A method for a probabilistic framework for real-time encoding and precursor inference of scanning SWATH data.

Abstract

To provide a favorable method regarding scanning SWATH data.SOLUTION: A precursor ion transmission window is moved in overlapping steps across a precursor ion mass range. The precursor ions transmitted at each overlapping step by the mass filter are fragmented or transmitted. The intensity or count is detected for each of the one or more resulting product ions or precursor ions for each overlapping window, and the intensity or count forms mass spectrum data for each overlapping window. Each unique product ion detected is encoded in real time during data acquisition. This encoding includes sums of counts or intensity values of respective unique ions detected for the overlapping windows and positions of the windows associated with the respective sums. The encoding for each unique ion is stored in a memory device rather than the mass spectral data. A deblurring algorithm or numerical method is used to determine a precursor ion of each unique ion from the encoded data.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] (Related Application) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 855,242, filed May 31, 2019, the content of which is incorporated herein by reference in its entirety.

[0002] The teachings herein relate to encoding and storing data from a scanning SWATH mass spectrometry method. More specifically, the teachings herein relate to systems and methods for reducing the file size required to store scanning SWATH data by applying real-time encoding of the scanning quadrupole dimension based on a quadrupole response function or a precursor ion speculation probability function.

Background Art

[0003] As described below, scanning SWATH is a tandem mass spectrometry method in which a precursor ion mass selection window or a precursor ion transmission window is scanned across a mass range, such that consecutive windows have large overlapping areas and small non-overlapping areas. This scanning makes the resulting product ions a function of the scanned precursor ion mass selection window. This additional information is useful in identifying one or more precursor ions involved in each product ion, which is sometimes difficult to do with conventional SWATH.

[0004] One problem with scanning SWATH is that it requires significantly more long-term (e.g., file) storage of data than conventional SWATH. The increased amount of file storage is approximately proportional to the amount of precursor ion transmission window overlap. Thus, if n precursor ion transmission windows are overlapped in a scanning SWATH, at least about n times more data needs to be stored in the file in a scanning SWATH experiment for the same precursor ion mass range being analyzed than in a conventional SWATH experiment.

[0005] While file storage itself has become consistently smaller and cheaper, the cost of post-processing such large files is significant in terms of the processing time and power required. Scanning SWATH data stored in files is post-processed, for example, to infer the precursor ions of the measured generated ions. Numerous numerical decomposition or probabilistic inference methods are available that can be applied to scanning SWATH data.

[0006] Traditionally, in scanning SWATH experiments, raw mass spectrometer detections (e.g., time-of-flight (TOF) mass spectrometer counts) for each generated ion produced from each overlapping precursor ion permeation window are stored in a file. Alternatively, intermediate types of data can also be used. For example, the spectra of each generated ion for each overlapping precursor ion permeation window can be stored in a file. Unfortunately, in both methods, the storage size is still at least about n times larger than the storage size required for conventional SWATH storing the same type of data, where n is the number of overlapping precursor ion permeation windows.

[0007] As a result, additional systems and methods are needed to reduce the file size required to store scanning SWATH data without losing any information necessary for post-processing of the data, such as precursor ion prediction. (Tandem mass spectrometry and scanning SWATH)

[0008] In general, tandem mass spectrometry, or MS / MS, is a well-known technique for analyzing compounds. Tandem mass spectrometry involves ionization of one or more compounds from a sample, selection of one or more precursor ions of one or more compounds, fragmentation of one or more precursor ions into a product ion, and mass spectrometry of the product ion.

[0009] Tandem mass spectrometry can provide both qualitative and quantitative information. The generated ion spectrum can be used to identify the molecule of interest. The intensity of one or more generated ions can be used to quantify the amount of compound present in the sample.

[0010] Numerous different types of experimental methods or workflows can be performed using tandem mass spectrometers. These workflows fall into three broad categories: targeted acquisition, information-dependent acquisition (IDA) or data-dependent acquisition (DDA), and data-independent acquisition (DIA).

[0011] In targeted acquisition methods, one or more transitions of a precursor ion to a product ion are predefined with respect to the compound of interest. When the sample is introduced into a tandem mass spectrometer, one or more of these transitions are investigated during each of several periods or cycles. In other words, the mass spectrometer selects and fragments the precursor ion for each transition and performs targeted mass spectrometry with respect to the product ion of the transition. As a result, a mass spectrum is generated for each transition. Targeted acquisition methods include, but are not limited to, multiple reaction monitoring (MRM) and selective reaction monitoring (SRM).

[0012] In the IDA method, the user can define criteria for performing targeted or untargeted mass spectrometry of the generated ions while the sample is introduced into the tandem mass spectrometer. For example, in the IDA method, a precursor ion or mass spectrometry (MS) survey scan is performed to generate a precursor ion peak list. The user can select criteria for filtering the peak list with respect to a subset of the precursor ions on the peak list. Then, MS / MS is performed on each of the subset of precursor ions. A generated ion spectrum is generated for each precursor ion. MS / MS is repeatedly performed on the subset of precursor ions while the sample is introduced into the tandem mass spectrometer.

[0013] However, in proteomics and many other sample types, the complexity and dynamic range of compounds are very large. This poses challenges to conventional targeting and IDA methods and requires very fast MS / MS acquisition to deeply investigate samples in order to both identify and quantify a wide range of specimens.

[0014] As a result, a third broad category of tandem mass spectrometry, the DIA method, was developed. These DIA methods were used to increase the reproducibility and comprehensiveness of data acquisition from complex samples. DIA methods can also be called nonspecific fragmentation methods. In conventional DIA methods, the action of the tandem mass spectrometer is not varied between MS / MS scans based on data obtained in previous precursor or generated ion scans. Instead, a precursor ion mass range is selected, and the precursor ion permeation window is shifted across the precursor ion mass range. All precursor ions within the precursor ion permeation window are fragmented, and all generated ions of all precursor ions within the precursor ion permeation window are mass-spectrated.

[0015] The precursor ion permeation window used to scan the mass range can be very narrow, so as to minimize the possibility of multiple precursors within the window. This type of DIA method is used, for example, in MS / MS. ALL It is called MS / MS. ALL In this method, a precursor ion permeability window of approximately 1 amu is scanned or shifted across the entire mass range. A generated ion spectrum is produced for each 1 amu precursor mass window. The generated ion spectrum for the entire precursor ion mass range is produced by combining the generated ion spectra for each mass selection window. The time required to analyze or scan the entire mass range once is referred to as one scan cycle. However, scanning a narrow precursor ion permeability window across a wide precursor ion mass range during each cycle is not practical for some instruments and experiments.

[0016] As a result, a larger precursor ion permeability window or a wider selection window is shifted across the entire precursor mass range. This type of DIA method is called, for example, SWATH acquisition. In SWATH acquisition, the precursor ion permeability window shifted across the precursor mass range in each cycle can have a width of 5–25 amu or even larger. MS / MS ALL As in the method, all precursor ions within each precursor ion permeation window are fragmented, and all generated ions of all precursor ions within each mass selection window are mass-spectrated. However, because a wider precursor ion permeation window is used, the cycle time is MS / MS. ALL The cycle time of the method can be significantly reduced compared to the cumulative time. Alternatively, with respect to liquid chromatography (LC), the cumulative time can be increased. Generally, with respect to LC, the cycle time is defined by the LC peak. Sufficient points (intensity as a function of cycle time) must be acquired across the LC peak to determine its shape. When the cycle time is defined by LC, the number of experiments or mass spectrometry scans that can be performed within the cycle defines the length of time each experiment or scan can accumulate ion observations. Consequently, a wider precursor ion permeability window can increase the cumulative time.

[0017] U.S. Patent No. 8,809,770 (Patent Document 1) describes a method in which SWATH acquisition may be used to provide quantitative and qualitative information about the precursor ions of a compound of interest. In particular, the generated ions found by fragmenting the precursor ion permeation window are compared with a database of known generated ions of the compound of interest. In addition, ion traces or extracted ion chromatograms (XICs) of the generated ions found by fragmenting the precursor ion permeation window are analyzed to provide quantitative and qualitative information.

[0018] However, identifying a compound of interest in a sample analyzed using, for example, SWATH acquisition can be difficult. This can be difficult because there is no precursor ion information provided to help determine the precursor ions that make up each generated ion, or because the provided precursor ion information derives from mass spectrometry (MS) observations with low sensitivity. In addition, because there is little or no specific precursor ion information provided to the precursor ion permeation window, it is also difficult to determine whether the generated ion is convoluted with or includes contributions from multiple precursor ions within the precursor ion permeation window.

[0019] As a result, a method called scanning SWATH was developed, which involves scanning the precursor ion permeation window during SWATH acquisition. Essentially, in scanning SWATH, the precursor ion permeation window is scanned over a mass range such that the continuous window has areas of large overlap and areas of small non-overlap. This scanning makes the resulting generated ions a function of the scanned precursor ion permeation window. This additional information can then be used to identify one or more precursor ions involved in each generated ion.

[0020] Scanning SWATH is described in International Publication No. WO2013 / 171459A2 (hereinafter referred to as "Application No. 459") (Patent Document 2). In Application No. 459, a precursor ion permeable window or a 25Da precursor ion permeable window is scanned over time such that the range of the precursor ion permeable window changes over time. The timing of detection of the generated ions is then related to the timing of the precursor ion permeable window through which those precursor ions were transmitted.

[0021] The correlation is first performed by plotting the mass-to-charge ratio (m / z) of each generated ion detected as a function of the precursor ion m / z value transmitted by the quadrupole mass filter. Since the precursor ion transmission window is scanned over time, the precursor ion m / z value transmitted by the quadrupole mass filter can also be considered as time. The start and end times when a particular generated ion is detected are related to the start and end times when its precursor is transmitted from the quadrupole. Consequently, the start and end times of the generated ion signals are used to determine the start and end times of their corresponding precursor ions.

[0022] Scanning SWATH is also described in U.S. Patent No. 10,068,753 (hereinafter referred to as "Patent No. '753") (Patent Document 3). Patent No. '753 improves the accuracy of the correlation of generated ions to their corresponding precursor ions by combining generated ion spectra from a successive group of overlapping precursor ion transmission windows. The generated ion spectra from the successive group are combined by successively summing the intensities of the generated ions in the generated ion spectra. This sum produces a function that may have a shape that is not constant with respect to the precursor mass. The shape represents the generated ion intensity as a function of the precursor mass. The precursor ions are identified from the function calculated with respect to the generated ions.

[0023] For scanning SWATH, a rectangular precursor ion permeable window is preferable. However, another advantage of scanning SWATH is that it can equally and efficiently handle any ion permeability function. The ion permeability function can even be calibrated from the data itself, without needing to be known in detail beforehand. In other words, while a rectangular precursor ion permeable window is preferred, a precursor ion permeable window of any shape can be used.

[0024] Application No. ‘459 and Patent No. ‘753 provide a method for identifying one or more precursor ions corresponding to product ions in a SWATH data set. However, Application No. ‘459 and Patent No. ‘753 do not address reducing the file size required to store SWATH data without losing any information required for post-processing of the data.

Prior Art Documents

Patent Documents

[0025]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0026] Systems, methods, and computer program products for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows according to various embodiments are disclosed. The system includes an ion source device, a mass filter, a fragmentation device, a mass analyzer, and a processor.

[0027] The ion source device converts a sample or a target compound from a sample into an ion beam. The mass filter receives the ion beam. The mass filter then filters the ions by moving a precursor ion transmission window with a precursor ion mass / charge ratio (m / z) width W in overlapping steps over a precursor ion mass range of R m / z with a step size of S m / z. A series of overlapping transmission windows are generated over the mass range. The mass filter transmits the precursor ions within the transmission window at each overlapping step.

[0028] The fragmentation device fragments or transmits precursor ions transmitted in each overlapping step by a mass filter. One or more resulting product ions are generated with respect to each overlapping window in a sequence. The mass spectrometer detects the intensity or count for each of the one or more resulting product ions with respect to each overlapping window in a sequence, and the intensity or count forms mass spectral data with respect to each overlapping window in a sequence.

[0029] The processor communicates with the ion source device, mass filter, fragmentation device, and mass spectrometer. Instead of storing the mass spectral data in a file in a memory device for each overlapping window in a sequence, the processor performs the encoding and storage steps.

[0030] The processor encodes and stores each unique generated ion detected by the mass spectrometer in real time during data acquisition by performing several substeps. First, the processor identifies the overlapping window of the first occurrence of each unique ion in a sequence of occurrences. The processor then selects a group of G overlapping windows in a sequence immediately preceding the overlapping window of the first occurrence such that the group extends at least to the width W of the transmission window. The number G of overlapping windows in the group is calculated, for example, according to G ≥ W / S. The width W of the transmission window by the group of G overlapping windows is, for example, the precursor ion uncertainty interval.

[0031] In various embodiments, the precursor ion likelihood can vary over an uncertainty interval W. For convenience, it is assumed to be constant (or a rectangular precursor ion likelihood function where the precursor ion likelihood function is equal to the mass filter transmission function). A constant precursor ion likelihood over an uncertainty interval W results in a triangular precursor ion uncertainty distribution function. However, another possible precursor ion uncertainty distribution function can be a Gaussian precursor ion uncertainty distribution function.

[0032] The processor calculates the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group. The processor associates the sums with the positions of the overlapping windows in the group. The processor shifts the selected group of G overlapping windows forward by one overlapping window, calculates the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group, associates the sums with the positions of the overlapping windows in the group, stores the sums and positions in a memory device, and repeats these steps until at least one of the overlapping windows of the group no longer overlaps with the overlapping window of the first occurrence.

[0033] These and other features of the applicant's teachings are described herein. This specification also provides, for example, the following items: (Item 1) A system for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, wherein the system is: A mass filter for a tandem mass spectrometer, wherein the mass filter has a step size S A mass filter comprises a mass filter that, in overlapping steps, moves a precursor ion permeation window with a precursor ion mass / charge ratio (m / z) width W over a precursor ion mass range with m / z, generating a continuous overlapping permeation window over the mass range, and transmits precursor ions within the permeation window in each overlapping step. A fragmentation device for the analyzer, wherein the fragmentation device fragments or transmits the precursor ions transmitted by the mass filter in each overlapping step, and generates one or more resulting product ions with respect to each overlapping window of the sequence, The mass spectrometer of the analyzer, wherein the mass spectrometer detects the intensity or count of each of the one or more resulting generated ions with respect to each of the overlapping windows of the sequence, and the intensity or count forms mass spectral data with respect to each of the overlapping windows of the sequence, A processor that communicates with the aforementioned mass spectrometer Equipped with, Instead of storing the mass spectral data in a memory device for each overlapping window of the sequence, the processor encodes each unique generated ion detected by the mass spectrometer in real time during data acquisition. The above encoding is Identifying the overlapping windows of the first appearances in the sequence, each of the unique ions, Select a group of G overlapping windows from the sequence immediately preceding the first overlapping window of appearance such that the group extends at least to the width W of the transmission window; calculate the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group; and associate the sum with the position of the overlapping windows of the group. The selected group of G overlapping windows is shifted forward by the width of one overlapping window; the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group is calculated; the sum is associated with the position of the overlapping windows of the group; the sum and the position are stored in the memory device; and these steps are repeated until at least one overlapping window of the group no longer overlaps with the overlapping window of the first occurrence. A system that performs this action. (Item 2) The system according to item 1, wherein the processor further compresses this data by reducing the number of stored totals and positions for each unique ion, while still maintaining the shape of the originally stored totals and positions for each unique ion. (Item 3) The system according to item 1, wherein the processor further removes the effects caused by encoding from the stored total and position of each unique ion by using a deblurring algorithm, the deblurring algorithm resolves with respect to the deblurred total and position of each unique ion by assuming that the convolution of the deblurred total and position with a probability distribution function dependent on the uncertainty interval of the mass filter is equal to the stored total and position. (Item 4) The system according to item 3, wherein the processor further determines the precursor ion of each unique ion using the debossed sum and position of each unique ion. (Item 5) The system according to item 3, wherein the processor further stores the deblended total and position of each unique ion instead of the stored total and position, thereby reducing the space required in the memory device. (Item 6) The stored total and position of each unique ion in the system described in item 1 have a triangular shape. (Item 7) The system according to item 4, wherein the processor further determines the precursor ions of each unique ion as precursor ions corresponding to the vertices of the triangular shape. (Item 8) The system according to item 1, wherein the processor further determines the precursor ion of each unique ion using a numerical method, the numerical method resolves with respect to the unknown precursor ion column matrix by arranging the stored sums and positions as a column matrix of length n, and assuming that the column matrix is ​​equal to a known n × m mass filter matrix for the mass filter multiplied by an unknown precursor ion column matrix of length m, thereby determining the precursor ion. (Item 9) The numerical method is the system described in item 6, which includes non-negative matrix factorization (NNMF). (Item 10) The numerical method is the system described in item 6, including the non-negative least squares method (NNLS). (Item 11) The system according to item 1, wherein the processor further compresses the stored totals and positions of each unique ion by removing some totals and positions while still maintaining the same shape of the stored totals and positions. (Item 12) The number of overlapping windows G in the group is calculated according to the system described in item 1, where G ≥ W / S. (Item 13) The system according to item 1, wherein the width W of the transmission window due to the group of G overlapping windows is the precursor ion uncertainty interval. (Item 14) A method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, wherein the method is: Using a mass filter of a tandem mass spectrometer, a precursor ion permeation window with a precursor ion mass / charge ratio (m / z) width W is moved in overlapping steps over a precursor ion mass range with a step size S m / z, thereby generating a continuous overlapping permeation window over the said mass range, wherein the mass filter transmits precursor ions within the permeation window in each overlapping step. Using the fragmentation device of the analyzer, the precursor ions transmitted in each overlapping step by the mass filter are fragmented or transmitted, and with respect to each overlapping window of the sequence, one or more resulting product ions are generated. Using the mass spectrometer of the analyzer, the intensity or count of each of the one or more resulting generated ions is detected with respect to each overlapping window of the sequence, wherein the intensity or count forms mass spectral data with respect to each overlapping window of the sequence. The processor is used to encode each unique generated ion detected by the mass spectrometer in real time during data acquisition. Includes, The above encoding is Identifying the overlapping windows of the first appearances in the sequence, each of the unique ions, Select a group of G overlapping windows from the sequence immediately preceding the first overlapping window of appearance such that the group extends at least to the width W of the transmission window; calculate the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group; and associate the sum with the position of the overlapping windows of the group. The selected group of G overlapping windows is shifted forward by the width of one overlapping window; the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group is calculated; the sum is associated with the position of the overlapping windows of the group; the sum and the position are stored in the memory device; and these steps are repeated until at least one overlapping window of the group no longer overlaps with the overlapping window of the first occurrence. A method that involves doing so. (Item 15) A computer program product comprising a non-transient and tangible computer-readable storage medium, the contents of which comprises a program with instructions executed on a processor to perform a method of encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, the method being: The present invention provides a system comprising one or more different software modules, each of which comprises a control module and an encoding and storage module. The mass filter of a tandem mass spectrometer is instructed, using the control module, to move a precursor ion permeation window with a precursor ion mass / charge ratio (m / z) width W in overlapping steps over a precursor ion mass range with a step size S m / z, thereby generating a continuous overlapping permeation window over the mass range, wherein the mass filter transmits precursor ions within the permeation window in each overlapping step. The fragmentation device of the analyzer is instructed, using the control module, to fragment or transmit the precursor ions transmitted by the mass filter in each overlapping step, and to generate one or more resulting product ions with respect to each overlapping window of the sequence. The mass spectrometer of the analyzer is instructed, using the control module, to detect the intensity or count of each of the one or more resulting generated ions with respect to each of the overlapping windows in the sequence, wherein the intensity or count forms mass spectral data with respect to each of the overlapping windows in the sequence. Using the encoding and storage module, each unique generated ion detected by the mass spectrometer is encoded in real time during data acquisition. Includes, The above encoding is Identifying the overlapping windows of the first appearances in the sequence, each of the unique ions, Select a group of G overlapping windows from the sequence immediately preceding the first overlapping window of appearance such that the group extends at least to the width W of the transmission window; calculate the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group; and associate the sum with the position of the overlapping windows of the group. The selected group of G overlapping windows is shifted forward by the width of one overlapping window; the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group is calculated; the sum is associated with the position of the overlapping windows of the group; the sum and the position are stored in the memory device; and these steps are repeated until at least one overlapping window of the group no longer overlaps with the overlapping window of the first occurrence. A computer program product produced by performing [this action]. [Brief explanation of the drawing]

[0034] Those skilled in the art will understand that the drawings provided below are for illustrative purposes only. The drawings are not intended to limit the scope of this instruction in any way.

[0035] [Figure 1] Figure 1 is a block diagram illustrating a computer system in which an embodiment of this teaching may be implemented.

[0036] [Figure 2] Figure 2 is a schematic diagram showing a system for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, according to various embodiments.

[0037] [Figure 3] Figure 3 is a schematic diagram illustrating how each unique generated ion detected during scanning SWATH data acquisition is encoded in real time according to various embodiments.

[0038] [Figure 4] Figure 4 is an illustrative schematic diagram illustrating how, in various embodiments, a set of summed counts or intensities for each unique ion is stored or encoded in real time during scanning SWATH data acquisition.

[0039] [Figure 5] Figure 5 is an illustrative plot showing that, in various embodiments, the summed counts or intensity compression after encoding still preserve the information necessary to infer the precursor ions.

[0040] [Figure 6] Figure 6 is an exemplary heatmap plot showing the total generated ion count, plotted as a function of the precursor ion transmission window position and generated ion m / z, before applying the deblurring algorithm to the data according to various embodiments.

[0041] [Figure 7] Figure 7 is an exemplary heatmap plot showing the total generated ion count plotted as a function of precursor ion transmission window position and generated ion m / z after applying the deblurring algorithm of Figure 6 to the data according to various embodiments.

[0042] [Figure 8] Figure 8 is an illustrative schematic diagram including heatmap plots showing the total generated ion counts plotted as a function of chromatograph time and precursor ion transmission window position before applying the deblurring algorithm to the data according to various embodiments, and a plot showing the XIC found from the heatmap regarding the precursor ion m / z values.

[0043] [Figure 9] Figure 9 is an illustrative schematic diagram including heatmap plots showing the total generated ion counts plotted as a function of chromatograph time and precursor ion transmission window position after applying the deblurring algorithm of Figure 8 to the data according to various embodiments, and a plot showing the XIC found from the heatmap with respect to the precursor ion m / z value.

[0044] [Figure 10] Figure 10 is a flowchart illustrating methods for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, according to various embodiments.

[0045] [Figure 11] Figure 11 is a schematic diagram of a system comprising one or more different software modules that implement a method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, according to various embodiments. [Modes for carrying out the invention]

[0046] Before one or more embodiments of this teaching are described in detail, those skilled in the art will understand that this teaching is not limited to the details of the structures, component arrangements, and step arrangements described or illustrated in the drawings for the following uses. It should also be understood that the expressions and terminology used herein are for illustrative purposes only and should not be considered limiting.

[0047] (Computer-implemented system) Figure 1 is a block diagram illustrating a computer system 100 in which embodiments of this teaching may be implemented. The computer system 100 includes a bus 102 or other communication mechanism for communicating information and a processor 104 coupled to the bus 102 for processing information. The computer system 100 also includes a memory 106, which may be 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 the 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 for storing information and instructions and is coupled to the bus 102.

[0048] The computer system 100 may be coupled to a display 112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), via a bus 102 to display information to the computer user. An input device 114, including alphanumeric and other keys, is coupled to the bus 102 to communicate information and command selections to the processor 104. Another type of user input device is a cursor control 116, such as a mouse, trackball, or cursor directional keys, to communicate directional information and command selections to the processor 104 and to control cursor movement on the display 112. This input device typically has two degrees of freedom, on two axes, namely a first axis (i.e., x) and a second axis (i.e., y), which allows the device to specify a position in a plane.

[0049] Computer system 100 can implement this teaching. According to one implementation of this teaching, the result is 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. The execution of the sequence of instructions contained in memory 106 causes processor 104 to carry out the process described herein. Alternatively, wired circuits may be used instead of, or in combination with, the software instructions for implementing this teaching. Thus, implementation of this teaching is not limited to any specific combination of hardware circuits and software.

[0050] As used herein, the term “computer-readable medium” refers to any medium involved in providing instructions to the processor 104 for execution. Such mediums can take many forms, but are not limited to, non-volatile mediums, volatile mediums, and precursor ion mass-selective mediums. Non-volatile mediums include, for example, optical or magnetic disks such as the storage device 110. Volatile mediums include dynamic memory such as the memory 106. Precursor ion mass-selective mediums include coaxial cables, copper wires, and optical fibers, including wiring with a bus 102.

[0051] Common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, or any other magnetic media, CD-ROMs, digital video discs (DVDs), Blu-ray discs, any other optical media, thumb drives, memory cards, RAM, PROMs, and EPROMs, flash-EPROMs, any other memory chips or cartridges, or any other tangible media that a computer can read.

[0052] Various forms of computer-readable media can be involved in transporting one or more sequences of one or more instructions to the processor 104 for execution. For example, the instructions may initially be transported on a magnetic disk of a remote computer. The remote computer may load the instructions into its dynamic memory and transmit them over a telephone line using a modem. A modem local to computer system 100 can receive the data over the telephone line and convert the data into an infrared signal using an infrared transmitter. An infrared detector coupled to bus 102 can receive the data transported by the infrared signal and place the data on bus 102. Bus 102 transports the data to memory 106, and the processor 104 reads the instructions from memory 106 and executes them. The instructions received by memory 106 may optionally be stored on storage device 110 either before or after execution by processor 104.

[0053] According to various embodiments, instructions configured to be executed by a processor to carry out the method are stored on a computer-readable medium. The computer-readable medium can be a device for storing digital information. For example, the computer-readable medium includes, as is well known in the art, a compact disc read-only memory (CD-ROM) for storing software. The computer-readable medium is accessed by a processor suitable for executing the instructions configured to be executed.

[0054] The following descriptions of various implementations of this instruction are presented for illustrative and explanatory purposes only. They are neither comprehensive nor limiting to the precise form in which this instruction is disclosed. Modifications and variations are possible in light of the above instruction, or they may be derived from the practice of this instruction. In addition, the implementations described include software, but this instruction may be implemented as a combination of hardware and software, or in hardware alone. This instruction may be implemented by both object-oriented and non-object-oriented programming systems.

[0055] (Encoding and storage of scanning SWATH data) As described above, scanning SWATH is a tandem mass spectrometry method in which a precursor ion permeable window is scanned over a mass range such that the continuous window has areas of high overlap and areas of low non-overlap. One problem with scanning SWATH is that it requires significantly more data for long-term (e.g., file) storage than conventional SWATH. As a result, additional systems and methods are needed to reduce the file size required to store scanning SWATH data without losing any information necessary for post-processing of the data, such as precursor ion estimation.

[0056] Application No. 459 and Patent No. 753 provide a method for identifying one or more precursor ions corresponding to generated ions in scanning SWATH data. However, Application No. 459 and Patent No. 753 do not address reducing the file size required to store scanning SWATH data without losing any information necessary for post-processing of the data.

[0057] In various embodiments, the file size required to store scanning SWATH data is reduced by real-time encoding of the scanning quadrupole dimension based on the quadrupole response function or the estimated probability function of the precursor ion. In other words, instead of storing all the raw detection data collected from each scan of the scanning SWATH for each detected product ion, a sequence of summed counts or intensities and their positions are stored, which describe how each detected product ion varies within the scanning quadrupole dimension or as the transmission window moves along the precursor ion mass range. This significantly reduces the file size required to store scanning SWATH data without losing any information needed for post-processing of the data, such as information about the precursor ion estimated.

[0058] (A system for encoding and storing scanning SWATH data) Figure 2 is a schematic diagram 200 showing a system for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows according to various embodiments. The system in Figure 2 includes an ion source device 210, a mass filter 220, a fragmentation device 230, a mass spectrometer 240, and a processor 250.

[0059] In various embodiments, the system shown in Figure 2 may further include a sample introduction device 260. The sample introduction device 260 introduces, for example, one or more compounds of interest from the sample into the ion source device 210 over time. The sample introduction device 260 can perform techniques including, but are not limited to, injection, liquid chromatography, gas chromatography, capillary electrophoresis, or ion mobility measurement.

[0060] In the system shown in Figure 2, the mass filter 220 and fragmentation device 230 are shown as different stages of a quadrupole, and the mass spectrometer 240 is shown as a time-of-flight (TOF) device. Those skilled in the art will understand that any of these stages may include, but are not limited to, other types of mass spectrometry devices, including ion traps, orbit traps, ion mobility devices, or Fourier transform ion cyclotron resonance (FT-ICR) devices.

[0061] The ion source device 210 converts a sample or a compound of interest from a sample into an ion beam. The ion source device 210 can perform ionization techniques including, but not limited to, matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI).

[0062] The mass filter 220 receives the ion beam. The mass filter 220 then filters the ions by moving a precursor ion transmission window with a precursor ion mass / charge ratio (m / z) width W in overlapping steps over a precursor ion mass range of R m / z with a step size S m / z. A series of overlapping transmission windows are generated over the mass range. The mass filter 220 transmits precursor ions through the transmission window in each overlapping step.

[0063] The fragmentation device 230 of the tandem mass spectrometer 201 fragments or transmits precursor ions transmitted in each overlapping step by the mass filter 220. One or more resulting product ions are generated with respect to each overlapping window in a sequence. The fragmentation device 230 fragments the precursor ions when sufficiently high collision energy is used to fragment the ions. The fragmentation device 230 transmits the precursor ions when insufficient low collision energy is used to fragment the ions. As a result, the resulting product ions may contain precursor ions.

[0064] The mass spectrometer 240 of the tandem mass spectrometer 201 detects the intensity or count for each of the one or more resulting generated ions for each overlapping window in a sequence that forms mass spectral data for each overlapping window in a sequence. If the mass spectrometer 240 is a TOF device, as shown, it detects the count. If the mass spectrometer 240 is, for example, a quadrupole, it detects the intensity.

[0065] The processor 250 can be, but is not limited to, a computer, a microprocessor, the computer system shown in Figure 1, or any device capable of transmitting and receiving control signals and data to and from the tandem mass spectrometer and processing the data. The processor 250 communicates with the ion source device 210, the mass filter 220, the fragmentation device 230, and the mass spectrometer 240. Although shown as a separate device, the processor 250 can be the processor or controller of the tandem mass spectrometer 201 or another device.

[0066] Instead of storing the mass spectral data for each overlapping window in a sequence in a file within a memory device (not shown), the processor 250 performs encoding and storage steps.

[0067] The processor 250 encodes and stores each unique generated ion detected by the mass spectrometer 240 in real time during data acquisition by performing several substeps. First, the processor 250 identifies the overlapping window of the first occurrence of each unique ion in a sequence of occurrences.

[0068] Figure 3 is a schematic diagram 300 illustrating how each unique generated ion detected is encoded in real time during scanning SWATH data acquisition, according to various embodiments. Plot 310 shows that the precursor ion 320 is present at m / z 321 within the precursor ion mass range. A precursor ion permeation window 330 with a certain m / z width W is shifted by a step size S m / z across the mass range to generate a series of overlapping permeation windows. In Figure 3, the first appearance of the unique generated ion 301 occurs, for example, in the overlapping window 331 of the first appearance.

[0069] Returning to Figure 2, the processor 250 then selects a group of G overlapping windows from the sequence immediately preceding the overlapping window of the first occurrence such that the group extends at least to the width W of the transmission window. The number G of overlapping windows in the group is calculated, for example, according to G ≥ W / S. The width W of the transmission window by the group of G overlapping windows is, for example, the precursor ion uncertainty interval. The processor 250 calculates the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group. The processor 250 associates the sum with the position of the overlapping window in the group.

[0070] In Figure 3, for example, a group of G overlapping windows 350 from a sequence immediately preceding the first overlapping window 331 is selected such that group 350 extends at least to a precursor ion uncertainty interval, which is the width W of the transmission window 330. The number of overlapping windows G in group 350 is calculated according to G ≥ W / S, which in this embodiment is 8. The sum of the counts or intensities of the unique generated ions 301 detected from each of the G overlapping windows of group 350 is calculated. The sum 351 calculated for group 350 is plotted in plot 360. The sum 351 is associated with the position of the first overlapping window of group 350 and plotted at the position of the first overlapping window of group 350 in plot 360.

[0071] Returning to Figure 2, the processor 250 shifts the selected group of G overlapping windows forward by one overlapping window. The processor 250 calculates the sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group. The processor 250 associates the sum with the position of the overlapping window in the group. The processor 250 stores the sum and position in a memory device. The processor 250 repeats these steps until at least one overlapping window of the group no longer overlaps with the overlapping window of the first occurrence.

[0072] In Figure 3, for example, group 350 is shifted forward by one overlapping window. The sum of the counts or intensities of the unique generated ions 301 detected from each of the G overlapping windows of group 350 is calculated. The sum is associated with the position of the first overlapping window of group 350. The sum and position are stored in a memory device. These steps are repeated until at least one overlapping window of group 350 no longer overlaps with the overlapping window 331 of the first occurrence. The sum and position are stored, for example, in a file on the memory device.

[0073] Figure 4 is an illustrative schematic diagram 400 showing how, in various embodiments, a set of summed counts or intensities for each unique ion is stored or encoded in real time during scanning SWATH data acquisition. In Figure 4, plot 360 from Figure 3 is shown again. A set 410 of the summed counts or intensities and their positions for the unique generated ions of plot 360 is stored in a memory device. Set 410 is stored, for example, as a sequence 412 of intensity and position (precursor ion m / z) pairs. Since set 410 contains 17 points, a sequence 412 contains 17 intensity and position pairs (I1, M1), (I2, M2), ..., (I 17 M 17 ) includes.

[0074] A comparison of the sets 410 or sequences 412 in Figure 4 with the precursor ion transmission windows 330 in Figure 3 shows that storing the sets 410 or sequences 412 in Figure 4 significantly reduces the memory requirements for each generated ion in a scanning SWATH. Each transmission window 330 in Figure 3 represents the generated ion mass spectrum that previously needed to be stored. In addition, each transmission window may contain the intensities of multiple generated ions. Figure 4 shows that by summing the generated ion intensities and storing only those intensities corresponding to the same precursor ion, the memory requirements for storing generated ions in a scanning SWATH are significantly reduced. In other words, the memory requirements for generated ion data are greatly reduced by using an encoding that shows the interrelationship of generated ion intensities by precursor ion m / z.

[0075] In plot 360 of Figure 3-4, the total count or intensity 351 has a triangular shape. This triangular shape is due to the quadrupole mass filter 220 of Figure 2, which transmits precursor ions with a uniform probability distribution function. Other mass spectrometers and mass filters may produce different shapes, for example. In other words, the shape of the probability distribution function depends on the uncertainty interval of each particular mass spectrometer. In addition, the uncertainty interval depends on the function of the mass filter in a tandem mass spectrometer.

[0076] An ideal tandem mass spectrometer with a mass filter that generates a rectangular precursor ion permeability function using rectangular precursor ion permeability then generates a triangular uncertainty function such as function 370 in plot 360 in Figure 3-4. Function 370 describes how the summed count or intensity of the unique generated ions 301 in Figure 3 varies with the position of group 350. Returning to Figure 4, function 370 in plot 360 can be described as function 420. A mathematical function 422 can then be used to describe function 420. Note that a is the length of the base of the triangle in mathematical function 422, and m is the slope or gradient over the interval of the sides of the triangle. Even more simply, function 420 can be described as the position 422 of the triangle and the width a of its base. Again, ideally, in various embodiments, the generated ions can most easily be encoded as the position and width of the triangular function with respect to this type of mass spectrometer.

[0077] Returning to Figure 2, in the storage step, the processor 250 stores the summed counts or pairs of intensity and location for each unique ion in a file within a memory device (not shown). In various embodiments, before or after storing the pairs, the processor 250 reduces the number of points in the pairs and compresses the data, while still maintaining the same shape of the pairs. Specifically, the processor 250 further reduces the number of stored sums and locations for each unique ion, while still maintaining the shape of the originally stored sums and locations for each unique ion and compressing this data.

[0078] Figure 5 is an exemplary plot 500 showing that, in various embodiments, compressing the summed counts or intensities after encoding still preserves the information necessary to infer the precursor ions. Point 510 represents the summed counts or intensities of the generated ions that remain after the encoded summed counts or intensities have been reduced to compress the data. An ideal triangle 520 is plotted with point 510, showing how the compressed point 510 still maintains its triangular shape. The vertices of the triangle are used, for example, to infer the precursor ions that generated the generated ions.

[0079] However, unfortunately, the encoding process can produce a blurred triangle function, resulting in a stretched base and a distorted triangular shape.

[0080] In various embodiments, the deblurring algorithm is therefore applied to the measured total count or intensity for the generated ions to remove the effects of the precursor ion uncertainty encoding. Returning to Figure 2, the processor 250 further removes the effects of strain (or uncertainty amplification) from the stored total and position of each unique ion by using the deblurring algorithm. The deblurring algorithm makes the stored total and position equal to the convolution of the deblurred total and position with the probability distribution function, which depends on the uncertainty interval of the scanning SWATH transmission function of the mass filter. The deblurring algorithm resolves the deblurred total and position of each unique ion.

[0081] In various embodiments, the deblurred sum and position of each unique ion can be used to "sharpen" the encoded data. In other words, the deblurred sum and position of each unique ion are written to a file in the memory device. Storing the deblurred data results in a smaller file size than writing the encoded data without deblurring. It also allows for storing the same amount of information and, for continuous data analysis, makes spectral and XIC extraction from such a file more accurate and less ambiguous. Specifically, the processor 250 in Figure 2 further stores each unique deblurred sum and position instead of the stored sum and position, reducing the space required in the memory device.

[0082] In various embodiments, the deblurred summed count or intensity can be further encoded as a function. More specifically, in the case of a triangle function, the deblurred summed count or intensity can be further encoded as the triangle's position and base width.

[0083] Deblurring algorithms are well known to those skilled in image processing. Typically, in imaging, blur is caused, for example, by camera vibration. Mathematically, camera vibration can be described as a function. The measured image is then modeled as a convolution of the deblurred image and the camera vibration function. The deblurring algorithm solves this equation for the deblurred image. If the blur in the image is caused by something that cannot be modeled as a function, a blind inverse convolution algorithm can be used.

[0084] In scanning SWATH, the blur, or blur probability distribution function, can be identified and modeled. This depends on the precursor ion transmission function performed by the mass filter. As a result, a blur removal algorithm can be used. Naturally, identifying the blur function makes solving the "blur problem" easier than when the blur function is not identified. However, in various alternative embodiments, blind deconvolution or blur removal without identifying the blur function can also be used.

[0085] In image processing, deblurring algorithms are typically applied in two dimensions (image length and width). In contrast, in various embodiments of scanning SWATH, the deblurring algorithm is applied to only one dimension: the precursor ion transmission window dimension. Applying the deblurring algorithm to only one dimension makes the problem solution more stable, for example. Any process introduces errors. If deconvolution does not exist, for example, in the time dimension, the errors are not introduced from all the uncertainties surrounding that dimension. In other words, understanding the blur function in the precursor ion transmission window dimension ensures that the deblurring error is minimized.

[0086] Scanning SWATH can generate, for example, four-dimensional data. The dependent dimension is the generated ion count or intensity. The three independent dimensions can include the generated ion m / z dimension, the precursor ion transmission window position dimension, and the chromatograph or separation time dimension. Generally, there is little uncertainty in the measurement of the generated ion m / z dimension.

[0087] The blur, probability distribution function, or uncertainty at the precursor ion position in the precursor dimension is independent of the compound being analyzed. Instead, it depends on the instrument or mass spectrometer and the method used. As a result, blur removal algorithms can be applied to this dimension.

[0088] However, blurring or uncertainty in the chromatographic or separation time dimension varies depending on the compound being analyzed. In other words, blurring in the chromatographic time dimension depends not only on the column used but also on the compound being analyzed. Consequently, deblurring algorithms are not applicable in the chromatographic or separation time dimension.

[0089] Figure 6 is an exemplary heatmap plot 600 showing the total generated ion counts plotted as a function of precursor ion permeation window position and generated ion m / z before applying the deblurring algorithm to the data according to various embodiments. Inset 610 shows that two different generated ions 611 and 612 have similar generated ion m / z values. It also shows that the uncertainty interval or triangular probability distribution function of these two generated ions in the precursor ion permeation window position dimension is highly overlapping.

[0090] Typically, the product ion spectrum for a particular precursor ion permeation window position is found by drawing a horizontal line, such as line 620, through plot 600. Inset 610 shows that the spectrum of line 620 contains both product ion 611 and product ion 612. In other words, due to a large overlap in the uncertainty interval between the two product ions, both product ions will be included with respect to the precursor ion permeation window position. Thus, due to this large overlap or blurring, both product ions will be found as product ions of a particular precursor ion, even if they are not both from the same precursor ion.

[0091] In various embodiments, deblurring algorithms are applied to the data in Figure 6. In particular, the Lucy-Richardson deblurring algorithm is applied in the precursor ion transmission window position dimension. The measured summed counts and their positions in the precursor ion transmission window position dimension are equal to the convolution of the removed summed counts and positions with respect to the uncertainty interval of the mass filter and the triangular probability distribution function. The deblurring algorithm solves this equation with respect to the deblurred summed counts and positions. Note that some deblurring algorithms require the first derivative at all points.

[0092] Figure 7 is an exemplary heatmap plot 700 showing the total generated ion counts plotted as a function of precursor ion permeation window position and generated ion m / z after applying the deblurring algorithm of Figure 6 to the data according to various embodiments. Inset 710 shows that two different generated ions 611 and 612 still have similar generated ion m / z values ​​after deblurring. However, the uncertainty interval or triangular probability distribution function of these two generated ions in the precursor ion permeation window position dimension shows much less overlap after deblurring. Essentially, the width of the base of the triangular probability distribution function of these two generated ions is greatly reduced.

[0093] This reduction in the triangular probability distribution function makes it easier to distinguish between the precursor ion and the generated ion. Inset 710 shows that, after deblurring, the spectrum of line 620 no longer contains both generated ion 611 and generated ion 612. In other words, due to the reduction in overlap in the uncertainty interval between the two generated ions, only one generated ion will be included due to the precursor ion transmission window position.

[0094] In various embodiments, after deblurring, the probability distribution function in the position dimension of the precursor ion permeation window can be stored as an encoding for each generated ion. For example, the positions and base widths of the triangles of generated ions 611 and 612 in Figure 7 can be stored in a memory device instead of the measured summation counts. In other words, storing only the positions and base widths of the triangle functions, such as function 420 in Figure 4, rather than the points of function 420, further reduces the memory requirements.

[0095] In various embodiments, a probability distribution function found after deblurring for each unique generated ion, or a measured set of summed counts or intensities, is read from a memory device. Both types of data are read, for example, from a file. A numerical decomposition method or a probabilistic inference method is then applied to the read data to determine the precursor ions of the unique generated ions. For example, with respect to a triangular function, the precursor ions of the unique generated ions are found at the vertices of the triangular function.

[0096] More specifically, returning to Figure 2, in various embodiments, the processor 250 uses the deblended sum and position of each unique ion to further determine the precursor ion of each unique ion. For example, the deblended sum and position of each unique ion have a triangular shape. The processor 250 further determines the precursor ion of each unique ion as the precursor ion corresponding to the vertices of the triangular shape.

[0097] Blur removal in the precursor ion transmission window position dimension also improves peak discovery in the chromatographic or separation time dimension. However, as described above, blur removal is not applied in the chromatographic or separation time dimension.

[0098] Figure 8 is an exemplary schematic diagram 800, which includes heatmap plots showing the total generated ion counts plotted as a function of chromatograph time and precursor ion permeation window position before applying the deblurring algorithm to the data according to various embodiments, and a plot showing the XIC found from the heatmap with respect to the precursor ion m / z value. Heatmap 810 shows the generated ion intensity regions at times 811 and 812. Before applying the deblurring algorithm, both intensity regions have a large width in the precursor ion permeation window position dimension.

[0099] As a result, with respect to a specific precursor ion permeation window position (precursor ion m / z value) represented by line 815, both intensity regions can be detected over time. In other words, due to the large range of uncertainty in the generated ions in the precursor ion permeation window position dimension, two different generated ions can be detected over time with respect to a specific precursor ion permeation window position.

[0100] XIC821 is shown in plot 820. XIC821 represents the intensity over time along line 815 in heatmap 810. XIC821 contains two distinct peaks that identify two different generated ions with respect to the precursor ion permeation window position before blur removal.

[0101] Figure 9 is an exemplary schematic diagram 900, which includes heatmap plots showing the total generated ion counts plotted as a function of chromatograph time and precursor ion permeation window position after applying the deblurring algorithm of Figure 8 to the data according to various embodiments, and a plot showing the XIC found from the heatmap with respect to the precursor ion m / z value. Heatmap 910 again shows the generated ion intensity regions at times 811 and 812. After applying the deblurring algorithm, both intensity regions have a narrower width in the precursor ion permeation window position dimension than before deblurring.

[0102] Here, for a specific precursor ion permeation window position (precursor ion m / z value) represented by line 815, only one intensity region can be detected over time. In other words, due to a narrower width of product ion uncertainty in the precursor ion permeation window position dimension, only one product ion is detected over time for a specific precursor ion permeation window position.

[0103] XIC921 is shown in plot 920. XIC921 represents the intensity over time along line 815 in heatmap 910. XIC921 contains a single peak that identifies only one generated ion at the precursor ion transmission window position of line 815 after blur removal.

[0104] In various embodiments, instead of using a deblurring algorithm, a numerical method may be applied to a sum of measured counts or intensities with respect to the generated ions to determine the precursor ions of the generated ions. U.S. Patent No. 10,651,019 (hereinafter, "Patent No. '019") discloses a method for determining the precursor ions of generated ions from scanning SWATH data, which is incorporated herein by reference as a whole. In Patent No. '019, the intensity of a selected generated ion is read from multiple generated ion spectra obtained from each scan of the precursor ion permeation window over the precursor ion mass range. A trace is generated that illustrates how the intensity of the selected generated ion changes with respect to the precursor ion permeation window.

[0105] A matrix multiplication equation is constructed that describes how one or more precursor ions correspond to traces with respect to selected product ions. The matrix multiplication equation includes a known n × m mass filter matrix multiplied by an unknown precursor ion column matrix of length m, which corresponds to a selected ion trace column matrix of length n. The matrix multiplication equation is solved for the unknown precursor ion column matrix, generally using numerical methods such as non-negative matrix factorization (NNMF), or in particular non-negative least squares (NNLS).

[0106] In various embodiments, a summed set of counts or intensities is used for a column matrix of length n. The matrix multiplication equation is solved with respect to the unknown precursor ion column matrix using a numerical method. As a result, the precursor ions of the generated ions are found.

[0107] More specifically, returning to Figure 2, processor 250 uses a numerical method to further determine the precursor ions for each unique ion. The numerical method arranges the stored sums and positions as a column matrix of length n. The numerical method equals a known n × m mass filter matrix for a mass filter, which is multiplied by an unknown precursor ion column matrix of length m. Finally, the numerical method resolves the unknown precursor ion column matrix to determine the precursor ions. The numerical method may include, but is not limited to, NNMF or NNLS.

[0108] (Method for encoding and storing scanning SWATH data) Figure 10 is a flowchart illustrating a method 1000 for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, according to various embodiments.

[0109] In step 1010 of method 1000, a precursor ion permeation window with a precursor ion mass / charge ratio (m / z) width W is moved in overlapping steps over a precursor ion mass range at a step size S m / z using a mass filter of a tandem mass spectrometer. A series of overlapping permeation windows over the mass range is generated. The mass filter transmits precursor ions through the permeation window in each overlapping step.

[0110] In step 1020, the precursor ions transmitted in each overlapping step by the mass filter are fragmented or transmitted using the fragmentation device of the tandem mass spectrometer. One or more resulting product ions or precursor ions are generated with respect to each overlapping window in the sequence.

[0111] In step 1030, the intensity or count is detected with respect to one or more resulting generated ions or precursor ions with respect to each overlapping window in a sequence, forming mass spectral data with respect to each overlapping window in a sequence.

[0112] In step 1040, each unique generated ion detected by the mass spectrometer is encoded in real time during data acquisition using a processor, according to steps 1050, 1060, and 1070.

[0113] In step 1050, the overlapping windows of the first appearances in a sequence, each involving the first appearance of a unique ion, are identified.

[0114] In step 1060, a group of G overlapping windows from the sequence immediately preceding the first overlapping window is selected such that the group extends at least to the width W of the transmission window. The sum of the counts or intensities of each unique ion detected from each of the G overlapping windows in the group is calculated and associated with the position of the overlapping window in the group.

[0115] In step 1070, the selected group of G overlapping windows is shifted forward by the width of one overlapping window, the sum of the unique ion counts or intensities detected from each of the G overlapping windows of the group is calculated, the sum is associated with the position of the overlapping window of the group, and the sum and position are stored in a memory device, and these steps are repeated until at least one of the overlapping windows of the group no longer overlaps with the overlapping window of the first occurrence.

[0116] (A computer program product for encoding and storing scanning SWATH data) In various embodiments, a computer program product includes a tangible computer-readable storage medium, the contents of which include a program with instructions executed on a processor to perform a method of encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows. This method is performed by a system comprising one or more different software modules.

[0117] Figure 11 is a schematic diagram of a system 1100 that includes one or more different software modules that implement a method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion permeation windows, according to various embodiments. The system 1100 includes a control module 1110 and an encoding and storage module 1120.

[0118] The control module 1110 instructs the mass filter of the tandem mass spectrometer to move a precursor ion permeation window with a precursor ion mass / charge ratio (m / z) width W in overlapping steps over a precursor ion mass range with a step size S m / z. A series of overlapping permeation windows over the mass range is generated. The mass filter transmits precursor ions within the permeation window in each overlapping step.

[0119] The control module 1110 instructs the fragmentation device of the tandem mass spectrometer to fragment or transmit precursor ions transmitted by the mass filter in each overlapping step. One or more resulting generated ions or precursor ions are produced with respect to each overlapping window in a sequence.

[0120] The control module 1110 instructs the mass spectrometer of the tandem mass spectrometer to detect the intensity or count of one or more resulting generated ions or each precursor ion for each overlapping window in a sequence that forms mass spectral data for each overlapping window in the sequence.

[0121] The encoding and storage module 1120 encodes each unique generated ion detected by the mass spectrometer in real time during data acquisition. The overlapping window of the first occurrence of each unique ion in a sequence is identified. A group of G overlapping windows in a sequence immediately preceding the overlapping window of the first occurrence is selected such that the group extends at least to the width W of the transmission window, and the sum of the counts or intensities of the unique ions detected from each of the G overlapping windows of the group is calculated and associated with the position of the overlapping window in the group.

[0122] A selected group of G overlapping windows is shifted forward by the width of one overlapping window. The sum of the unique ion counts or intensities detected from each of the G overlapping windows in the group is calculated, and the sum is associated with the position of the overlapping window in the group. The sum and position are stored in a memory device. These steps are repeated until at least one of the overlapping windows in the group no longer overlaps with the overlapping window of the first occurrence.

[0123] This instruction will be described in conjunction with various embodiments, but it is not intended to be limited to such embodiments. In contrast, this instruction includes various alternatives, modifications, and equivalents, as will be understood by those skilled in the art.

[0124] Furthermore, in the description of various embodiments, this specification may present methods and / or processes as a specific sequence of steps. However, to the extent that a method or process does not depend on a specific order of steps described herein, the method or process should not be limited to a specific sequence of steps described. Other sequences of steps may also be possible, as those skilled in the art will understand. Therefore, a specific order of steps described herein should not be construed as a limitation on the claims. In addition, claims relating to methods and / or processes should not be limited to the execution of those steps in the written order, and those skilled in the art will readily understand that the sequence may be varied and still remain within the spirit and scope of various embodiments.

Claims

1. A system for encoding and storing mass spectral data obtained using data-independent acquisition, wherein the system is Processor and A non-transient memory that communicates with the processor, wherein the non-transient memory stores instructions, and when an instruction is executed, it communicates to the processor, To obtain the mass spectral data including the count or intensity of one or more generated ions within each window of a continuous permeable ion window, To detect unique generated ions in the aforementioned mass spectral data, Identifying one or more windows in the sequence from which the unique generated ions appear, For each of the one or more windows mentioned above, record the location of the window and the total count or intensity for a group of windows that extend to that window. This executes using non-transient memory and Equipped with, With respect to each window in the group of windows, the operation of recording the sum of the count or intensity and the position is as follows: Record the position of the first window among the one or more windows, Selecting a group of windows that start from the first window and extend sequentially forward, Record the total count or intensity for the group of windows in relation to the aforementioned position. Includes, The mass spectral data is obtained using scanning SWAT (Sequential Window Acquisition of All Theoretical Mass Spectra), where the number of windows is at least equal to the width divided by the step size.

2. The system according to claim 1, wherein encoding the mass spectral data occurs in real time as the mass spectral data is acquired.

3. The system according to claim 2, wherein encoding the mass spectral data occurs in real time as the mass spectral data is obtained by the mass spectrometer.

4. The system according to claim 1, wherein the one or more windows include a first appearance window, and the first appearance window is one of the one or more windows in which the unique generated ion is first identified.

5. With respect to each window in the group of windows, the operation of recording the sum of the count or intensity and the position is as follows: To record the total and position of the unique generated ions with respect to the first group preceding the first appearance window, Moving at least one window forward from the aforementioned group of windows, To record the total and position of the unique generated ions with respect to the second group including the first appearance window. The system according to claim 4, including the system described in claim 4.

6. The operation of moving at least one window forward from the aforementioned group of windows, The operation of recording the total and position for subsequent groups and The system according to claim 5, further comprising repeating the step.

7. The mass spectral data is obtained using a scanning SWAT (Sequential Window Acquisition of All Theoretical Mass Spectra), and the operation of moving the window of one group forward by at least one window and recording the sum and position for the subsequent group is repeated until at least one window of the subsequent group no longer overlaps with the first appearance window, according to claim 6.

8. A method for encoding and storing a scanning SWAT data file, wherein the method is: Encoding the scanning ion transmission window dimension of mass spectral data, wherein the encoding includes, with respect to a unique generated ion, the position of the window in which the unique generated ion appears, and the sum of counts or intensities for a group of windows extending to the window in which the unique generated ion appears. To store the aforementioned position and the aforementioned total. Includes, Each total and position is stored as an ordered pair, and the instruction further tells the processor, Identifying each ordered pair associated with the unique generated ion as a set, The shape of the set is determined based on the distribution of the sum of counts or intensities across the location of the unique generated ions, By preserving the peak position and base width of the distribution, the number of ordered pairs within the set is reduced while maintaining the shape of the set. A method to make it happen.

9. A method for encoding and storing a scanning SWAT data file, wherein the method is: Encoding the scanning ion transmission window dimension of mass spectral data, wherein the encoding includes, with respect to a unique generated ion, the position of the window in which the unique generated ion appears, and the sum of counts or intensities for a group of windows extending to the window in which the unique generated ion appears. To store the aforementioned position and the aforementioned total. Includes, Each total and position is stored as an ordered pair, and the instruction further tells the processor, A method for removing the blurring of each sum and position before storing the ordered pairs.

10. The aforementioned instruction further instructs the processor, The method according to claim 9, wherein the sum is encoded as a function.

11. The method according to claim 10, wherein the function is a triangular function, and the encoding includes the width and position of the base of the triangular function.

12. The method according to claim 9, wherein each sum and position is blurred only in the ion transmission window dimension.

13. A method for identifying precursor ions of generated ions in data-independent acquisition mass spectrometry experiments, To obtain mass spectral data including the position of the ion permeable window where the generated ions appear and the total count or intensity of the generated ions with respect to a group of ion permeable windows extending to the ion permeable window at that position, The sum is arranged as a matrix of generated ions of length m, Obtaining a known n×m mass filter matrix, The method involves applying a numerical method to a matrix multiplication formula to determine the precursor ions of the generated ions, with respect to a precursor ion matrix of length n, wherein the matrix multiplication formula includes multiplying the known n × m mass filter matrix by the precursor ion matrix, thereby generating the generated ion matrix. Methods that include...

14. The method according to claim 13, wherein the numerical method includes non-negative matrix factorization (NNMF).

15. The method according to claim 14, wherein the numerical method includes non-negative least squares (NNLS).

16. The method according to claim 13, wherein the known n × m mass filter matrix is ​​determined according to the movement of the ion permeation window during the data-independent acquired mass spectrometry experiment.

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