Data-independent acquisition of product ion spectra and reference spectral library matching

Abstract

To provide systems and methods for data independent acquisition of product ion spectra and reference spectra library matching.SOLUTION: Systems and methods are used to store an electronic record of all product ion spectra of all detectable compounds of a sample. A plurality of product ion scans are performed on a tandem mass spectrometer one or more times in a single sample analysis across a mass range using a plurality of mass selection windows. All sample product ion spectra of all detectable compounds for each mass selection window are produced. All sample product ion spectra for each mass selection window are received from the tandem mass spectrometer using a processor. All sample product ion spectra for each mass selection window are stored as an electronic record of all detectable compounds of the sample using the processor. The electronic record is used to characterize compounds known at the time the electronic record is stored or to characterize compounds that became known after the electronic record was stored.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] (Citation of Related Application) This application claims the benefit of U.S. Provisional Patent Application No. 61 / 383,137, filed September 15, 2019, and European Patent Application No. 10009595.9, filed September 15, 2010, which are incorporated herein by reference in their entireties. [Background technology]

[0002] Mass spectrometry has been used for many years to identify and quantify compounds in complex mixtures. Typical compounds may include, but are not limited to, proteins, peptides, pharmaceutical compounds, and derivatives such as metabolites, drugs of abuse, pesticides, etc. A common mass spectrometry technique is tandem mass spectrometry. In tandem mass spectrometry, precursor ions are selected by a mass analyzer, fragmented in a certain way, and the fragments are analyzed in a second mass analyzer or in a second scan of the first analyzer. The fragments generated can be used for identification or quantification.

[0003] A common technique for quantification is selected reaction monitoring (SRM). SRM has long been used to quantify small molecules, but in recent years has also been applied to peptides, proteins, and other biological compounds, such as lipids and carbohydrates. SRM is typically performed on a triple quadrupole instrument, where the first and second mass analyzers have a mass separation peak width of about 0.7, and one or more combinations of precursor and fragment masses (known as transitions) are monitored during liquid chromatography-mass spectrometry (LC-MS).

[0004] Despite its sensitivity and robustness, SRM has at least the following challenges that limit its applications:

[0005] 1. The compound to be measured must be defined prior to data acquisition, and the transitions to be monitored must be determined from an experimental fragment spectrum of the compound of interest, or from a library of such spectra.

[0006] 2. The information obtained is incomplete, since the number of transitions, and therefore compounds, that can be measured during a single analysis is limited for several reasons. Chromatographic peaks must be clearly defined, since quantification is based on the height or area of ​​the peak in the chromatogram of the transition, i.e., on a plot of the response to the transition versus time. Therefore, the time spent measuring a set of transitions needs to be kept as short as possible. Sensitivity (the minimum amount of material that can be detected) depends on the amount of time spent monitoring the transitions (retention time), and therefore the more sensitive it is, the longer it takes, meaning that fewer compounds can be analyzed. Confidence that the correct compound has been identified usually requires that several transitions are measured and their response compared to that expected from a standard spectrum. This is especially true when the mass transmission window is relatively wide, such that in a complex mixture, more than one precursor or fragment may be selected at the same time, interfering with the measurement of the target fragment ion. Precise and accurate quantification requires the inclusion of confirmatory standard material (typically an isotopically labeled form of the target compound) that provides different transitions that can be distinguished from the target compound. That is, they too must be monitored, thus reducing the overall number of compounds that can be analyzed.

[0007] 3. Furthermore, the available data is limited to what was defined by the time the analysis was performed. Thus, in many cases, if different or additional transitions must be monitored to improve the accuracy of the confidence in quantification, or if additional data are required to detect different compounds or modified forms of the target compound, it is necessary to reanalyze the samples and generate additional data.

[0008] 4. Only a limited number of compounds can be analyzed at one time, so obtaining data for all compounds present in a sample requires many separate analyses.

[0009] An alternative acquisition method involves alternating scans with high and low fragmentation, which are then processed to determine groupings of precursors (low energy) and fragments (high energy). Quantification based on ion traces extracted from this data (as with SRM) is prone to interferences, since in complex mixtures many ions can be fragmented simultaneously.

[0010] Another alternative acquisition method selects small mass windows that are stepped across the mass range of interest, but complete coverage of the entire mass range requires multiple analyses and takes a significant amount of time. Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there is a trade-off between the number of compounds that can be analyzed in the same analysis, and thus the sample throughput if full coverage is required, and the sensitivity and the possibility of detecting interferences that degrade the quantification behavior. [Means for solving the problem]

[0012] The method allows for the determination of dynamic quantitative target transitions and modified forms of target compounds (such as metabolites or post-translational modifications) without reacquiring data on the sample.

[0013] In various embodiments, the systems and methods provide a method for creating a record of all detectable compounds, which includes a novel combination of data acquisition methods for creating fragment spectra of all compounds and targeted data analysis methods. The present specification also provides, for example, the following items: (Item 1) 1. A system for storing an electronic record of all product ion spectra of all detectable compounds of a sample, comprising: a tandem mass spectrometer that performs one or more multiple product ion scans in a single sample analysis across a mass range using multiple mass selection windows, the tandem mass spectrometer generating a full sample product ion spectrum of all detectable compounds for each mass selection window; a processor in communication with the tandem mass spectrometer, the processor receiving the total sample product ion spectrum for each mass selection window from the tandem mass spectrometer and storing the total sample product ion spectrum for each mass selection window as an electronic record of all detectable compounds in the sample; Equipped with The system wherein the electronic record is used to characterize known compounds at the time the electronic record is stored, and is also used to characterize compounds that become known after the electronic record is stored. (Item 2) 2. The system of claim 1, wherein each mass selection window of the plurality of mass selection windows has a width greater than 15 amu. (Item 3) 2. The system of claim 1, wherein the processor further receives a predetermined product ion spectrum corresponding to a known compound, receives the full sample product ion spectrum for one or more mass selection windows from the electronic record, and compares predetermined product ions of the predetermined product ion spectrum with sample product ions of the full sample product ion spectrum for one or more mass selection windows, wherein one or more matching sample product ions from the comparison characterize the known compound detectable in the sample. (Item 4) 4. The system of claim 3, wherein the known compounds comprise one or more of peptides, proteins, the complete proteome, endogenous metabolites, lipids, carbohydrates, or combinations thereof. (Item 5) 4. The system of claim 3, wherein the known compounds comprise one or more compounds of pharmaceutical, environmental, forensic, or industrial importance. (Item 6) The system of claim 3, wherein the predetermined product ion spectrum is determined from corroborative standards of the known compound, analysis of a sample containing the compound, or a pre-existing spectral library, or the predetermined product ion spectrum is computationally generated by applying empirical or spectroscopic fragmentation or modification rules to the known compound. (Item 7) 4. The system of claim 3, wherein the processor compares predetermined product ions of the predetermined product ion spectrum to sample product ions of the total sample product ion spectrum for one or more mass selection windows by calculating a score representing how well the predetermined product ions and the sample product ions match. (Item 8) 8. The system of claim 7, further comprising a separation device that separates sample compounds of the single sample analysis over time, wherein the tandem mass spectrometer performs multiple product ion scans of the single sample analysis as the sample compounds are separated. (Item 9) 9. The system of claim 8, wherein the score is further calculated based on how well peaks in the given product ion trace match peaks in the sample product ion trace. (Item 10) 10. The system of claim 9, wherein the processor further uses one or more matching sample product ions from the comparison to calculate a quantitative value for a compound of the sample. (Item 11) 4. The system of claim 3, wherein the processor further uses one or more matching sample-produced ions from the comparison to identify modified forms of one or more of the known compounds. (Item 12) 12. The system of claim 11, wherein the processor characterizes the type and location of the modification by comparing the matching sample ions to masses predicted from the predetermined ions and the modification. (Item 13) 12. The system of claim 11, wherein the processor further extracts spectra of the identified modification forms from the electronic record to characterize the type and location of the modification in the identified modification forms. (Item 14) 1. A method for storing an electronic record of all product ion spectra of all detectable compounds of a sample, comprising: performing one or more multiple product ion scans in a single sample analysis across a mass range using a tandem mass spectrometer, the single sample analysis using multiple mass selection windows to generate a full sample product ion spectrum of all detectable compounds for each mass selection window; receiving, using a processor, the full sample product ion spectrum for each mass selection window from the tandem mass spectrometer; storing, using said processor, all of said sample product ion spectra for each mass selection window as an electronic record of all detectable compounds of said sample; Including, The method of claim 1, wherein the electronic record is used to characterize known compounds at the time the electronic record is stored, and is also used to characterize compounds that become known after the electronic record is stored. (Item 15) 1. A computer program product comprising a tangible computer readable storage medium the contents of which include a program, the program having instructions for execution on a processor to perform a method for storing an electronic record of all product ion spectra of all detectable compounds of a sample, the method comprising: providing a system comprising one or more separate software modules, the separate software modules comprising a measurement module and a storage module; receiving, using the measurement module, a full sample product ion spectrum of all detectable compounds for each mass selection window from a tandem mass spectrometer, the tandem mass spectrometer performing one or more product ion scans in a single sample analysis across a mass range using multiple mass selection windows; storing, using the storage module, all of the sample product ion spectra for each mass selection window as an electronic record of all detectable compounds of the sample; Including, The computer program product, wherein the electronic record is used to characterize known compounds at the time the electronic record is stored, and also is used to characterize compounds that become known after the electronic record is stored.

[0014] Those skilled in the art will understand that the drawings, described below, are for illustration purposes only and are not intended to limit the scope of the present teachings in any way. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a block diagram illustrating a computer system upon which embodiments of the present teachings may be implemented. [Diagram 2] FIG. 2 is a schematic diagram showing how data is acquired for a complete mass range using a stepped precursor ion selection window in a mass analyzer, according to various embodiments. [Diagram 3] FIG. 3 is an exemplary plot showing mass traces extracted from the ion traces of FIG. 2 for fragments determined from a reference spectral library, according to various embodiments. [Figure 4] FIG. 4 is a schematic diagram showing a system for storing an electronic record of all product ion spectra of all detectable compounds of a sample, according to various embodiments. [Diagram 5] FIG. 5 is an exemplary flow diagram showing a method for storing an electronic record of all product ion spectra of all detectable compounds of a sample according to various embodiments. [Figure 6] FIG. 6 is a schematic diagram of a system including one or more individual software modules that implements a method for storing an electronic record of all product ion spectra of all detectable compounds of a sample, according to various embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Before describing one or more embodiments of the present teachings in detail, those skilled in the art will understand that the present teachings are not limited in their application to the details of construction, the arrangement of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0017] (Computer Implemented Systems) FIG. 1 is a block diagram illustrating a computer system 100 in which an embodiment of the present teachings may be implemented. The computer system 100 includes a bus 102 or other communication mechanism for communicating information and a processor 104 coupled with the bus 102 for processing information. The computer system 100 also includes a memory 106, which may be a random access memory (RAM) or other dynamic storage device, coupled to the bus 102 for storing instructions executed by the processor 104. The memory 106 may also be used to store temporary variables or other intermediate information during execution of instructions executed by the processor 104. The computer system 100 further includes a read-only memory (ROM) 108 or other static storage device coupled to the bus 102 for storing static information and instructions for the processor 104. A storage device 110, such as a magnetic disk or optical disk, is provided and coupled to the bus 102 for storing information and instructions.

[0018] 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 a cursor control 116, such as a mouse, trackball, or cursor direction keys, for communicating directional 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 allow the device to specify a position in a plane.

[0019] The computer system 100 is capable of implementing the present teachings. According to one implementation of the present teachings, results are provided by the computer system 100 in response to the processor 104 executing one or more sequences of one or more instructions contained in the memory 106. Such instructions may be read into the memory 106 from another computer-readable medium, such as the storage device 110. Execution of the sequences of instructions contained in the memory 106 causes the processor 104 to perform the processes described herein. Alternatively, hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementation of the present teachings is not limited to any specific combination of hardware circuitry and software.

[0020] The term "computer-readable medium" as used herein refers to any medium that participates in providing instructions to processor 104 for execution. Such media 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 include dynamic memory, such as memory 106. Transmission media include coaxial cables, copper wire and fiber optics, including the wires that comprise bus 102.

[0021] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape or any other magnetic medium, a CD-ROM, a digital video disk (DVD), a Blu-ray disk, any other optical medium, a thumb drive, a memory card, RAM, PROM, and EPROM, Flash-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0022] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to the processor 104 for execution. For example, the instructions may initially be carried on a 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 the computer system 100 can receive the data on the telephone line and use an infrared transmitter to convert the data to an infrared signal. An infrared detector coupled to the bus 102 can receive the data carried in the infrared signal and place the data on the bus 102. The bus 102 carries the data to memory 106, from which the processor 104 reads and executes the instructions. The instructions received by memory 106 may optionally be stored on storage device 110 before or after execution by the processor 104.

[0023] According to various embodiments, instructions configured to be executed by a processor to perform the method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, the computer-readable medium includes a compact disk read-only memory (CD-ROM), as is well known in the art, for storing software. The computer-readable medium is accessed by a suitable processor to execute the instructions configured to be executed.

[0024] The following description of various implementations of the present teachings is presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the present teachings to the precise forms disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the present teachings. In addition, while the described implementations include software, 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.

[0025] (Data processing system and method) As mentioned above, in conventional mass spectrometry methods, there is a trade-off between the number of compounds that can be analyzed in the same analysis, and the sensitivity, and the possibility of detecting interferences that degrade the quantification performance.

[0026] Thus, there is a need for a method that provides a complete record of all detectable compounds present in a complex mixture that can be used to quantify known compounds, determine and quantify modified forms of known compounds, or determine the type and location of unknown modifications. Furthermore, the record should be stored so that these operations can be performed at the time the data is acquired or at some point thereafter. Such a record allows for a complete analysis of all compounds that can be detected by the separation device and mass spectrometer system used.

[0027] Current selected reaction monitoring (SRM) methodologies require extensive preparatory work prior to sample injection to devise the method (including reference spectral library creation), i.e., they allow monitoring of a limited number of peptide transitions per injection, they require extensive data analysis with reference library matching to confirm the identity of the monitored peptides, they require the acquisition of new data to improve quantification accuracy (by replacing contaminating transitions with new ones) and / or to extend quantification to new transitions and / or analytes not described in the original data set.

[0028] In various embodiments, the systems and methods overcome the current limitations of SRM approaches and allow proteome-wide probing, iteratively and recursively, from a single sample injection. These systems and methods overcome the current limitations of SRM approaches but are not limited in any way to SRM or the type of experiment to which SRM is applied. These systems and methods include a combination of a novel LC-MS acquisition set up together with a bioinformatics pipeline for data analysis. Details of various embodiments are presented below.

[0029] (Method setting) In contrast to SRM, various embodiments do not require any pre-method design prior to sample injection. Because LC-MS acquisition can cover the complete analyte content of the sample across the recorded mass and retention time ranges (see below), the data can be mined after the fact for any compounds of interest. Retention times and mass ranges can be set to generate information for a particular range of interest.

[0030] (LC-MS acquisition method) In various embodiments, the acquisition method covers all compounds detectable by chromatography and the mass ranges used. That is, they can be broad and general to detect as many compounds as possible, or they can be adjusted to focus on a particular compound or class of compounds of interest. A wide window of precursor ions, e.g., >10, >15, >20 amu, is selected and fragmented to create a fragment spectrum of all precursors present in the window. The window is moved stepwise to cover the rest of the precursor space, e.g., for a window width of 25 amu, the first window can cover 100-125, the second window 125-150, and the third window 150-175.

[0031] The windows can overlap to ensure that all isotopic forms of a compound are present together in at least one window. Beneficially, the windows have a relatively square shape so that the overlap can be kept small and the number of windows required can be minimized.

[0032] Thus, the time required to acquire data for the entire mass range depends on the number of windows and their accumulation times, rather than on the number of precursors fragmented, and is short enough to maintain the fidelity of the peaks produced by the separation.

[0033] Creating product ion spectra for the entire mass range is repeated one or more times depending on whether a separation system is used. A mass spectrum of all unfragmented precursor ions can be included as part of the cycle. All acquired data is stored for later mining. The window width can be fixed or variable.

[0034] 2 is a schematic diagram 200 showing how data is acquired for a complete mass range using a stepped precursor ion selection window of a mass analyzer according to various embodiments. The schematic diagram 200 depicts an LC-MS method in which data-independent acquisition of a full fragment ion spectrum is obtained by panning the mass analyzer isolation window stepwise over the entire mass range 220 repeatedly during the entire chromatography 230. The schematic diagram 200 is, for example, an LC-MS map. Note that the dashed lines before the start of each cycle in the schematic diagram 200 depict the optional acquisition of a high-resolution accurate mass check (MS1) scan, which can also be used to reassociate fragment ions to the precursors from which they originate, if required for the analytical process.

[0035] The data in diagram 200 can be interpreted by combining the product ion spectra acquired for each isolation window into a separate MS2 map. MS2 map 240 is an exemplary combination of all product ion spectra for isolation window 210. MS2 map 240 includes ion traces 250 plotted as a function of mass-to-charge ratio (m / z), retention time, and signal intensity. Symbols 260 identify ion traces 250 of fragments belonging to the corresponding analyte. Symbols 270 identify ion traces 250 of fragments determined from a reference spectral library.

[0036] FIG. 3 is an exemplary plot 300 showing mass traces extracted from the ion traces of FIG. 2 for fragments determined from a reference spectral library, according to various embodiments.

[0037] In various embodiments, the LC-MS acquisition method comprises monitoring of product ions resulting from fragmentation of ion precursors as follows.

[0038] (Complete coverage of all specimens) 1) Independent data acquisition based on stepping of the separation window of the mass analyzer Instead of monitoring a small number of individual precursors / transitions per run, MS information is acquired in a data-independent manner for the entire mass range and throughout the entire chromatography, regardless of the sample content. In various embodiments, this can be achieved by stepping the precursor ion selection window of the mass analyzer stepwise through the complete mass range (see FIG. 2) instead of focusing on a given or target precursor. The cycle time (or dwell time) of these measurements is therefore determined by the number of steps required to cover the complete mass range (FIG. 2), not by the number of transitions monitored, as in SRM. Such stepwise data-independent fragmentation measurements allow the acquisition of complete information about the analytes / precursors contained in the sample in one single run. In fact, the data acquisition method can create a complete fragment ion map for all analytes present in the sample and relate the fragment ion spectra back to the precursor ion selection window from which the fragment ion spectra were acquired.

[0039] 2) Expanding the selection window of the first mass analyzer 2a) Even in the narrowest selection window achievable by a mass analyzer, it is nearly impossible to ensure that only uncontaminated precursors of interest are selected for fragmentation. Therefore, in various embodiments, the opposite approach is used: the precursor isolation window of the mass analyzer is expanded, thus including multiple precursors that co-elute and contribute to the stored fragmentation pattern during the analysis. Interpretation of composite product ion spectra resulting from fragmentation of multiple precursors is described below in the data analysis section.

[0040] 2b) As practiced in various embodiments, a positive side effect of widening the selection window of the mass analyzer is (i) the reduction in cycle time described in (1), and thus the acquisition of a more clearly defined and resolved chromatographic elution profile for the monitored precursors, and (ii) increased signal intensity for the fragments, since now the entire isotopic pattern of the precursor is involved in the fragmentation, rather than just the monoisotopic peak, as in SRM.

[0041] Increased confidence for peptide identification 3) Acquisition of full product ion spectra instead of monitoring a few product ions for a given precursor as in classical SRM experiments. A series of full product ion (MS2) spectra are acquired over the elution of the precursor, allowing greater confidence in precursor identification to be achieved by matching the complete fragmentation pattern of the full MS2 spectrum, rather than just a few ion fragments, to reference spectra from a library.

[0042] In summary, in various embodiments, the LC-MS method can cover the complete mass range and chromatographic profile instead of a few transitions per precursor, resulting in the acquisition of a series of MS2 maps that can be treated as pseudo-SRM traces acquired over a wide isolation window (Figures 2-3).

[0043] Successive MS2 maps can be acquired by overlapping several precursor separation windows, ensuring the transfer of the complete isotopic pattern of any given precursor ion in at least one separation window, thereby maintaining optimal correlation between parent and fragment isotopic peaks at any LC time point. This overlap can be reduced to a minimum value that can be experimentally determined to best match the fragment ion transmission profile achievable on the ion selection device used in the mass spectrometer. Reducing the overlap between successive separation windows allows maintaining a minimum size for the window, a minimum number of windows to cover a given m / z range, and a minimum dwell time between periodic separation window acquisitions.

[0044] Various embodiments include the following MS acquisition methods in various combinations, ie, alone and with various combinations of data analysis principles as further described below.

[0045] 1) Periodic acquisition of full fragmentation (MS2) spectra of precursor ions based on stepping the precursor isolation window of a mass analyzer in a content-independent manner (see FIG. 2). Acquisition does not include, for example, focusing the precursor isolation window on a predetermined (e.g., data-dependent acquisition / shotgun) or targeted (e.g., target list or SRM) precursor ion mass.

[0046] 2) A thorough search of these MS2 spectra for multiple parent precursors simultaneously selected within the parent ion precursor isolation window and simultaneously responsible for the observed fragmentation pattern, according to various embodiments of the invention described in the Data Analysis section. In other words, in various embodiments, the search is not performed on the precursor ion. The search is performed on fragments within the window that are expected to contain the precursor ion.

[0047] 3) Use of overlapping windows for precursor selection: Overlapping windows can be used in various embodiments to ensure that (i) all precursor ions are properly selected even in the case of a non-ideal mass analyzer, and (ii) precursor ions at the mass analyzer / selection window boundary have their entire or substantially entire isotope pattern selected for fragmentation within the same separation window.

[0048] 4) Use of fixed and / or variable widths for precursor separation windows during the same acquisition run. In various embodiments, fixed and / or variable widths are used for precursor separation windows during the same cycle (i.e., a set of scans across the mass range). Use of larger windows allows for shorter cycle times of acquisition in less crowded parts of the mass / chromatographic space (i.e., where the fewest analytes are expected). Use of narrower windows can allow for increased dynamic range of analysis in the most complex parts of the mass / chromatographic space. In effect, narrower windows contain fewer precursor ions to fragment, and therefore have a lower chance of containing precursors with large differences in abundance.

[0049] 5) Use of single and / or multiple (variable or discrete) collision energies per precursor selection window during the same acquisition run. Increases or decreases in fragment ion intensities acquired during such multiple collision energy experiments can be checked for co-elution and correlated with reference fragment ion intensities from a spectral library, enhancing the identification of fragment ion peaks arising from the same parent ion (see Data Analysis section).

[0050] 6) Use of fixed and / or variable times per precursor isolation window for acquisition of MS2 spectra during the same acquisition run. Variable acquisition times can still be used for quantification purposes (as in "dynamic" or "periodic" SRM) since the signal can be reported as counts (e.g., cts / msec) over the acquisition time. Longer acquisition times can allow monitoring of low abundance precursors with higher sensitivity.

[0051] Various embodiments of the bioinformatics / data analysis pipeline Data analysis comprises the use of fragment ion elution information and data mining of reference spectral libraries. Reference spectral libraries of proteotypic peptides (MS observable peptides that are found uniquely in a protein and therefore unambiguously characterize that protein qualitatively and quantitatively) can be created for whole organisms, using pools of synthetic peptides (Picottietal, Nat Method 2010), and / or from previous extensive MS sequencing proteomic analyses performed on those organisms. Similarly, reference spectral libraries for other analytes can be created from synthetic analyte references and / or from previous analyte MS analyses. Importantly, once a reference fragment ion library is created, it can be used permanently.

[0052] Since the LC-MS data comprise full product ion spectra acquired from a wide precursor selection window, data processing is modified to account for the presence of multiple precursors, and all fragment ions, potentially responsible for the fragmentation patterns observed in the recorded MS2 spectra.

[0053] (Search for post-mortem precursors) In contrast to SRM approaches, where a precursor of interest must be selected prior to sample injection, the "full content coverage" acquisition approach allows for the post-hoc searching and quantification of any analyte present in the LC-MS / MS data set, in various embodiments within a spectral library. Data analysis comprises the extraction of the fragment mass trace of the precursor of interest (determined from a reference spectral library and / or computational predictions) from the set of all product ion spectra acquired within the expected selection window (m / z) of that precursor (see Figures 2-3).

[0054] Confidence in precursor identification can be scored based on, for example, mass accuracy and / or relative intensity of acquired product ion fragments compared to that of a reference (or expected) fragmentation spectrum, the number of matched fragments, similar chromatographic characteristics of the extracted ion traces of these fragments (co-elution, peak shape, etc.). Probability of identification can be determined, for example, by similarly searching (and scoring) decoy precursor fragment ions from the same LC-MS data set. Relative quantification can be performed by integration of product ion traces over the chromatographic elution of the precursor. In various embodiments, differentially isotopically labeled reference analytes (similarly identified, quantified, and scored) are utilized to achieve absolute quantification of the corresponding precursors of interest.

[0055] Increased confidence in peptide identification To achieve greater confidence in precursor identification by matching the complete fragmentation pattern of the entire MS2 spectrum, rather than just a few ion fragments, to a reference spectrum from a library, a set of entire product ion (MS2) spectra can be extracted around the elution of the best scoring fragment ion peak candidates.

[0056] The aforementioned data mining approach uses unbiased extraction of reference fragment ion traces (from a spectral library) from the LC-MS / MS data set. Thus, the entire product ion map is mined for identification of multiple precursors, since they are extracted by matching fragment ion traces independent of the library. Therefore, this does not limit the search number of co-eluting precursors within the selection window of the mass analyzer, and can allow identification of multiple precursors within the same product ion spectrum.

[0057] (Alternative data processing using MS2 feature extraction) Another data processing embodiment comprises de novo feature extraction of all or substantially all of the fragment ion signals from the reconstructed MS2 map (FIG. 2). The co-eluting fragment ion signals can then be grouped and searched by reference spectral library matching (or ultimately against a database of pre-calculated theoretical fragment ions for the analyte) to determine their original precursor. In various embodiments, the method proceeds by iteration of precursor identification and removal of product ion signals of that precursor over its elution, increasing the sensitivity of the analysis and removing lower abundance ion fragments from interest.

[0058] Various embodiments include the following data analysis principles in various combinations, ie, alone and in conjunction with various combinations of the MS acquisition principles further described above.

[0059] 1) Extraction of fragment mass traces of a precursor of interest (as determined from a reference spectral library or computational predictions) from the set of all product ion spectra (see Figures 2-3) acquired in the expected selection window of that precursor (or a window for a modified or multiple charge state peptide).

[0060] 2) Identification of analytes by scoring extracted fragment ion traces based on the following parameters, such as (i) coelution of extracted fragment ion traces, (ii) correlation of their peak shapes, (iii) correlation of their relative intensities with those from a reference spectral library (or computational predictions), (iv) proximity to expected reference chromatographic retention times, (v) coelution and peak shape correlation of fragment ion traces of multiple charge states of the same precursor, (vi) coelution, peak shape, and relative intensity correlation with one or more differentially isotopically labeled reference fragment ion traces (e.g., heavy or light reference analytes for light or heavy endogenous samples, respectively), (vii) coelution and peak shape correlation of fragment ion traces obtained from windows acquired at different collision energies, (viii) correlation of relative intensities of fragment ions obtained from windows acquired at different collision energies with those from a reference spectral library (or computational predictions), and (ix) combinations of two or more of the foregoing.

[0061] 3) Distinguishing true from false positive identifications by false discovery rate assessment, also based on searching (and scoring) the same LC-MS / MS data set for decoy precursor fragment ions. The decoy hits can be subsequently used to optimize a combination of one or more of the aforementioned scores using machine learning techniques (e.g., semi-supervised learning) and to predict the false discovery rate by assuming that they are similar to the null distribution of identifications.

[0062] 4) Use of coeluting fragment ion intensities to quantify identified analytes contained in a sample.

[0063] 5) "Refining" and re-searching (e.g., in multiple replicates) of the acquired data by substantially removing contaminating fragment ion traces, or those of already identified analytes, over their chromatographic elution.

[0064] 6) Extraction of pre-calculated theoretical fragment mass traces of any precursor of interest from a set of all product ion spectra acquired in the expected selection window of that precursor (or a window for modified or multiple charge state peptides) (e.g., for the acquisition and refinement of spectral libraries of natural or synthetic compounds). 7) Novel "feature extraction" of fragment ion signals from the reconstructed MS2 map (Figure 2). For example, grouping and scoring of those fragment ion signals as described above in (2). Searching them by reference spectral library matching to determine their original precursors (or finally against a database of pre-calculated fragment ions of the analyte). Quantification of identified analytes based on their co-eluting fragment ion intensities.

[0065] In summary, the various embodiments can enable (i) the comprehensive acquisition of product ion spectra of all analytes present in a sample in a single LC-MS injection or analysis, (ii) their complete identification and quantitative analysis by specific data mining techniques, and (iii) the refinement and / or complementation of their analysis by iterative data mining. The present combined LC-MS acquisition and data processing methodology thus constitutes a significant improvement over conventional approaches in terms of data consistency, identification rate, and quantification speed. These inventions enable methods for the acquisition of complete proteome maps and their qualitative and quantitative data mining.

[0066] The potential applications of these inventions are essentially the same as those of SRM quantitative proteomics, including any biotechnological, biomedical, pharmaceutical, and biological applications that rely on qualitative and quantitative LC-MS analysis. For example, the approach is, in various embodiments, particularly suitable for performing analyses of large numbers of candidate precursors of interest (e.g., peptides) within complex samples (e.g., whole organisms, cells, organs, body fluids, etc.) that may be available only in limited amounts.

[0067] Various embodiments include, among others, the following applications:

[0068] - Rapid acquisition and refinement of spectral libraries of natural or synthetic compounds (e.g. peptides).

[0069] - Qualitative and quantitative analysis of natural or synthetic compounds of interest (e.g., in the context of specific analyte or biomarker measurements, or to analyze the composition of protein complexes).

[0070] - Qualitative and quantitative analysis of naturally or artificially modified analytes by sharing fragment ions with their unmodified counterparts (e.g., proteins / peptides or chemically crosslinked proteins / peptides with post-translational modifications reacted with activity-based probes), or by their modifications sharing fragment ions (e.g., ubiquitin or ubiquitin-like molecules) or common reporter ions, or by using the mass difference (positive or negative) that the modification imparts to the fragment ions of those analytes.

[0071] -Qualitative and quantitative analysis (e.g. in the context of partial or complete proteome analysis) of all detectable analytes present in the spectral library or newly identified (see Data Analysis section).

[0072] - Iterative data mining of the obtained datasets to identify their qualitative and quantitative Ability to refine and / or complement the analysis.

[0073] These various embodiments can pave the way for complete qualitative and quantitative analysis of the entire metabolome / proteome of complex samples in a high-throughput manner.

[0074] (Quantitative Data Processing) In various embodiments, all of the fragment data acquired from a single precursor mass window can be processed together, even though the data may contain fragments from more than one precursor ion (compound), to quantify a compound of interest or to search for modified forms of such a compound.

[0075] The precursor mass of the compound of interest and a set of expected fragments at high resolution and mass accuracy are obtained from a library or by analyzing a confirmatory standard form of the compound, or from a previous analysis (whether the compound is known or unknown) or by prediction using known fragmentation rules. The set of fragments can be selected based on their expected intensity, the likelihood that they are unique to the compound of interest, or other characteristics. For a window that includes the expected precursor mass, the set of fragment masses is used to generate an ion trace, e.g., a chromatogram that includes one or more peaks.

[0076] The traces are scored to determine the correct or most likely peak. The score can be based on information from the mass spectrum, such as how well the detected masses of the sample fragment ions match the expected masses of the given product ions, how well the relative intensities of the sample fragment ions match the relative intensities of the given product ions, that the measured sample ions are of the correct isotopic form, usually monoisotopic, that the precursor and fragment ions have the expected charge states, etc.

[0077] If a separation step is included, the score can be based on additional information, such as how well the detected ion traces match each other in shape and position. If different isotopic forms of a sample are analyzed, such as a combination of labeled and native forms, data from the different forms can be used to further refine the score. If one or more fragments in the collection receive a poor score due to the presence of interferences, they can be removed from the collection and replaced with another fragment from a given spectrum, if desired.

[0078] Ions that receive acceptable scores can be used to generate a quantitative value for the target compound that can be compared to similar values ​​from other samples, such as components of a time course study, groups of samples that have been treated or prepared differently, groups of samples from healthy or diseased subjects, etc. Because all fragment ions from all detected precursors are present in the data, in various embodiments, optimal quantification can be performed by using surrogate fragment ions that reduce error in the measurement.

[0079] The acquired data includes fragments from all detectable compounds and can be mined for any number of compounds, and scoring can produce a quantitative value.

[0080] (Qualitative Data Processing) In various embodiments, the data can be further mined to extract qualitative information about the compounds present in the sample. Modification forms can be detected, for example, by locating the same set of fragment ions at unexpected retention times in the same precursor window or in different windows. The windows can be determined based on the expected mass difference caused by the modification. In various embodiments, modification forms can be detected by locating ions that are characteristic of the modification.

[0081] Once a modification is detected, the type and location of the modification can be determined in several ways. For example, the type and location of the modification can be determined by predicting ions that depend on the position or type of the modification, and creating and scoring traces extracted from the data for those predicted masses. In various embodiments, the type and location of the modification can be determined by creating a spectrum from the data and interpreting the spectrum.

[0082] Further, the data from each window can be processed to extract spectra of known or unknown compounds that can be interpreted to determine or identify the associated ions and thereby determine the identity of the compound.

[0083] (Tandem Mass Spectrometer System) 4 is a schematic diagram showing a system 400 for storing an electronic record of all product ion spectra of all detectable compounds of a sample, according to various embodiments. System 400 includes a tandem mass spectrometer 410 and a processor 420. Processor 420 can be, but is not limited to, a computer, microprocessor, or any device capable of sending control signals, receiving data from mass spectrometer 410, and processing the data.

[0084] The tandem mass spectrometer 410 can include one or more physical mass analyzers that perform two or more mass analyses. The mass analyzers of the tandem mass spectrometer can include, but are not limited to, a time-of-flight (TOF), quadrupole, ion trap, linear ion trap, orbitrap, or Fourier transform mass analyzer. The tandem mass spectrometer 410 can also include a separation device (not shown). The separation device can perform separation techniques including, but not limited to, liquid chromatography, gas chromatography, capillary electrophoresis, or ion mobility. The tandem mass spectrometer 410 can include separation of mass analysis stages or steps in space or time, respectively.

[0085] The tandem mass spectrometer 410 performs multiple product ion scans, one or more times, across a mass range using multiple mass selection windows. The multiple product ion scans are performed in a single sample analysis. The single sample analysis is, for example, a single sample injection. From the multiple product ion scans, the tandem mass spectrometer 410 generates a full sample product ion spectrum of all detectable compounds for each mass selection window.

[0086] The processor 420 is in communication with the tandem mass spectrometer 410. The processor 420 receives from the tandem mass spectrometer 410 the full sample product ion spectrum for each mass selection window. The processor 420 then stores the full sample product ion spectrum for each mass selection window as an electronic record of all detectable compounds of the sample. The electronic record is used to characterize known compounds at the time the electronic record is stored, or to characterize compounds that become known after the electronic record is stored.

[0087] In various embodiments, each mass selection window of the plurality of mass selection windows has a width greater than 10 atomic mass units (amu), or a width greater than 15 amu.

[0088] In various embodiments, at least two mass selection windows of the plurality of mass selection windows have different widths.

[0089] In various embodiments, the entire sample product ion spectrum for one or more mass selection windows from the electronic record is searched against a predetermined product ion spectrum from a reference library. For example, the processor 420 receives a predetermined product ion spectrum corresponding to a known compound. The processor 420 receives an entire sample product ion spectrum for one or more mass selection windows from the electronic record. The processor 420 then compares the predetermined product ions of the predetermined product ion spectrum with the sample product ions of the entire sample product ion spectrum for one or more mass selection windows. One or more matching sample product ions from the comparison characterize a known compound detectable in the sample. A known compound includes, for example, any compound that results in a reproducible product ion spectrum.

[0090] In various embodiments, the known compounds include one or more of peptides, proteins, the complete proteome, endogenous metabolites, lipids, or carbohydrates.

[0091] In various embodiments, the known compounds include one or more compounds of biological, pharmaceutical, environmental, forensic, or industrial importance, which may include, but are not limited to, one or more of a pesticide, a herbicide, a fungicide, an industrial chemical, a drug of abuse, a dopant, or an explosive.

[0092] In various embodiments, a given product ion spectrum is computationally generated by applying empirical or speculative fragmentation or modification rules to known compounds.

[0093] In various embodiments, the predetermined product ion spectrum is obtained from the analysis of authentic standard compounds that are isolated from naturally occurring sources or chemically synthesized.

[0094] In various embodiments, the predetermined product ion spectra are obtained from public or proprietary spectral libraries.

[0095] In various embodiments, the predetermined product ion spectrum is obtained from a pre-analysis of a representative sample, with or without identification of the compound that corresponds to the predetermined product ion spectrum.

[0096] In various embodiments, the search for the given product ion spectrum from the reference library includes the calculation of a score. For example, the processor 420 compares the given product ions of the given product ion spectrum to the sample product ions of the full sample product ion spectrum for one or more mass selection windows by calculating a score that represents how well the given product ions and the sample product ions match. The score can include, for example, a comparison of ion masses and relative intensities. In various embodiments, the score includes a determination that the sample precursor ions have the expected isotopic form. In various embodiments, the score can include information regarding the expected charge states of the precursor and fragment ions.

[0097] In various embodiments, the separation device separates the sample compounds of a single sample analysis over time. The tandem mass spectrometer 410 performs multiple product ion scans for the single sample analysis as the sample compounds are separated. In various embodiments, the processor 420 further calculates a score for the match based on a comparison of the peak shapes of the ion traces or the detection time similarity of the matching sample product ions.

[0098] In various embodiments, the processor 420 further uses one or more matching sample-produced ions from the search comparison to calculate a quantitative value for the compound of the sample. The quantitative value is calculated, for example, using the intensity of one or more matching sample-produced ions in the sample-produced ion spectrum. In various embodiments, the quantitative value is calculated using the intensity or area of ​​one or more matching ion trace peaks.

[0099] In various embodiments, the processor 420 further uses one or more matching sample-generated ions from the search comparison to identify modified forms of one or more compounds of known compounds. The modified forms are identified, for example, by finding one or more matching sample-generated ions in the same mass selection window or in a different mass selection window. The different mass selection window is determined, for example, from the mass of the expected modification.

[0100] In various embodiments, the modified form is identified by finding the mass corresponding to one or more matching sample-produced ions adjusted by the mass of the modification, e.g., the modification is a known modification, occurs by a known reaction, or is suggested by other experiments.

[0101] In various embodiments, the modified forms are identified by finding the mass signature of the modified moiety of a known compound.

[0102] In various embodiments, the modified forms are identified by finding a composite synthetic spectrum from the binding molecules.

[0103] In various embodiments, the processor 420 further extracts spectra of the identified modifications from the electronic record to characterize the type and location of modifications in the identified modifications.

[0104] In various embodiments, the processor 420 further uses the given product ions and modifications to predict masses that will represent sites of modification, generate a score for each mass, and determine the location of the modification.

[0105] (Tandem mass spectrometry method) FIG. 5 is an exemplary flow diagram showing a method 500 for storing an electronic record of all product ion spectra of all detectable compounds of a sample, according to various embodiments.

[0106] In step 510 of method 500, multiple product ion scans are performed on a tandem mass spectrometer one or more times in a single sample analysis, using multiple mass selection windows across a mass range, and a full sample product ion spectrum of all detectable compounds for each mass selection window is generated.

[0107] In step 520, the full sample product ion spectrum for each mass selection window is received from the tandem mass spectrometer using a processor.

[0108] In step 530, the entire sample product ion spectrum for each mass selection window is recorded using a processor as an electronic record of all detectable compounds of the sample. The electronic record is used to characterize compounds that were known at the time the electronic record was stored, or that become known after the electronic record is stored.

[0109] (Tandem Mass Spectrometry Computer Program Product) In various embodiments, a computer program product includes a tangible computer readable storage medium, the contents of which include a program with instructions for execution on a processor to perform a method for storing an electronic record of all product ion spectra of all detectable compounds of a sample, the method being performed by a system that includes one or more individual software modules.

[0110] 6 is a schematic diagram of a system 600 including one or more separate software modules that implement a method for storing an electronic record of all product ion spectra of all detectable compounds of a sample, according to various embodiments. System 600 includes a measurement module 610 and a storage module 620.

[0111] The measurement module 610 receives from the tandem mass spectrometer a total sample product ion spectrum of all detectable compounds for each mass selection window in a mass range, the tandem mass spectrometer generating the sample product ion spectrum by performing multiple product ion scans across the mass range, one or more times in a single sample analysis, using multiple mass selection windows.

[0112] The storage module 620 stores the entire sample product ion spectrum for each mass selection window as an electronic record of all detectable compounds of the sample. The electronic record is used to characterize compounds that are known when the electronic record is stored, or that become known after the electronic record is stored.

[0113] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0114] Furthermore, in describing various embodiments, the present specification may present the method and / or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps described herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would understand, other sequences of steps may be possible. Thus, the particular order of steps described herein should not be construed as a limitation on the claims. In addition, claims directed to methods and / or processes should not be limited to the execution of its steps in the order written, and one of ordinary skill in the art can readily understand that the sequence may be varied and still be within the spirit and scope of various embodiments.

Claims

1. A system for analyzing a sample using two or more graduated overlapping precursor separation windows, comprising: a tandem mass spectrometer including a mass analyzer that allows two or more overlapping precursor isolation windows to be stepped across a continuous precursor ion mass range of the sample; a processor in communication with the tandem mass spectrometer; Equipped with The system, wherein the processor instructs the tandem mass spectrometer to stepwise select and fragment each of the two or more overlapping precursor separation windows across the continuous precursor ion mass range of the sample to cover the continuous precursor ion mass range of the sample, and instructs the mass analyzer to mass analyze fragment ions resulting from the fragmentation of each of the two or more overlapping precursor separation windows resulting in a fragment ion spectrum for each of the two or more overlapping precursor separation windows, at least two of the two or more overlapping precursor separation windows having different widths.

2. The system described in claim 1, wherein the two or more overlapping precursor separation windows are overlapped to ensure that all isotopic forms of the compound are present within at least one precursor separation window.

3. The system described in claim 1, wherein the two or more overlapping precursor separation windows are overlapped to ensure that all precursor ions across the precursor ion mass range are selected.

4. The system described in claim 1, wherein each of the two or more overlapping precursor separation windows has a width greater than 10 amu.

5. The system described in claim 1, wherein at least one of the two or more precursor separation windows is selected and fragmented more than once in the same acquisition of the mass range using different collision energies for fragmentation.

6. The system of claim 1, wherein the tandem mass spectrometer enables the two or more overlapping precursor separation windows to be stepped across consecutive precursor ion mass ranges of a sample by moving a single precursor separation window in a stepped manner across the consecutive precursor ion mass ranges of the sample such that there is overlap of the precursor separation window between the steps, resulting in consecutive overlapping precursor separation windows.

7. The system described in claim 6, wherein the overlap of the precursor separation windows of the consecutive overlapping precursor separation windows is reduced to an experimentally determined minimum value that matches the fragment ion transmission profile achievable by the mass analyzer.

8. The system described in claim 7, wherein the overlap of the precursor separation windows of the successive overlapping precursor separation windows is reduced to the minimum value so as to maintain a minimum size for the single precursor separation window, a minimum number of successive overlapping precursor separation windows to cover the successive precursor ion mass range of the sample, and a minimum dwell time between periodic acquisitions of the successive precursor ion mass range of the sample.

9. A method for analyzing a sample using two or more graduated overlapping precursor separation windows, comprising: instructing the tandem mass spectrometer, using a processor, to stepwise select and fragment each of two or more overlapping precursor isolation windows across a continuous precursor ion mass range of the sample to cover the continuous precursor ion mass range of the sample; instructing a mass analyzer of the tandem mass spectrometer, using the processor, to mass analyze fragment ions resulting from fragmentation of each of the two or more overlapping precursor isolation windows resulting in a fragment ion spectrum for each of the two or more overlapping precursor isolation windows; Including, the tandem mass spectrometer allowing two or more overlapping precursor isolation windows to be stepped across the continuous precursor ion mass range of the sample; The method, wherein at least two of the two or more overlapping precursor separation windows have different widths.

10. The method of claim 9, wherein the two or more overlapping precursor separation windows are overlapped to ensure that all isotopic forms of the compound are present within at least one precursor separation window.

11. The method of claim 9, wherein the two or more overlapping precursor separation windows are overlapped to ensure that all precursor ions across the precursor ion mass range are selected.

12. The method of claim 9, wherein each of the two or more overlapping precursor separation windows has a width greater than 10 amu.

13. The method of claim 9, wherein at least one of the two or more precursor separation windows is selected and fragmented more than once in the same acquisition of the mass range using different collision energies for fragmentation.

14. The method of claim 9, wherein the tandem mass spectrometer selects each of the two or more overlapping precursor separation windows in a stepwise manner across the continuous precursor ion mass range of the sample, such that there is overlap of precursor separation windows between steps, resulting in continuous overlapping precursor separation windows.

15. The method of claim 14, wherein the overlap of the precursor separation windows of the successive overlapping precursor separation windows is reduced to an experimentally determined minimum value that matches the fragment ion transmission profile achievable by the tandem mass spectrometer.

16. The method of claim 15, wherein the overlap of the precursor separation windows of the successive overlapping precursor separation windows is reduced to the minimum value so as to maintain a minimum size for the single precursor separation window, a minimum number of successive overlapping precursor separation windows to cover the successive precursor ion mass range of the sample, and a minimum dwell time between periodic acquisitions of the successive precursor ion mass range of the sample.

17. A non-transitory tangible computer readable storage medium, the contents of which include a program, the program having instructions to be executed on a processor to perform a method for analyzing a sample using two or more graduated overlapping precursor separation windows, the method comprising: providing a system including one or more separate software modules, the separate software modules comprising a measurement module; instructing the tandem mass spectrometer to use a measurement module to stepwise select and fragment each of two or more overlapping precursor isolation windows across a continuous precursor ion mass range of the sample to cover the continuous precursor ion mass range of the sample; instructing a mass analyzer of the tandem mass spectrometer to mass analyze, using the measurement module, fragment ions resulting from fragmentation of each of the two or more overlapping precursor isolation windows resulting in a fragment ion spectrum for each of the two or more overlapping precursor isolation windows; Including, the tandem mass spectrometer allowing two or more overlapping precursor isolation windows to be stepped across the continuous precursor ion mass range of the sample; 13. A non-transitory tangible computer readable storage medium, wherein at least two of the two or more overlapping precursor separation windows have different widths.

18. The non-transitory tangible computer-readable storage medium of claim 17, wherein the two or more overlapping precursor separation windows are overlapped to ensure that all isotopic forms of the compound are present within at least one precursor separation window.

19. A non-transitory tangible computer-readable storage medium as described in claim 17, wherein the two or more overlapping precursor separation windows are overlapped to ensure that all precursor ions are selected across the precursor ion mass range.

20. The non-transitory tangible computer-readable storage medium of claim 17, wherein each of the two or more overlapping precursor separation windows has a width greater than 10 amu.

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