in Table 3, the fragment at m / z 657.32 is associated with this compound with a confidence of 80%.

[0114] P2023-3015-WO (PLG Ref. 4277-0407W001)

[0115] Table 3

[0116] Table 4 below in turn provides the intensities of the fragment ion signals that can be associated with the second compound (i.e., AASGTQBBVLR) and a degree of confidence (certainty) for each such association. In this example, the QI m / z for each fragment ion signal is computed in a manner discussed above as the maximum of the variation of the intensity of each fragment ion mass peak as a function of an m / z of the ion transmission window (e.g., the m / z associated with the leading edge of the scanning transmission window) of the mass filter (QI) as the transmission window is scanned. Again, the dark grey bars identify those fragment ions that were matched to a single compound and the light grey bars identify those fragment ions that were matched to more than one compound. The degree of confidence of the association of each fragment ion with the respective compound is shown via the length of the respective shaded bar. By way of example, in Table 4, the fragment ions at m / z values of 630.41 and 712.39 are associated with this compound with a confidence of 80%. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0117] Table 4

[0118] A-A-M-E-P-FV-I-S-A-K

[0119] Finally, Table 5 below provides the intensities of the fragment ion signals that can be associated with the third compound (i.e., AAMEPIVISAK) and a degree of confidence (certainty) for each such association. In this example, the QI m / z for each fragment ion signal is computed in a manner discussed above as the maximum of the variation of the intensity of each fragment ion mass peak as a function of an m / z of the ion transmission window (e.g., the m / z associated with the leading edge of the scanning transmission window) of the mass filter (QI) as the transmission window is scanned. Again, the dark grey bars identify those fragment ions that were matched to a single compound and the light grey bars identify those fragment ions that were matched to more than one compound. The degree of confidence for the association of each fragment ion with the respective compound is shown via the length of the respective shaded bar. By way of example, in Table 5, the fragment ion at m / z 615.54 is associated with this compound with a confidence of 20%. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0120] Table 5

[0121] At the bottom of each of the above Tables 3, 4, and 5, the sequence coverage map shows the evidence denoted by anchor above and below each amino acid in the compound. This schematic includes all ions from the respective tables.

[0122] When the spectra shown in FIGS. 7A and 7B are searched against putative list of compounds, multiple candidates are feasible, as shown in Table 2. Generally, the most abundant compound can be confidently identified due to the presence of more respective fragment ions and / or higher intensity of fragment ions present in the MS2 spectrum. For other compounds, the precursor level attributes (factors) (such as mass accuracy), number of matched fragments and intensity of the matched fragments can influence the ranking of the candidates. As seen in Table 2, the second compound has fewer matched fragment ions and lower mass error compared to the third compound; subsequently, the intensity of the fragment ions as seen in Table 5 is higher than the fragment ions in Table 4.

[0123] To discriminate between the 2ndand 3rdcandidates, the degree of certainty of the fragment ions contributing to the identification of the specific compound can be considered. These degrees of certainty are determined based on the apex of the QI profiles for each fragment (FIGS. 7C-1 to 7C-7 show profiles for select fragments). Based on Table 4, the confidence in P2023-3015-WO (PLG Ref. 4277-0407W001) the 2ndcandidate is lowered (i.e. 3 of 10 fragments have a high uncertainty). The downweighting of the fragment ions that are contributing to 1stcandidate, based on Table 3, would further decrease (i.e. 5 of 10) the confidence in the identity of the second compound.

[0124] Alternatively, the exclusion of the uncertain and shared fragment ions evidenced from sequence coverage map will display two anchors, which can also be inferred at less likely identification.

[0125] Alternatively, for the 3rdcandidate, the degree of certainty of the fragment ions contributing to the identification, based on Table 4, can be used to increase the confidence of the identification. Additionally, QI m / z for each high confidence fragment can be used to define the uncertainty on the precursor m / z and thus reducing the influence of “fixed” error tolerance and / or precursor peak properties (charge, intensity) on the confidence of the identification.

[0126] Example 2

[0127] When analyte L- Aspartic acid is eluted at a given cycle time, there are other analytes that closely elute at the same cycle time and have close m / z ratios. This generates a complex MS / MS spectrum, which can lead to incorrect identification (with score of 0.7 or higher) by library search algorithm.

[0128] FIG. 8A shows fit scores of analytes in a given cycle before and after weighting various degrees of certainty and after deconvolution. FIG. 8B shows purity scores of analytes at a given cycle before and after weighting various degrees of certainty and after deconvolution (filtered).

[0129] The incorrect identification is exemplified by high score of 2-aminobezimidazole in original fit in FIG. 8A and no analytes with score > 0.7 in FIG. 8B.

[0130] After filtering, higher scores (>0.7) are observed for some of the analytes (top n). With weighting, the scores of the top n analytes can be discriminated sufficiently to annotate the correct identity, i.e., L-aspartic acid. This is shown by the scores in filtered and original weighted markers on FIGS. 8A and 8B. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0131] The above descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.

[0132] Depending on certain implementation requirements, embodiments of the present teachings, the controller can be implemented in hardware, firmware and / or in software.

[0133] In some embodiments, the instructions for operating the optical system can be stored using a non-transitory storage medium such as a digital storage medium, for example a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable.

[0134] While various embodiments have been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; embodiments of the present disclosure are not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing embodiments of the present disclosure, from a study of the drawings, the disclosure, and the appended claims.

[0135] In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single processor or other processing unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. P2023-3015-WO (PLG Ref. 4277-0407W001)

[0136] Those having ordinary skill in the art will appreciate that various changes can be made to the above embodiments without departing from the scope of the present teachings.

Claims

P2023-3015-WO (PLG Ref. 4277-0407W001)What is claimed is:

1. A method for compound identification using mass spectrometry, comprising: passing a plurality of different precursor ions through an ion dissociation device to cause fragmentation thereof to generate a plurality of fragment ions, acquiring one or more fragment ion signals associated with said plurality of fragment ions, assigning to at least one of said fragment ion signals a corresponding experimentally-derived uncertainty associated with at least one ion attribute such that said assigned uncertainty is independent from a respective uncertainty assigned to any of the other ones of said plurality of fragment ion signals, and generating at least one candidate for identity of a compound corresponding to at least one of said precursor ions by utilizing said at least one ion attribute and said corresponding experimentally-derived uncertainty.

2. The method of Claim 1, wherein said experimentally-derived uncertainty includes an uncertainty interval and one or more uncertainty values over said interval, wherein optionally said uncertainty values are distributed uniformly or non-uniformly over said interval.

3. The method of any one of Claims 1 and 2, further comprising defining said experimentally-derived uncertainty by evaluating the at least one of said fragment ion signals.

4. The method of Claim 3, wherein the step of evaluating the at least one of said fragment ion signals comprises evaluating a shape of that ion signal relative to an expected signal shape.

5. The method of any one of Claims 1 - 4, wherein said ion attributes comprises any of m / z ratio of any of a precursor and a fragment ion, an association between a fragment ion and an m / z of a respective precursor ion, an ion mobility of a precursor ion, and a retentionP2023-3015-WO (PLG Ref. 4277-0407W001) time associated with passage of an analyte corresponding to a precursor ion through an LC column.

6. The method of Claim 5, further comprising determining an intensity variation of the at least one of said fragment ion signals as a function of a change in a parameter of a mass filter through which the precursor ions pass prior to reach said dissociation device and utilizing said intensity variation to determine an association between said fragment ion signal and a precursor m / z and an uncertainty corresponding to said association. .

7. The method of any one of Claims 5 and 6, wherein the parameter of the mass filter is a transmission m / z range associated with the mass filter.

8. The method of Claim 7, wherein said intensity variation function corresponds to a variation of the intensity of said fragment ion signal as a function of a change of said transmission m / z range of the mass filter, wherein optionally said intensity variation function has a substantially triangular shape and said m / z associated with said at least one of the precursor ions corresponds to an apex of the substantially triangular shape.

9. The method of Claim 8, wherein said corresponding experimentally-derived uncertainty corresponds to an uncertainty in an m / z position of said apex.

10. The method of any one of Claims 1 - 9, wherein the step of generating the at least one candidate comprises identifying a compound in a library database as the candidate based on said at least one ion attribute and its corresponding experimentally-derived uncertainty.

11. The method of Claim 1, wherein said at least one candidate comprises a plurality of candidates for the identity of said compound.

12. The method of Claim 11, further comprising ranking said candidates to identify the most probable candidate.P2023-3015-WO (PLG Ref. 4277-0407W001)13. The method of Claim 12, wherein the ranking of said candidates is performed based on one or more precursor level attributes and uncertainties associated with those attributes.

14. The method of Claim 13, wherein said one or more precursor level attributes comprise any of mass accuracy, associated fragment m / z values and an intensity associated with one or more of the fragment ion signals.

15. The method of Claim 13, wherein said one or more precursor level attributes comprise a combination of attributes derived from MS2 and MSI spectra.

16. The method of any one of the preceding claims, wherein the fragment ion signals are generated via a data independent acquisition (DIA) mass spectrometric method.

17. The method of any one of the preceding claims, further comprising presenting an identity of said compound to a user.

18. The method of any one of the preceding claims, further comprising utilizing a programmed computer to perform said step of assigning to at least one of said fragment ion signals the corresponding experimentally-derived uncertainty associated with the at least one ion attribute and said step of generating at least one candidate for the identity of the compound.

19. A mass spectrometer, comprising: an ion source for ionizing a sample to generate a plurality of different precursor ions, a mass filter configured to allow passage of at least one subset of the plurality of precursor ions having one or more m / z ratios within an m / z transmission window of the mass filter therethrough, a controller in communication with the mass filter to cause scanning the m / z transmission window of the mass filter over an m / z range so as to generate a plurality of overlapping m / z transmission windows,P2023-3015-WO (PLG Ref. 4277-0407W001) an ion dissociation device for receiving ions exiting the mass filter and causing dissociation thereof to generate a plurality of fragment ions, a mass analyzer configured to receive said fragment ions and generate one or more fragment ion signals, a data analysis module including a microprocessor and configured to: process said fragment ion signals to derive a plurality of ion attributes, assign to at least one of said fragment ion signals a corresponding experimentally-derived uncertainty associated with at least one of said ion attributes, wherein the experimentally-derived uncertainty assigned to the at least one of said fragment ion signals is independent from a respective uncertainty assigned to any of the other ones of said plurality of fragment ion signals, and generate at least one candidate for identity of a compound corresponding to at least one of said precursor ions by utilizing said at least one ion attribute and said corresponding experimentally-derived uncertainty.

20. The mass spectrometer of Claim 19, wherein said at least one of the ion attributes corresponds to an association between a fragment ion signal and an m / z of one of the precursor ions.

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