Method for real-time encoding of scanning swath data and probabilistic framework for precursor inference

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 Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 855,242, filed May 31, 2019, the contents of which are incorporated by reference herein in their entirety.

[0002] The teachings herein relate to encoding and storing scanning SWATH mass spectrometry data. 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 precursor ion guess probability function. [Background technology]

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

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

[0005] While file storage itself is constantly becoming smaller and cheaper, the cost of post-processing such large files is large in terms of the processing time required and the processing power required. The scanned SWATH data stored in files is post-processed, for example, to infer precursor ions of the measured product ions. A number of numerical decomposition or probabilistic inference methods are available that can be applied to the scanned SWATH data.

[0006] Conventionally, for a scanning SWATH experiment, the raw mass analyzer detection values ​​(e.g., time-of-flight (TOF) mass analyzer counts) for each product ion generated from each overlapping precursor ion transmission window are stored in a file. Alternatively, intermediate types of data can also be used. For example, each product ion spectrum for each overlapping precursor ion transmission window can be stored in a file. Unfortunately, in both methods, the storage size is still at least about n times larger, where n is the number of overlapping precursor ion transmission windows, than the storage size required for conventional SWATH to store the same type of data.

[0007] As a result, additional systems and methods are needed to reduce the file size required to store scanned SWATH data without losing any information required for post-processing of the data for information such as precursor ion guesses. (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 ionizing one or more compounds from a sample, selecting one or more precursor ions of the one or more compounds, fragmenting the one or more precursor ions into product ions, and mass analyzing the product ions.

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

[0010] Many different types of experimental methods or workflows can be performed using tandem mass spectrometers. Three broad categories of these workflows are 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 precursor ions to product ions are predefined for a compound of interest. When a sample is introduced into a tandem mass spectrometer, the one or more transitions are investigated during each period or cycle of a plurality of periods or cycles. In other words, the mass spectrometer selects and fragments the precursor ions of each transition, and performs targeted mass analysis on the product ions 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 selected reaction monitoring (SRM).

[0012] In the IDA method, a user can define criteria for performing targeted or non-targeted mass analysis of product ions while the sample is being 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 for a portion of the precursor ions on the peak list. MS / MS is then performed on each precursor ion of the portion of precursor ions. A product ion spectrum is generated for each precursor ion. MS / MS is repeatedly performed on precursor ions of the portion of precursor ions as the sample is being introduced into the tandem mass spectrometer.

[0013] However, in proteomics and many other sample types, the compound complexity and dynamic range is very large, which challenges traditional targeted and IDA methods and requires very fast MS / MS acquisition to deeply interrogate the sample to both identify and quantify a wide range of analytes.

[0014] As a result, a third broad category of tandem mass spectrometry, DIA methods, was developed. These DIA methods were used to increase the reproducibility and comprehensiveness of data collection from complex samples. DIA methods can also be called non-specific fragmentation methods. In traditional DIA methods, the action of the tandem mass spectrometer is not varied between MS / MS scans based on data acquired in previous precursor or product ion scans. Instead, a precursor ion mass range is selected. Then, the precursor ion transmission window is shifted across the precursor ion mass range. All precursor ions within the precursor ion transmission window are fragmented, and all product ions of the precursor ions within the precursor ion transmission window are mass analyzed.

[0015] The precursor ion transmission window used to scan the mass range can be very narrow so that the likelihood of multiple precursors within the window is small. This type of DIA method is also useful for, for example, MS / MS ALL It is called MS / MS. ALL In the method, a precursor ion transmission window of about 1 amu is scanned or shifted across the mass range. A product ion spectrum is generated for each 1 amu precursor mass window. A product ion spectrum for the entire precursor ion mass range is generated by combining the product ion spectra for each mass selection window. The time it takes to analyze or scan the entire mass range once is referred to as one scan cycle. However, scanning a narrow precursor ion transmission window across a wide precursor ion mass range during each cycle is impractical for some instruments and experiments.

[0016] As a result, a larger precursor ion transmission window or a selection window with a larger width 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 transmission window that is shifted across the precursor mass range in each cycle can have a width of 5 to 25 amu or even larger. MS / MS ALL As in the previous method, all precursor ions within each precursor ion transmission window are fragmented and all product ions of all precursor ions within each mass selection window are mass analyzed. However, because a wider precursor ion transmission window is used, the cycle time is longer than in the MS / MS ALL The cycle time of the method can be significantly shortened compared to the cycle time of the method. Or, for liquid chromatography (LC), the accumulation time can be increased. Generally, for LC, the cycle time is defined by the LC peak. Enough 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 a cycle defines the length of time that each experiment or scan can accumulate ion observations. As a result, a wider precursor ion transmission window can increase the accumulation time.

[0017] US Patent No. 8,809,770 describes how SWATH acquisition can be used to provide quantitative and qualitative information about precursor ions of a compound of interest. In particular, product ions found from fragmenting the precursor ion transmission window are compared to a database of known product ions of the compound of interest. In addition, ion traces or extracted ion chromatograms (XICs) of product ions found from fragmenting the precursor ion transmission window are analyzed to provide quantitative and qualitative information.

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

[0019] As a result, a method of scanning the precursor ion transmission window in a SWATH acquisition, called Scanning SWATH, was developed. Essentially, in Scanning SWATH, the precursor ion transmission window is scanned across the mass range such that successive windows have large areas of overlap and small areas of non-overlapping. This scanning makes the resulting product ions a function of the scanned precursor ion transmission window. This additional information can then be used to identify one or more precursor ions associated with each product ion.

[0020] Scanning SWATH is described in International Publication WO 2013 / 171459 A2 (hereinafter "the '459 Application"). In the '459 Application, a precursor ion transmission window, or a precursor ion transmission window of 25 Da, is scanned over time such that the extent of the precursor ion transmission window changes over time. The timing at which product ions are detected then correlates to the timing of the precursor ion transmission window through which those precursor ions were transmitted.

[0021] The correlation is performed by first plotting the mass-to-charge ratio (m / z) of each detected product ion as a function of the precursor ion m / z value transmitted by the quadrupole mass filter. Because the precursor ion transmission window is scanned over time, the precursor ion m / z values ​​transmitted by the quadrupole mass filter can also be thought of as times. The start and end times at which a particular product ion is detected correlate to the start and end times at which that precursor is transmitted from the quadrupole. As a result, the start and end times of the product 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. Pat. No. 10,068,753 (hereinafter "the '753 patent"). The '753 patent improves the accuracy of the correlation of product ions to their corresponding precursor ions by combining product ion spectra from successive groups of overlapping precursor ion transmission windows. Product ion spectra from successive groups are combined by successively summing the intensities of the product ions in the product ion spectra. This sum produces a function that may have a non-constant shape with precursor mass. The shape represents product ion intensity as a function of precursor mass. Precursor ions are identified from the function calculated for the product ions.

[0023] For scanning SWATH, it is preferred to have a rectangular precursor ion transmission window. However, another advantage of scanning SWATH is that it can equally and efficiently handle any ion transmission function. The ion transmission function does not even need to be known in detail beforehand, but can be calibrated from the data itself. In other words, although a rectangular precursor ion transmission window is preferred, a precursor ion transmission window of any shape can be used.

[0024] The '459 application and the '753 patent provide methods for identifying one or more precursor ions that correspond to product ions in scanned SWATH data, but the '459 application and the '753 patent do not address reducing the file size required to store the scanned SWATH data without losing any information required for post-processing of the data. [Prior art documents] [Patent documents]

[0025] [Patent Document 1] U.S. Patent No. 8,809,770 [Patent Document 2] International Publication No. 2013 / 171459 [Patent Document 3] U.S. Patent No. 10,068,753 Summary of the Invention [Means for solving the problem]

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

[0027] An ion source device converts the sample or compounds of interest from the sample into an ion beam. A 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-to-charge ratio (m / z) width W in overlapping steps across a precursor ion mass range of R m / z with a step size S m / z. A series of overlapping transmission windows are created across the mass range. The mass filter transmits precursor ions within the transmission window in each overlapping step.

[0028] A fragmentation device fragments or transmits precursor ions transmitted by the mass filter in each overlapping step. One or more resultant product ions are generated for each overlapping window in the series. A mass analyzer detects intensities or counts for each of the one or more resultant product ions for each overlapping window in the series, the intensities or counts forming mass spectral data for each overlapping window in the series.

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

[0030] The processor encodes and stores each unique product ion detected by the mass analyzer in real time during data acquisition by performing several substeps. First, the processor identifies the overlapping window of the first occurrence of the sequence with the first occurrence of each unique ion. The processor then selects a group of G overlapping windows in the sequence immediately preceding the overlapping window of the first occurrence such that the group spans at least a 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 of the group of G overlapping windows is, for example, the precursor ion uncertainty interval.

[0031] In various embodiments, the precursor ion likelihood may vary over the uncertainty interval W. For convenience, it is considered 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 the 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 a 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 sum with the position of the overlapping window of the group. The processor shifts the group of G overlapping windows of the selected series forward by one overlapping window, calculates a sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group, associates the sum with the position of the overlapping window of the group, stores the sum and the position in a memory device, and repeats these steps until at least one overlapping window of the group no longer overlaps with the overlapping window of the first occurrence.

[0033] These and other features of the applicants' teachings are set forth herein. The present specification also provides, for example, the following items: (Item 1) 1. A system for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows, the system comprising: A mass filter for a tandem mass spectrometer, the mass filter having a step size S a mass filter moving a precursor ion transmission window with a precursor ion mass / charge ratio (m / z) width W across a precursor ion mass range with m / z in overlapping steps to generate a series of overlapping transmission windows across the mass range, the mass filter transmitting precursor ions within the transmission window in each overlapping step; a fragmentation device of the spectrometer, the fragmentation device fragmenting or transmitting the precursor ions transmitted by the mass filter in each overlapping step to generate one or more resultant product ions for each overlapping window in the series; a mass analyzer of the spectrometer, the mass analyzer detecting an intensity or count for each of the one or more resultant product ions for each overlapping window in the series, the intensities or counts forming mass spectral data for each overlapping window in the series; a processor in communication with the mass spectrometer; Equipped with the processor encodes each unique product ion detected by the mass analyzer in real time during data acquisition instead of storing the mass spectral data for each overlapping window in the series in a memory device; The encoding step comprises: identifying an overlapping window of a first occurrence of said sequence with a first occurrence of each of said unique ions; selecting a group of G overlapping windows in said series immediately preceding the overlapping window of said first occurrence such that said group spans at least a width W of said transmission window, calculating a sum of counts or intensities of each unique ion detected from each window of said G overlapping windows in said group, and relating said sum to the position of the overlapping window in said group; shifting a group of G overlapping windows of said selected series forward by one overlapping window, calculating a sum of counts or intensities of each unique ion detected from each of said G overlapping windows of said group, relating said sum to a position of an overlapping window of said group, storing said sum and said position in said memory device, and repeating these steps until at least one overlapping window of said group no longer overlaps with an overlapping window of said first occurrence. The system performs the above steps. (Item 2) 2. The system of claim 1, wherein the processor further compresses this data by reducing the number of stored sums and positions for each unique ion while still maintaining the shape of the original stored sums and positions for each unique ion. (Item 3) 2. The system of claim 1, wherein the processor further removes effects caused by encoding from the stored sum and position of each unique ion by using a deblurring algorithm, the deblurring algorithm resolving for each unique ion's deblurred sum and position a convolution of the deblurred sum and position with a probability distribution function that depends on the mass filter uncertainty interval as equal to the stored sum and position. (Item 4) 4. The system of claim 3, wherein the processor further determines a precursor ion for each unique ion using the deblurred sum and position of each unique ion. (Item 5) 4. The system of claim 3, wherein the processor further stores a deblurred sum and location of each unique ion in place of the stored sum and location, reducing space required in the memory device. (Item 6) 2. The system of claim 1, wherein the stored sums and positions of each unique ion have a triangular shape. (Item 7) 5. The system of claim 4, wherein the processor further determines a precursor ion for each unique ion as a precursor ion corresponding to a vertex of the triangular shape. (Item 8) 2. The system of claim 1, wherein the processor further determines a precursor ion for each unique ion using a numerical method, the numerical method arranging the stored sums and positions as a column matrix of length n, and solving for the unknown precursor ion column matrix as equal to a known n×m mass filter matrix for the mass filter multiplied by an unknown precursor ion column matrix of length m to determine the precursor ion. (Item 9) 7. The system of claim 6, wherein the numerical method comprises non-negative matrix factorization (NNMF). (Item 10) 7. The system of claim 6, wherein the numerical method comprises non-negative least squares (NNLS). (Item 11) 2. The system of claim 1, wherein the processor further compresses the stored sums and locations for each unique ion by removing some sums and locations while still maintaining the same shape of the stored sums and locations. (Item 12) 2. The system of claim 1, wherein the number G of overlapping windows in the group is calculated according to G≧W / S. (Item 13) 2. The system of claim 1, wherein a width W of the transmission window of the group of G overlapping windows is the precursor ion uncertainty interval. (Item 14) 1. A method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows, the method comprising: using a mass filter of a tandem mass spectrometer to move a precursor ion transmission window with a precursor ion mass / charge ratio (m / z) width W across a precursor ion mass range with a step size S m / z in overlapping steps to generate a series of overlapping transmission windows across the mass range, the mass filter transmitting precursor ions within the transmission window in each overlapping step; using a fragmentation device of the spectrometer to fragment or transmit the precursor ions transmitted by the mass filter in each overlapping step to produce one or more resultant product ions for each overlapping window in the series; detecting, using a mass analyzer of the spectrometer, an intensity or count for each of the one or more resultant product ions for each overlapping window in the series, the intensities or counts forming mass spectral data for each overlapping window in the series; using a processor to encode each unique product ion detected by said mass analyzer in real time during data acquisition; Including, The encoding step comprises: identifying an overlapping window of a first occurrence of said sequence with a first occurrence of each of said unique ions; selecting a group of G overlapping windows in said series immediately preceding the overlapping window of said first occurrence such that said group spans at least a width W of said transmission window, calculating a sum of counts or intensities of each unique ion detected from each window of said G overlapping windows in said group, and relating said sum to the position of the overlapping window in said group; shifting a group of G overlapping windows of said selected series forward by one overlapping window, calculating a sum of counts or intensities of each unique ion detected from each of said G overlapping windows of said group, relating said sum to a position of an overlapping window of said group, storing said sum and said position in said memory device, and repeating these steps until at least one overlapping window of said group no longer overlaps with an overlapping window of said first occurrence. A method for performing the above. (Item 15) A computer program product comprising a non-transitory tangible computer readable storage medium, the contents of which include a program with instructions for execution on a processor to perform a method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows, the method comprising: providing a system comprising one or more distinct software modules, the distinct software modules comprising a control module and an encoding and storage module; instructing a mass filter of a tandem mass spectrometer to move a precursor ion transmission window with a precursor ion mass-to-charge ratio (m / z) width W across a precursor ion mass range with a step size S m / z in overlapping steps to generate a series of overlapping transmission windows across the mass range, the mass filter transmitting precursor ions within the transmission window in each overlapping step; instructing a fragmentation device of the spectrometer using the control module to fragment or transmit the precursor ions transmitted by the mass filter in each overlapping step to generate one or more resultant product ions for each overlapping window in the series; instructing a mass analyzer of the spectrometer using the control module to detect an intensity or count for each of the one or more resultant product ions for each overlapping window in the series, the intensity or count forming mass spectral data for each overlapping window in the series; using said encoding and storage module to encode each unique product ion detected by said mass analyzer in real time during data acquisition; Including, The encoding step comprises: identifying an overlapping window of a first occurrence of said sequence with a first occurrence of each of said unique ions; selecting a group of G overlapping windows in said series immediately preceding the overlapping window of said first occurrence such that said group spans at least a width W of said transmission window, calculating a sum of counts or intensities of each unique ion detected from each window of said G overlapping windows in said group, and relating said sum to the position of the overlapping window in said group; shifting a group of G overlapping windows of said selected series forward by one overlapping window, calculating a sum of counts or intensities of each unique ion detected from each of said G overlapping windows of said group, relating said sum to a position of an overlapping window of said group, storing said sum and said position in said memory device, and repeating these steps until at least one overlapping window of said group no longer overlaps with an overlapping window of said first occurrence. A computer program product comprising: [Brief description of the drawings]

[0034] 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.

[0035] [Figure 1] FIG. 1 is a block diagram illustrating a computer system on which embodiments of the present teachings may be implemented.

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

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

[0038] [Figure 4] FIG. 4 is an exemplary diagram illustrating how the set of summed counts or intensities for each unique ion are stored or encoded in real time during scanning SWATH data acquisition, according to various embodiments.

[0039] [Diagram 5] FIG. 5 is an example plot showing that compression of the summed counts or intensities after encoding still preserves the information necessary to infer precursor ions, according to various embodiments.

[0040] [Figure 6] FIG. 6 is an exemplary heat map plot showing summed product ion counts plotted as a function of precursor ion transmission window position and product ion m / z before applying a deblurring algorithm to the data in accordance with various embodiments.

[0041] [Figure 7] FIG. 7 is an exemplary heat map plot showing summed product ion counts plotted as a function of precursor ion transmission window position and product ion m / z after applying the deblurring algorithm of FIG. 6 to the data in accordance with various embodiments.

[0042] [Figure 8] FIG. 8 is an exemplary diagram including a heat map plot showing summed product ion counts plotted as a function of chromatographic time and precursor ion transmission window position before applying a deblurring algorithm to the data, and a plot showing the XIC found from the heat map for precursor ion m / z values, in accordance with various embodiments.

[0043] [Figure 9] FIG. 9 is an exemplary diagram including a heat map plot showing summed product ion counts plotted as a function of chromatographic time and precursor ion transmission window position after applying the deblurring algorithm of FIG. 8 to the data, according to various embodiments, and a plot showing the XIC found from the heat map for precursor ion m / z values.

[0044] [Figure 10] FIG. 10 is a flow chart illustrating a method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows according to various embodiments.

[0045] [Figure 11] FIG. 11 is a schematic diagram of a system including one or more different software modules implementing a method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows, according to various embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Before one or more embodiments of the present teachings are described 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. It is also to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0047] (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.

[0048] 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.

[0049] 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.

[0050] The term "computer readable medium" as used herein refers to any medium that participates in providing instructions to the processor 104 for execution. Such media may take many forms including, but not limited to, non-volatile media, volatile media, and precursor ion mass selection media. Non-volatile media include, for example, optical or magnetic disks, such as the storage device 110. Volatile media include dynamic memory, such as the memory 106. Precursor ion mass selection media include coaxial cables, copper wire, and fiber optics, including the wiring that comprises the bus 102.

[0051] 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.

[0052] 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, and the processor 104 reads the instructions from memory 106 and executes them. The instructions received by memory 106 may optionally be stored on a storage device 110 either before or after execution by the processor 104.

[0053] According to various embodiments, the instructions configured to be executed by the 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 disc 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.

[0054] 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 form 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.

[0055] (Scanning SWATH Data Encoding and Storage) As explained above, scanning SWATH is a tandem mass spectrometry method in which a precursor ion transmission window is scanned across a mass range such that successive windows have large areas of overlap and small areas of non-overlapping. One problem with scanning SWATH is that it requires long-term (e.g., file) storage of significantly more data than traditional 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 required for post-processing of the data for information such as precursor ion predictions.

[0056] The '459 application and the '753 patent provide methods for identifying one or more precursor ions that correspond to product ions in scanned SWATH data, but the '459 application and the '753 patent do not address reducing the file size required to store the scanned SWATH data without losing any information required for post-processing of the data.

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

[0058] (SYSTEM FOR ENCODING AND STORING SCANNED SWATH DATA) 2 is a schematic diagram 200 illustrating a system for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows, according to various embodiments. The system of FIG. 2 includes an ion source device 210, a mass filter 220, a fragmentation device 230, a mass analyzer 240, and a processor 250.

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

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

[0061] The ion source device 210 converts the sample or compounds of interest from the 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-to-charge ratio (m / z) width W in overlapping steps across a precursor ion mass range of R m / z with a step size S m / z. A series of overlapping transmission windows is created across the mass range. The mass filter 220 transmits precursor ions within the transmission window at each overlapping step.

[0063] The fragmentation device 230 of the tandem mass spectrometer 201 fragments or transmits the precursor ions transmitted by the mass filter 220 in each overlapping step. One or more resultant product ions are generated for each overlapping window of the series. The fragmentation device 230 fragments the precursor ions when a collision energy high enough to fragment the ions is used. The fragmentation device 230 transmits the precursor ions when a collision energy low enough to fragment the ions is used. As a result, the resultant product ions can include the precursor ions.

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

[0065] The processor 250 can be, without limitation, a computer, a microprocessor, the computer system of Figure 1, or any device capable of sending and receiving control signals and data to and processing data from the tandem mass spectrometer. The processor 250 is in communication with the ion source device 210, the mass filter 220, the fragmentation device 230, and the mass analyzer 240. Although shown as a separate device, the processor 250 can be a 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 series in a file in a memory device (not shown), the processor 250 performs the encoding and storing steps.

[0067] Processor 250 encodes and stores each unique product ion detected by mass analyzer 240 in real time during data acquisition by performing several substeps: First, processor 250 identifies an overlapping window of the first occurrence of a sequence involving the first occurrence of each unique ion.

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

[0069] Returning to FIG. 2, processor 250 then selects a group of G overlapping windows in the series immediately preceding the overlapping window of the first occurrence such that the group spans at least a 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 of the group of G overlapping windows is, for example, the precursor ion uncertainty interval. Processor 250 calculates a sum of the counts or intensities of each unique ion detected from each of the G overlapping windows of the group. Processor 250 associates the sum with the position of the overlapping window in the group.

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

[0071] Returning to FIG. 2, processor 250 shifts the selected group of G overlapping windows in the series forward by one overlapping window. 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. Processor 250 associates the sum with the position of the overlapping window of the group. Processor 250 stores the sum and the position in a memory device. 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 FIG. 3, for example, group 350 is shifted forward by one overlapping window. A sum of the counts or intensities of the unique product 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 the 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 first occurrence overlapping window 331. The sum and the position are stored, for example, in a file in the memory device.

[0073] 4 is an exemplary diagram 400 illustrating how a set of summed counts or intensities for each unique ion are stored or encoded in real-time during scanning SWATH data acquisition according to various embodiments. In FIG. 4, plot 360 of FIG. 3 is shown again. A set 410 of summed counts or intensities of the unique product ions of plot 360 and their positions is stored in a memory device. Set 410 is stored, for example, as a series 412 of intensity and position (precursor ion m / z) pairs. Because set 410 includes 17 points, series 412 is stored as a series of 17 intensity and position pairs (I1, M1), (I2, M2), ..., (I 17 , M 17 ).

[0074] A comparison of the set 410 or sequence 412 of FIG. 4 with the precursor ion transmission window 330 of FIG. 3 shows that storing the set 410 or sequence 412 of FIG. 4 greatly reduces the storage requirements for each product ion of the scanning SWATH. Each transmission window 330 of FIG. 3 represents a product ion mass spectrum that previously had to be stored. In addition, each transmission window may include intensities of multiple product ions. FIG. 4 greatly reduces the storage requirements for storing product ions of the scanning SWATH by summing the product ion intensities and storing only those intensities that correspond to the same precursor ion. In other words, the storage requirements for product ion data are greatly reduced by using an encoding that correlates the intensities of the product ions with the precursor ion m / z.

[0075] In plot 360 of Figures 3-4, the summed counts or intensities 351 have a triangular shape. This triangular shape is due to the quadrupole mass filter 220 of Figure 2 transmitting precursor ions with a uniform probability distribution function. Other mass analyzers and mass filters, for example, may produce different shapes. In other words, the shape of the probability distribution function depends on the uncertainty interval of each particular mass analyzer. In addition, the uncertainty interval depends on the function of the mass filter of the tandem mass analyzer.

[0076] An ideal tandem mass analyzer with a mass filter using a rectangular precursor ion transmission to generate a rectangular precursor ion transmission function would then generate a triangular uncertainty function, such as function 370 of plot 360 in FIG. 3-4. Function 370 describes how the summed counts or intensities of the unique product ions 301 in FIG. 3 vary with the position of the group 350. Returning to FIG. 4, function 370 of plot 360 can be described as function 420. 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 for the interval of the triangle's sides. 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 product ions can most simply be encoded as the position and width of the triangular function for this type of mass analyzer.

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

[0078] 5 is an example plot 500 illustrating that compressing the summed counts or intensities after encoding still preserves the information necessary to infer precursor ions, according to various embodiments. Points 510 represent the summed counts or intensities of product ions remaining after the encoded summed count or intensity set has been reduced to compress the data. An ideal triangle 520 is plotted along with points 510 to show how the compressed points 510 still maintain a triangular shape. The vertices of the triangular shape are used, for example, to infer the precursor ions that generated the product ions.

[0079] Unfortunately, however, the encoding process can produce blurred triangle functions, which result in elongated bases and distorted triangle shapes.

[0080] In various embodiments, a deblurring algorithm is therefore applied to the measured summed counts or intensities for product ions to remove the effects of precursor ion uncertainty encoding. Returning to FIG. 2, processor 250 further removes the effects of distortion (or uncertainty spreading) from the stored sums and positions of each unique ion by using a deblurring algorithm. The deblurring algorithm equates the stored sums and positions to a convolution of the deblurred sums and positions with a probability distribution function that depends on the uncertainty interval of the scanning SWATH transmission function of the mass filter. The deblurring algorithm resolves the deblurred sums and positions of each unique ion.

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

[0082] In various embodiments, the deblurred summed counts or intensities can be further encoded as a function, more specifically, in the case of a triangular function, the deblurred summed counts or intensities can be further encoded as the position and base width of the triangle.

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

[0084] In scanning SWATH, the blur, or blur probability distribution function, can be known and modeled. It depends on the precursor ion transmission function implemented by the mass filter. As a result, a deblurring algorithm can be used. Of course, knowing the blurring function makes it easier to solve the "blurring problem" than when the blurring function is not known. However, in various alternative embodiments, blind deconvolution or deblurring that does not know the blurring 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, deblurring algorithms are applied to only one dimension, which is the precursor ion transmission window dimension. Deblurring algorithms are applied to only one dimension, for example, making the problem solution more stable. Any processing introduces errors. If there is no deconvolution, for example, in the time dimension, errors will not be introduced from all of the uncertainties surrounding that dimension. In other words, knowing the blur function in the precursor ion transmission window dimension ensures that deblurring errors are minimized.

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

[0087] The blur, probability distribution function, or uncertainty in precursor ion positions in the precursor dimension does not depend on the compound being analyzed. Instead, it depends on the instrument or mass spectrometer and the method used. As a result, de-blurring algorithms can be applied to this dimension.

[0088] However, the blur or uncertainty in the chromatographic or separation time dimension varies with the compound being analyzed. In other words, the blur in the chromatographic time dimension depends not only on the column used but also on the compound being analyzed. As a result, de-blurring algorithms are not applied in the chromatographic or separation time dimension.

[0089] 6 is an exemplary heat map plot 600 showing summed product ion counts plotted as a function of precursor ion transmission window position and product ion m / z before applying a deblurring algorithm to the data, according to various embodiments. Inset 610 shows that two different product ions 611 and 612 have similar product ion m / z values. It also shows that the uncertainty intervals or triangular probability distribution functions of these two product ions in the precursor ion transmission window position dimension are highly overlapped.

[0090] Typically, the product ion spectrum for a particular precursor ion transmission 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 includes both product ion 611 and product ion 612. In other words, due to the large overlap in the uncertainty intervals of the two product ions, both product ions will be included for the precursor ion transmission window position. Therefore, due to this large overlap or blurring, both product ions will be found as product ions of a particular precursor ion, even though they are not both from the same precursor ion.

[0091] In various embodiments, a deblurring algorithm is applied to the data of FIG. 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 equated to the convolution of a triangular probability distribution function with the removed summed counts and positions for the mass filter uncertainty interval. The deblurring algorithm solves this equation for the deblurred summed counts and positions. Note that some deblurring algorithms require the first derivative at every point.

[0092] 7 is an exemplary heat map plot 700 showing summed product ion counts plotted as a function of precursor ion transmission window position and product ion m / z after applying the deblurring algorithm of FIG. 6 to the data, according to various embodiments. Inset 710 shows that two different product ions 611 and 612 still have similar product ion m / z values ​​after deblurring. However, the uncertainty intervals or triangular probability distribution functions of these two product ions in the precursor ion transmission window position dimension show much less overlap after deblurring. In essence, the width of the bases of the triangular probability distribution functions of these two product ions has been greatly reduced.

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

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

[0095] In various embodiments, the probability distribution function found after deblurring for each unique product ion, or the measured set of summed counts or intensities, is read from a memory device. Either type of data may be read, for example, from a file. Numerical decomposition or probabilistic inference methods are then applied to the read data to determine the precursor ions of the unique product ions. For example, for a triangular function, the precursor ions of the unique product 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 deblurred sum and position of each unique ion to further determine precursor ions for each unique ion. For example, the deblurred sum and position of each unique ion have a triangular shape. The processor 250 further determines precursor ions for each unique ion as precursor ions corresponding to the vertices of the triangular shape.

[0097] Deblurring in the precursor ion transmission window position dimension also improves peak finding in the chromatographic or separation time dimension, however, as explained above, deblurring is not applied in the chromatographic or separation time dimension.

[0098] 8 is an exemplary diagram 800 including a heat map plot showing summed product ion counts plotted as a function of chromatographic time and precursor ion transmission window position before applying a deblurring algorithm to the data, and a plot showing the XIC found from the heat map for precursor ion m / z values, according to various embodiments. Heat map 810 shows product ion intensity regions at times 811 and 812. Before applying the deblurring algorithm, both intensity regions have a large width in the precursor ion transmission window position dimension.

[0099] As a result, both intensity regions may be detected over time for a particular precursor ion transmission window position (precursor ion m / z value) represented by line 815. In other words, due to the large width of product ion uncertainty in the precursor ion transmission window position dimension, two different product ions may be detected over time for a particular precursor ion transmission window position.

[0100] XIC 821 is shown in plot 820. XIC 821 represents the intensity over time along line 815 of heat map 810. XIC 821 contains two separate peaks that identify two different product ions for the precursor ion transmission window position before deblurring.

[0101] 9 is an exemplary diagram 900 including a heat map plot showing summed product ion counts plotted as a function of chromatographic time and precursor ion transmission window position after applying the deblurring algorithm of FIG. 8 to the data, according to various embodiments, and a plot showing the XIC found from the heat map for precursor ion m / z values. Heat map 910 again shows product ion intensity regions at times 811 and 812. After applying the deblurring algorithm, both intensity regions have a narrower width in the precursor ion transmission window position dimension than before deblurring.

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

[0103] XIC 921 is shown in plot 920. XIC 921 represents the intensity over time along line 815 of heat map 910. XIC 921 contains only one peak identifying only one product ion for the precursor ion transmission window position of line 815 after deblurring.

[0104] In various embodiments, instead of using a deblurring algorithm, a numerical method can be applied to the summed counts or intensities measured for product ions to determine the precursor ions of the product ions. U.S. Patent No. 10,651,019 (hereinafter, the "'019" patent) discloses a method for determining precursor ions of product ions from scanned SWATH data and is incorporated herein by reference in its entirety. In the '019 patent, the intensities of selected product ions are read from multiple product ion spectra obtained from each scan of the precursor ion transmission window over the precursor ion mass range. A trace is generated that describes how the intensity of the selected product ion varies with the precursor ion transmission window.

[0105] A matrix multiplication equation is created that describes how one or more precursor ions correspond to traces for 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 using a numerical method such as non-negative matrix factorization (NNMF) in general, or non-negative least squares (NNLS) in particular.

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

[0107] More specifically, returning to FIG. 2, the processor 250 further determines the precursor ion for each unique ion using a numerical method. The numerical method arranges the stored sums and positions as a column matrix of length n. The numerical method equates the column matrix to the known n×m mass filter matrix for the mass filter multiplied by the unknown precursor ion column matrix of length m. Finally, the numerical method solves the unknown precursor ion column matrix to determine the precursor ion. The numerical method can include, but is not limited to, NNMF or NNLS.

[0108] (Method of encoding and storing scanned SWATH data) FIG. 10 is a flow chart illustrating a method 1000 for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows, according to various embodiments.

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

[0110] In step 1020, the precursor ions transmitted by the mass filter in each overlapping step are fragmented or transmitted using a fragmentation device of the tandem mass spectrometer. One or more resultant product or precursor ions are generated for each overlapping window in the series.

[0111] In step 1030, intensities or counts are detected for one or more resulting product ions or each precursor ion for each overlapping window in the series using a mass analyzer of the tandem mass spectrometer to form mass spectral data for each overlapping window in the series.

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

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

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

[0115] In step 1070, the group of G overlapping windows of the selected series is shifted forward by one overlapping window, a sum of the counts or intensities of unique ions detected from each of the G overlapping windows of the group is calculated, the sum is related to the position of the overlapping window of the group, the sum and the position are stored in a 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.

[0116] (Computer program product for encoding and storing scanned 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 for execution on a processor to perform a method for encoding and storing tandem mass spectrometry data measured from overlapping precursor ion transmission windows, the method being performed by a system including one or more distinct software modules.

[0117] 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 transmission 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 commands the mass filter of the tandem mass spectrometer to move a precursor ion transmission window with a precursor ion mass-to-charge ratio (m / z) width W in overlapping steps across the precursor ion mass range with a step size S m / z. A series of overlapping transmission windows spanning the mass range is created. The mass filter transmits precursor ions within the transmission window in each overlapping step.

[0119] The control module 1110 commands the fragmentation device of the tandem mass spectrometer to fragment or transmit precursor ions transmitted by the mass filter in each overlapping step, and one or more resultant product ions or precursor ions are generated for each overlapping window in the series.

[0120] The control module 1110 instructs the mass analyzer of the tandem mass spectrometer to detect intensities or counts for one or more resulting product ions or each precursor ion for each overlapping window in the series forming mass spectral data for each overlapping window in the series.

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

[0122] The group of G overlapping windows of the selected series is shifted forward by one overlapping window, a sum of the counts or intensities of unique ions detected from each of the G overlapping windows of the group is calculated, the sum is related to the position of the overlapping window of the group, and the sum and the position are stored in a 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.

[0123] 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.

[0124] 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 skilled 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 those steps in the order written, and one skilled in the art can readily understand that the sequence can be varied and still remain within the spirit and scope of the various embodiments.

Claims

1. A system for encoding and storing mass spectral data acquired using data independent acquisition, the system comprising: A processor; a non-transitory memory in communication with the processor, the non-transitory memory storing instructions that, when executed, cause the processor to: acquiring said mass spectral data including counts or intensities for one or more product ions in each window of a series of transmission ion windows; detecting unique product ions within the mass spectral data; identifying one or more windows within said sequence in which said unique product ions appear; for each window of the one or more windows, recording a location of the window and a count or intensity sum for a group of windows spanning the window; A non-transient memory and A system comprising:

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

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

4. The act of recording the count or intensity sum and the location for each window in the group of windows comprises: recording a position of a first window of the one or more windows; selecting a group of windows beginning with the first window and extending sequentially forward; recording a count or intensity sum for said group of windows in association with said location; The system of claim 1 , comprising:

5. The system of claim 4, wherein the mass spectral data is obtained using Scanning SWATH (Sequential Window Acquisition of All Theoretical Mass Spectra), and the number of windows is at least equal to the width divided by the step size.

6. The system described in claim 1, wherein the one or more windows include a first emergence window, the first emergence window being the window among the one or more windows in which the unique product ion is first identified.

7. The act of recording the count or intensity sum and the location for each window in the group of windows comprises: recording the sum and position for a first group preceding the first appearance window of the unique product ions; moving the group of windows forward by at least one window; recording the sum and position for a second group of unique product ions that includes the first window of occurrence; The system of claim 6 , comprising:

8. An operation of moving the group of windows forward by at least one window; Recording the totals and positions for the following groups; The system of claim 7 , further comprising: repeating:

9. The system of claim 8, wherein the mass spectral data is obtained using Scanning SWATH (Sequential Window Acquisition of All Theoretical Mass Spectra), and the operations of moving the group of windows forward by at least one window and recording sums and positions for subsequent groups are repeated until at least one window of the subsequent group no longer overlaps with the first emergence window.

10. A method for encoding and storing a scanned SWATH data file, the method comprising: encoding a scanning ion transmission window dimension of the mass spectral data, said encoding including, for a unique product ion, a position of the window in which the unique product ion appears and a count or intensity sum for a group of windows spanning the window in which the unique product ion appears; storing said location and said sum; A method comprising:

11. The system of claim 10, wherein each sum and position is stored as an ordered pair.

12. The instructions further cause the processor to: identifying each ordered pair associated with said unique product ions as a set; determining a shape of the set; reducing the number of ordered pairs in the set while maintaining the shape of the set; The system of claim 11 , further comprising:

13. The instructions further cause the processor to: The system of claim 11 , further comprising deblurring each sum and position before storing the ordered pairs.

14. The instructions further cause the processor to: The system of claim 13 , further comprising: encoding the sum as a function.

15. The system of claim 14, wherein the function is a triangular function and the encoding includes a width and a position of a base of the triangular function.

16. The system of claim 13, wherein each sum and position is deblurred in the ion transmission window dimension only.

17. A method for identifying precursor ions of product ions in a data independent acquisition mass spectrometry experiment, comprising: acquiring mass spectral data including a position in an ion transmission window in which the product ion appears and a count or intensity sum for the product ion for a group of ion transmission windows spanning the ion transmission window at the position; arranging the sum as a generator ion column matrix of length m; Obtaining a known n×m mass filter matrix; solving for a precursor ion column matrix of length n to determine precursor ions of said product ions by applying a numerical method to a matrix multiplication equation, said matrix multiplication equation including said known n×m mass filter matrix multiplied by said precursor ion column matrix to generate said product ion column matrix; A method comprising:

18. The method of claim 17, wherein the numerical method includes non-negative matrix factorization (NNMF).

19. The method according to claim 18, wherein the numerical method includes non-negative least squares (NNLS).

20. The method of claim 17, wherein the known n×m mass filter matrix is ​​determined according to movement of the ion transmission window during the data-independent acquisition mass spectrometry experiment.

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