WO2025104656A1 - Mass spectrometer automatic tuning feedback - Google Patents

Abstract

Systems and methods for evaluating the operational state of a mass measuring system, including its tuning include a first mass separator in series with a second mass separator, with the second mass separator being faster than the first mass separator. Unprocessed mass data of detection of a set of ions is received from the second mass separator. A known ion is identified in the unprocessed mass data, and an actual appearance and an actual disappearance of the known ion is mapped to determine an appearance period of the known ion. The appearance period is compared with an intended appearance period for the known ion, and, based on the comparing, a match status between the appearance period and the intended appearance period is determined for the unfragmented mass.

Description

MASS SPECTROMETER AUTOMATIC TUNING FEEDBACK

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is being filed as a PCT International application and claims the benefit of and priority to U.S. Provisional Application No. 63/598,868, filed November 14, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Mass spectrometry is a powerful analytical technique used to identify and quantify molecules based on their mass and charge. It works by ionizing a sample, separating the resulting ions based on their mass-to-charge ratio, and then detecting and measuring the abundance of these ions. This information can be used to determine the composition and structure of molecules, making mass spectrometry a valuable tool in various scientific fields, including chemistry, biochemistry, and environmental science.

[0003] Tandem mass spectrometry (MS/MS) uses two sequential stages of mass analysis to first isolate a specific ion or group of ions from a sample. The isolated ion is subjected to further fragmentation, generating a range of smaller ions. Another mass spectrometer analyzes these fragments, providing detailed information about the structure and composition of the original molecule. MS/MS is widely used for precise identification and characterization of complex molecules in fields such as proteomics, metabolomics, and environmental analysis.

[0004] Selection of the ion of group of ions in the first stage, sometimes referred to as precursor ions, is controlled by configuring a precursor ion transmission window. The process of specific adjustments made to optimize the selection of precursor ions through a mass filter is referring to as “tuning” the mass separator of the first stage.

[0005] Tuning the mass separator involves optimizing its parameters to achieve desired mass resolution and selectivity for a particular analysis. For example, in a quadrupole mass separator, the radiofrequency (RF) and direct current (DC) voltages are set to create a stable and specific RF/DC field to determine which ions are transmitted through the quadrupole and which are not. These voltage settings can be adjusted to optimize mass resolution, referring to the separation of ions with different mass-to- charge ratios (m/z). Tuning also involves fine-tuning the settings to achieve the desired selectivity for the target ions. Ions of interest need to be efficiently transmitted through the mass separator while minimizing the transmission of unwanted ions or contaminants. Selectivity is important in filtering out interference and improving signal-to-noise ratios. [0006] Tuning may also include adjustments to enhance the stability and robustness of the mass separator’s operation. Ensuring that the mass separator maintains its settings over time is essential for reliable and consistent results. Proper tuning ensures that ions are effectively focused as they pass through the mass separator, minimizing the spread of ion trajectories and improving sensitivity. Tuning is a critical step to optimize the performance of a mass spectrometer for a specific analytical task. The instrument' s settings are tailored to achieve the best possible mass resolution, selectivity, and ion transmission efficiency for particular target compounds.

SUMMARY

[0007] Examples presented herein relate to a method for evaluating the operation of mass measuring system, including the tuning state of a mass measuring system. The mass measuring system includes a first mass separator in series with a second mass separator, with the second mass separator being faster than the first mass separator. The method includes receiving unprocessed mass data of detection of a set of ions from the second mass separator, identifying a known ion in the unprocessed mass data, and mapping an actual appearance and an actual disappearance of the known ion to determine an appearance period of the known ion. The method further includes comparing the appearance period with an intended appearance period for the known ion, and determining, based on the comparing, a match status between the appearance period and the intended appearance period for the unfragmented mass.

[0008] In other examples presented herein, the method further includes processing the unprocessed mass data into a standardized file format, and incorporating the match status into the standardized file format. In still other examples presented herein, the method further includes determining a mass of the known ion. In further examples presented herein, the intended appearance period for the known ion is based on the mass of the known ion.

[0009] In other examples presented herein, the mapping characterizes an actual transmission window of the first mass separator. In further examples presented herein, the match status comprises a width of the actual transmission window. In still further examples presented herein, the match status comprises a function of a shape of the transmission window. In yet further examples presented herein, the method further includes moving the transmission window in a series of overlapping steps across a mass range, wherein the mapping characterizes a series of transmission windows of the series of overlapping steps across the mass range.

[0010] In other examples presented herein, the match status comprises a match between the appearance period and the intended appearance period for the known ion. In other examples presented herein, the match status comprises a mismatch between the appearance period and the intended appearance period for the known ion. In further examples presented herein, the further includes generating, in response to the mismatch, a mismatch alert. In still further examples presented herein, the method further includes determining, in response to the mismatch, a degree of mismatch between the appearance period and the intended appearance period for the known ion. In other further examples presented herein, the method further includes adjusting, based on the degree of mismatch, the transmission window of the first mass separator.

[0011] Other examples presented herein relate to a system for tuning a mass spectrometer. The system includes a first mass separator, a second mass separator in series with the first mass separator, the second mass separator being faster than the first mass separator. Each of the first and second mass separator is configured to: receive a set of ions; perform a detection of the set of ions; and generate a set of detection signals corresponding to detection of the set of ions. The system further includes a controller configured to receive unprocessed mass data from second mass separator including the set of detection signals, identify an known ion in the unprocessed mass data, map an actual appearance and an actual disappearance of the known ion to determine an appearance period of the known ion, compare the appearance period with an expected appearance period for the unfragmented mass, and determine, based on the comparing, a match status between the appearance period and the expected appearance period for the known ion.

[0012] In other examples presented herein, the controller is further configured to process the mass data into a standardized file format and incorporate the match status into the standardized file format. In further examples presented herein, the controller is further configured to store the standardized file format. In still further examples presented herein, the controller is further configured to transmit the standardized file format. In other examples presented herein, the second mass separator is a time of flight mass analyzer. In further examples presented herein, the first mass separator is a quadrupole.

[0013] In examples, presented herein, a method for processing data of a mass measuring system including a first mass separator in series with a second mass separator is described where the second mass separator is faster than the first mass separator. The method involves receiving unprocessed mass data of detection of a set of ions from the second mass separator, identifying a known ion in the unprocessed mass data, mapping an actual appearance and an actual disappearance of the known ion to determine an actual transmission window of the known ion. In some examples, the method can further include the steps of encoding the mass data using the actual transmission window as an input parameter. In some examples, the method can further include encoding the mass data using a shape of the actual transmission window as an input parameter. In embodiments, the mass data is stored in a file or in memory.

[0014] Other examples presented herein relate to a system for tuning a first mass separator with a scanning transmission window. The system includes a second mass separator, in series with the first mass separator and configured to receive a set of ions, perform a detection of the set of ions, and generate a set of detection signals corresponding to the detection of the set of ions. The system further includes a controller configured to receive unprocessed mass data including the set of detection signals, identify an known ion in the unprocessed mass data, map an actual appearance and an actual disappearance of the known ion to determine an appearance period of the known ion, compare the appearance period with an expected appearance period for the known ion, and determine, based on the comparing, a match status between the appearance period and the expected appearance period for the known ion, wherein the match status characterizes the scanning transmission window.

[0015] Yet other examples presented herein relate to a method for processing data of a mass measuring system including a first mass separator in series with a second mass separator, wherein the second mass separator is faster than the first mass separator. The method includes receiving unprocessed mass data of detection of a set of ions from the second mass separator and identifying a known ion in the unprocessed mass data. The method further includes mapping an actual appearance and an actual disappearance of the known ion to determine an actual transmission window of the known ion. In other examples presented herein, the method further includes encoding the mass data using the actual transmission window as an input parameter and/or encoding the mass data using a shape of the actual transmission window as an input parameter.

[0016] A variety of additional inventive aspects will be set forth in the description that follows. The inventive aspects can relate to individual features and to combinations of features. It is to be understood that both the forgoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of the description, illustrate several aspects of the present disclosure. A brief description of the drawings is as follows:

[0018] FIG. 1 is a block diagram of an example system for integrated tuning feedback for a mass spectrometer.

[0019] FIG. 2 is block diagram of an example data processing system for tuning of a mass spectrometer.

[0020] FIG. 3 is a graph showing percent gain as a function of window size for an example with an actual ion transmission window that is wider than the acquisition value set by the user for the experiment.

[0021] FIG. 4A is an example of the detrimental effects on data quality for an example with an actual ion transmission window that is wider than the acquisition value set by the user for the experiment.

[0022] FIG. 4B is another example of the detrimental effects on data quality for the example of FIG. 4A.

[0023] FIG. 4C is another example of the detrimental effects on data quality for the example of FIG. 4A.

[0024] FIG. 5 is a flowchart of a method for evaluating tuning of a mass measuring system.

[0025] FIG. 6 illustrates an example computing system with which aspects of the present disclosure may be implemented.

DETAILED DESCRIPTION

[0026] Disclosed herein are methods and systems for automatic tuning feedback for a mass spectrometer. The present disclosure describes the extraction of relevant tuning data from raw mass separator output data and incorporation of tuning analysis and feedback into existing data processing processes. The parameters of an actual transmission window are automatically determined according to the raw output data of a mass spectrometer experiment and can be compared with the intended transmission window’s parameters to assess proper tuning of the mass separator.

[0027] With the development of more complex mass spectrometer protocols, such as data independent acquisition analysis including for example, Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) and scanning-SWATH, data produced during a mass spectrometry scan is becoming more complex. With this increasing complexity, the negative impact of an improperly tuned mass separator can have an increasingly significant adverse impact on generated data. However, evaluating effective tuning of the mass spectrometer presently relies on independently run methods that can only be conducted before or after an experiment. Evaluation of the tuning of a mass spectrometer can only be applied retroactively to confirm the results, and may often require an experiment to be entirely reperformed.

[0028] While raw mass spectrum data includes sufficient information to enable determination of the tuning status of the mass spectrometer from the mass spectrum data, current data processing methods either discard or otherwise render inaccessible the necessary information. Systems and methods disclosed herein enable integration of tuning evaluation directly into post-experiment data processing. By incorporating tuning analysis and feedback into existing data processing protocols, tuning can be immediately assessed for a given experiment and without requiring initiation of additional protocols by a user.

[0029] Mass spectrometer data acquisition generally produces a raw data format that requires conversion to another format, such as a wiff file, for example, for further processing. Conversion of mass spectrometer data is generally enabled by a converter, which may form part of an overall controller and include code and algorithms in the form of an executable, that may be immediately triggered post-acquisition of the raw data in a mass spectrometer operation system. During the process of conversion, the raw file is read and then translated into the post-processing format.

[0030] The proposed invention provides additional functionality within existing raw data converter applications. The addition consists of methods, systems, devices, and algorithms to measure the actual transmission window width during acquisition. These width measurements are recorded, such as into a log file. Inspection of the log file provides a quick output to understand if there is a deviation from the expected transmission window set by the user prior to acquisition. If there is a deviation from expected values, this is an indication that the instrument needs tuning of the mass separator before further data collection. By incorporating the tuning feedback directly into existing post-acquisition processing, tuning is immediately assessed and an alert may be generated well before data associated with an improperly tuned separator is relied upon or before samples analyzed with the improperly tuned separator are removed or discarded.

[0031] The principles of the present disclosure enable easy assessment of the state of the mass spectrometer, such as the tuning state and specifically the mass separator tuning, and ensures optimal performance while acquiring data independent acquisition data including scanning SWATH or any other experimental data, thereby saving instrument time and samples. Examples presented herein discuss tuning of a first quadrupole (QI) and, in some cases, within the context of performing a data independent acquisition analysis including scanning SWATH. However, those of skill in the art will understand that the principles of the present disclosure will be widely applicable to other mass separators and mass spectrometry methods. For example, principles of the present disclosure may be applicable to mass separators including, as non-limiting examples, quadrupole mass analyzers, time of flight mass analyzers, magnetic sector mass analyzer, electrostatic sector mass analyzer, quadrupole ion trap mass analyzers, and ion cyclotron resonance. Non-limiting examples of mass spectrometry methods to which the principles of the present disclosure will readily apply include data-independent acquisition (DIA) methods such as broadband DIA, including forms of collision-induced dissociation (CID), and various SWATH-MS methods, including scanning SWATH, and data- dependent acquisition (DDA) methods, where a fixed number of precursor ions are selected and analyzed by tandem mass spectrometry.

[0032] FIG. 1 is a block diagram of an example system 100 for integrated tuning feedback for a mass spectrometer. Example system 100 includes an ion source 110, a first mass separator 120, a fragmentation device 130, a second mass separator or a mass analyzer 140, and a computing system 150.

[0033] In embodiments, system 100 further includes a sample introduction device 170. Sample introduction device 170 introduces one or more compounds of interest from a sample to ion source 110 over time. Sample introduction device 170 performs techniques that include, but are not limited to, direct injection, liquid chromatography, gas chromatography, capillary electrophoresis, or ion mobility.

[0034] Mass filter 120 and fragmentation device 130 are shown as different stages of a quadrupole and mass analyzer 140 is shown as a time-of-flight (TOF) device. Those of ordinary skill in the art will appreciate that either of mass filter 120 and mass analyzer 140 may include other types of mass separator and analysis devices including, but not limited to, ion traps, orbitraps, ion mobility devices, time-of-flight (TOF) devices, or Fourier transform ion cyclotron resonance (FT-ICR) devices. In embodiments, mass filter 120 and mass analyzer 140 are respective examples of a first and a second mass separator, arranged in a series. The second mass separator may configured to be faster than the first mass separator. For example, a system may be configured according to the present disclosure with a quadrupole for the first mass separator, or mass filter 120, and a TOF device for the second mass separator, or mass analyzer 140. Each mass separator is configured to receive a set of ions, perform a detection of the set of ions, and generate a set of detection signals corresponding to detection of the set of ions.

[0035] Ion source device 110 transforms a sample or compounds of interest from a sample into an ion beam. Ion source device 110 can perform ionization techniques that include, but are not limited to, matrix assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI).

[0036] Mass filter 120 receives the ion beam. In embodiments, mass filter 120 is configured by a user for a particular precursor ion transmission window based on the experimental goals for the sample being run. The precursor ion transmission window, as discussed herein, refers to the range of precursor or parent ions that are allowed to pass through a specific selection step and into the subsequent stages of mass analysis or fragmentation. In many tandem mass spectrometry (MS/MS) experiments, the precursor ions are first selected based on their m/z (mass-to-charge ratio) in order to isolate a specific ion of interest for further analysis or fragmentation. The precursor ion selection process employs a mass filter or a specific set of voltages that allow only ions within a certain m/z range (the precursor ion transmission window) to pass through to the next stage.

[0037] The precursor ion transmission window is typically defined by setting specific parameters within the mass spectrometer's control software. The width of the precursor ion transmission window affects the specificity of the analysis with a narrower window providing higher specificity, while a wider window may allow more ions to pass through but with potentially less selectivity. Balancing these factors is crucial for achieving the desired level of analytical sensitivity and specificity in a given experiment. Further, ensuring accurate execution of the selected window by the mass spectrometer is essential to accurate results, as discussed in further detail below.

[0038] In embodiments, such as those implementing a scanning data independent acquisition analysis including a scanning SWATH method, mass filter 120 filters the ions by moving a precursor ion transmission window with a precursor ion mass-to-charge ratio (m/z) width 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 produced across the mass range. Mass filter 120 transmits precursor ions within the transmission window at each overlapping step.

[0039] Fragmentation device 130 of tandem mass spectrometer 102 fragments or transmits the precursor ions transmitted at each overlapping step by mass filter 120. In examples related to scanning SWATH, one or more resulting product ions are produced for each overlapping window of the series. Fragmentation device 130 fragments the precursor ions when a collision energy high enough to fragment ions is used. Fragmentation device 130 transmits the precursor ions when a collision energy low enough not to fragment ions is used. As a result, the resulting product ions can include precursor ions.

[0040] Mass analyzer 140 of tandem mass spectrometer 102 detects intensities or counts for each of the one or more resulting product ions for each overlapping window of the series that form mass spectrum data for each overlapping window of the series.

[0041] Computing system 150 can be, but is not limited to, a computer, a microprocessor, the computing system of FIG. 6, or any device capable of sending and receiving control signals and data from a tandem mass spectrometer and processing data. Computing system 150 is in communication with ion source device 110, mass filter 120, fragmentation device 130, and mass analyzer 140. Computing system 150 is shown as a separate device but can be a processor or controller of tandem mass spectrometer 102 or another device. Computing system 150 may store in a memory device (not shown) mass spectrum data for each precursor ion window analysis is performed for, including for each overlapping window of the series in examples performing scanning SWATH. In embodiments, computing system 150 instead performs an encoding and storing step, and encodes and stores each unique product ion detected by mass analyzer 140 in real-time during data acquisition. Prior to storing mass spectrum data, computing system 150 performs one or more processing steps on the raw mass spectrum data received to prepare the data for viewing, analysis, and storage. Raw mass spectrum data includes the counts or intensities of product ions at different m/z ratios over time.

[0042] FIG. 2 is block diagram of an example data processing system 200 for tuning of a mass spectrometer. Data processing system 200 is implemented, in embodiments, by computing system 150. Data processing system 200 may be among multiple subsystems or software executed by computing system 150 in operating mass spectrometer 102 and handling of data output by the mass spectrometer. The present disclosure is directed to analysis and feedback of the tuning of the mass spectrometer, but those of skill in the art will readily under that other subsystems and/or modules may exist within and be executed by computing system 150. Computing system 150 is presented in examples herein as a single device, but in embodiments may be one or more processing devices networked or otherwise in communication. Functions may be divided among individual devices or shared across the collective processing capability of the one or more processing devices. In embodiments, data processing system 200 forms part of or serves as a controller. The controller may be configured to transmit operation commands to the mass spectrometer and/or to receive unprocessed mass data from second mass separator including the set of detection signals and perform one more data processing actions on the unprocessed mass data.

[0043] Raw mass spectrometer data is typically large and complex, and it undergoes extensive data processing and analysis to extract meaningful information. Data processing may be carried out in a series or collection of actions. Actions may be performed collectively by the data processing system 200 or individual steps or portions of the processing may be executed by individual components or modules of the data processing system 200. In the example of FIG. 2, data processing system 200 contains a number of subcomponents with dedicated functions. In this example, data processing system 200 includes preprocessor 204, peak detector 206, peak identifier 208, tuner 210, and file formatter 212. Together, preprocessor 204, peak detector 206, peak identifier 208, tuner 210, and file formatter 212 may form a converter application.

[0044] Preprocessor 204 performs one or more preprocessing actions on the raw data as it is received from the mass spectrometer 102. Preprocessing of the raw data includes baseline corrections, which removes any constant offset or baseline noise from the raw data, smoothing out of random noise in the data, and normalization, which scales the intensity values to a common scale and can be useful for comparing spectra from different samples. In some examples, normalization may be performed separately from preprocessing.

[0045] Peak detector 206 quantifies individual peaks and/or groups of peaks in the mass spectrum. Peak identifier 208 annotates the mass data to assign m/z values to the detected peaks, which may involve comparing them to a known database of compounds. Deconvolution may be performed in some embodiments to separate overlapping peaks, which may be particularly important in complex samples where multiple compounds can contribute to a single peak. Integration quantifies the area under each peak, which is proportional to the abundance of the corresponding ion. Alignment is performed in some cases, particularly in studies involving multiple samples, to ensure that spectra from different samples are aligned correctly. In some cases, statistical methods may be applied to compare mass spectra from different samples or conditions.

[0046] Tuner 210 performs processing and analysis to automatically assess the tuning of the mass spectrometer based on the mass data. As disclosed herein, automatic instrument diagnostic analysis of tuning of the mass spectrometer provide for rapid recognition of an improperly tuned mass spectrometer, leading to fast troubleshooting. This conserves samples and instrument time otherwise wasted running suboptimal acquisitions.

[0047] Tuner 210 identifies a known ion in the mass data, maps an actual appearance and an actual disappearance of the known ion to determine an appearance period of the known ion, compares the appearance period with an expected appearance period for the unfragmented mass, and determines, based on the comparing, a match status between the appearance period and the expected appearance period for the known ion. As disclosed herein, the processing further includes measuring of an actual transmission ion window, determined based on the appearance period of the known ion, and may further execute one or more responsive actions based on the measured actual precursor ion window.

[0048] The processed data can be visualized using various plots, such as mass spectra, chromatograms, heatmaps, etc. Chemical identities may be assigned to the detected peaks based on databases or spectral libraries. The specific steps and techniques used in processing mass spectrometry data can vary depending on the type of experiment, the instrument used, and the nature of the samples.

[0049] File formatter 212 converts the final processed mass spectrum data into a standardized file format for storage and transmittal. In embodiments, the standardized file format is a wiff file. [0050] Proper tuning is necessary to ensure accurate data is collected, as is demonstrated with examples below in the context of a proteomics workflow executed on a system using a quadrupole as a first mass separator (“QI”). This example presents the consequences to data quantitation and quality in proteomics workflows if a QI transmission window used by the instrument in an experimental run is not equal to the acquisition value set by a user. As discussed herein, the acquisition value, or expected Q 1 window width, refers to the Q 1 window width set by the user, according to the desired experiment parameters, to be used during acquisition.

[0051] The post-acquisition converter code, and associated algorithms, may assume a “close to square” QI trace, sometimes referred to as a QI pulse, in processing calculations. In embodiments, tuner 210 may represent a portion of the post-acquisition converter code, responsible for determining the QI trace and performing the subsequent calculations. In some embodiments, knowledge of the QI shape, if not “square” QI trace, may also be determined or assumed. In embodiments, the shape of the QI trace may be parabolic, triangular, an irregular multipoint shape, etc. The examples discussed below relates to a system assuming a close to square QI trace. In example, the close to square QI trace may be characterized using a width and a position.

[0052] An exact QI window width is also assumed during post-acquisition conversion and used in processing calculations. The exact QI window width, which may also be referred to as an actual QI window width or measured QI window width, as discussed herein, refers to the QI window width applied by the mass spectrometer during the course of the experiment from which the data was collected. At present, typical data processing systems for mass spectrometers perform the processing calculations without verifying the exact QI window width or verifying tuning, the actual QI window width is generally assumed to the acquisition value set by the user. However, if the actual QI pulse is not close to square or the width is not set properly (e.g., the actual QI window width deviates from the acquisition value), the encoded QI trace cannot be processed accurately.

[0053] For instance, if the actual QI window width used is wider than the acquisition value, this will affect quantitation results. During processing, the QI window width is assumed to be the acquisition value. However, if the actual QI window width is wider, the data processing will produce encoded peaks with a smaller area than is accurate for the data acquired, and which therefore include a smaller number of ions. Further, specificity will be affected. Another scenario is if the actual QI window is narrower than the acquisition value set by the user. This results in loss of specificity with no sensitivity improvement.

[0054] FIG. 3 is a graph 300 showing percent gain as a function of window size for an example with an actual QI window that is wider than the acquisition value set by the user for the experiment and used during post-acquisition data processing. In the example of FIG. 3, the effects on quantitation can be seen in the percent gain.

[0055] In this example, an expected QI window width is set to 1 Da by the user during acquisition. Due to improper QI tuning, the actual QI width value used by the mass spectrometer during acquisition was 2.5 Da. This results in a 1.5 Da difference between the actual value and the acquisition value the user had set the QI width to be and intended the experiment to be conducted at. In examples, the actual QI window width may be measured manually with research tools after data acquisition.

[0056] Bar 302 depicts the number of ions identified based on the assumed QI window width of 1 Da based on the set acquisition value. Bar 304 depicts the number of ions identified based on an actual QI window width of 2.5 Da. In comparing the two values, bar 304 demonstrates a gain 306 of identified ions of about 40% based on the change in the QI acquisition window. Thus, having an improper QI tuning is detrimental to the quantitation results.

[0057] FIGS. 4A, 4B, and 4C are collectively an example of the detrimental effects on data quality for an example with an actual QI window that is narrower than the acquisition value set by the user for the experiment. FIGS. 4A, 4B and 4C together depict a visualization of processed data in a research microapp.

[0058] At 402, data processed with an expected QI window width of 7.5 Da is shown. At 404, data processed with an actual QI window width 11 Da is shown. For each of 402 and 404, the extracted-ion chromatogram (XIC) is observed at 402a and 404a, followed by the QI trace at 402b and 404b, and the spectrum at 402c and 404c. In this example, run conditions such as a 45 minute gradient, 50 nanogram load, and Zeno trapping being utilized may be assumed.

[0059] In this example the expected QI window width was set to 7.5 Da during acquisition by the user. However, due to improper QI tuning, the actual QI width value used by the instrument was 11 Da. There is a discrepancy of 3.5 Da between the expected and actual QI width value. Data quality is poor when QI width is wider than set QI window width in a number of ways. The QI traces are noisy. XIC and spectrum each show decreased sensitivity. This in turn will have consequences on quantitation. [0060] To overcome these and other problem associated with improper tuning, methods are described herein to automatically determine an actual ion transmission window width. In embodiments, the determination of the actual ion transmission window width may be performed automatically as part of post-acquisition data processing. The measured actual ion transmission window width is compared with an expected ion transmission window width, based on a user’s set acquisition value. The comparison may be performed automatically as part of post-acquisition data processing or may be initiated in response to a user action or may be triggered based on a system status or a feature of the data.

[0061] Automatic determination of the actual ion transmission window and associated tuning operations and features provide a number of advantages of the present state of tuning processes. For example, tuning can be automated, reducing time spent on tuning specific operations and time and samples wasted on experimental runs which produce inaccurate data due to the mass spectrometer being improperly tuned. Data acquisition is more reliable both because tuning is more consistently maintained and improper tuning is more quickly identified and corrected. Troubleshooting time is reduced and a user has ready access to feedback in order to effectively address issues related to the ion transmission window and other issues related to a mass separator.

[0062] FIG. 5 is a flowchart of a method 500 for evaluating tuning of a mass measuring system. In embodiments, the mass measuring system takes the form of a tandem mass spectrometry including a first mass separator in series with a second mass separator. Each mass separator may represent any appropriate mass separating or mass filtering device. Some non-limiting examples of mass separators include a quadrupole mass filter, a time-of-flight (TOF) mass analyzer, a magnetic sector analyzer, an orbitrap mass analyzer, an ion cyclotron resonance (ICR) analyzer, a quadrupole ion trap, a linear ion trap, and a FT-ICR (Fourier transform ion cyclotron resonance) analyzer. The mass measuring system may be a mass spectrometer, such as mass spectrometer 102 of FIG. 1. Method 500 may be performed independently or as part of post-acquisition data processing, such as by tuner 210 of FIG. 2.

[0063] In embodiments, the second mass separator in the series is faster than the first mass separator. When discussing one mass separator being “faster” than another herein, the first and second mas separate are generally understood to operate at different time scales. The faster of the mass separators is configured to separate and analyze ions more quickly or with a higher throughput. The speed of a mass separator can vary depending on the specific design and technology used. For example, TOF mass analyzers are known for their high-speed capabilities, making them suitable for applications requiring rapid data acquisition. On the other hand, instruments like magnetic sector analyzers may have slower scan rates but offer high mass resolution.

[0064] At 502, raw or otherwise unprocessed mass data is received from a second mass separator in a series. The mass data may include counts or intensities related to detection of a set of ions. The specific contents of raw mass spectrometer data may vary depending on the type of mass spectrometer and the experimental setup. In embodiments, the mass data may include mass-to-charge ratio (m/z) data for each ion detected, an intensity or abundance indicating how many ions of a particular m/z ratio were detected, a time or scan number, an ionization mode, instrument parameters, noise and bassline signals, chromatographic data, TOF data, fragmentation patterns, spectral metadata, and a data format.

[0065] At 504, a known ion is identified in the unprocessed mass data. A known ion may be an representative candidate selected based on predetermined characteristics to define a reliable representative candidate. For example, the known ion may be selected based on a most intense peak identified. Other factors which may be considered include whether the ion is fragmented, whether the ion has collision energy, or whether the ion is a background ion. In embodiments, an unfragmented ion is preferable for the known ion. An ion with low or no collision energy or which is not a background ion may also be preferred in some cases. In embodiments, the known ion may be identified during the processing of the unprocessed mass data. In embodiments, identifying the known ion includes determining a mass of the known ion from the mass data.

[0066] In some cases, a tuning standard is run for selection as the known ion. A sample may be run through without fragmentation for tuning purposes, ensuring a significant number of reliable candidates for the known ion. While an unfragmented ion may generally be preferable for selection as a known ion, in some cases a known fragment ion is selected as the known ion.

[0067] At 506, an actual appearance and an actual disappearance of the known ion is mapped to determine an appearance period of the known ion. An actual appearance of the known ion refers to the first appearance of the known ion in the unprocessed mass data. An actual disappearance of the known ion refers to the last appearance of the known ion in the unprocessed mass data. By mapping each of the actual appearance and the actual disappearance of the known ion, an appearance period, or a measured window during which the known ion appears in the mass data, is determined. This measured window, with the known ion as a reference point, enables determination of the width of the actual ion transmission window applied by the mass spectrometer in the course of the experiment from which the unprocessed mass data is generated. In embodiments, the mapping characterizes an actual transmission window of the first mass separator. Due to the relationship between the actual appearance and disappearance of the known ion and the ion transmission window applied during data acquisition, the appearance period can be used to characterize the actual transmission window applied by the mass spectrometer during data acquisition.

[0068] At 508, the appearance period is compared with an intended appearance period for the known ion. In embodiments, the intended appearance period for the known ion is based on the mass of the known ion. Based on the mass of the known ion, the known ion is expected to appear and disappear at a particular time or point in the mass spectrum, providing an intended appearance window as a reference. The length of the intended appearance window will vary with the width of the ion transmission window. [0069] In embodiments, method 500 is performed in the context of scanning SWATH with the transmission window moving in a series of overlapping steps across a mass range. An actual appearance and an actual disappearance of the known ion, and an appearance period, may be determined for each transmission window of the series. Those of skill in the art will understand that the principles of the method 500 are also applicable in other DIA methods, in which multiple ion transmission windows are stepped across a mass range including those involving overlapping steps.

[0070] When method 500 is performed during a scanning SWATH experiment, the mapping characterizes the series of transmission windows in overlapping steps across the mass range. Each transmission window of the series has an associated intended appearance period for the known ion. The measured appearance period and the intended appearance period may be compared for each transmission window of the series or a portion of the transmission windows of the series. The portion may be a predetermined number of transmission window or a percentage or interval of the total series. For example, the system may be configured to verify tuning be verifying half of the windows of a series, such that every other window of the series is analyzed for consistency between the appearance period and the intended appearance period. In another example, the system is configured to analyze the appearance period for every third, every fourth, every fifth, etc. window of the series. [0071] At 510, a match status between the appearance period and the intended appearance period for the unfragmented mass is determined based on the comparison. A match or mismatch, or a degree of match or mismatch, between the actual appearance period and the intended appearance period will indicate whether the actual ion transmission window is consistent with the acquisition value input by the user. In some cases, the match status is a match between the appearance period and the intended appearance period for the known ion. In other cases, the match status is a mismatch between the appearance period and the intended appearance period for the known ion.

[0072] In embodiments, match status is based on a predetermined threshold. For example, a minimum degree of match, such as 60%, 70%, 75%, 80% 90%, 95%, 99%, etc., is required to determine sufficient consistency exists between the appearance window and the intended appearance window to validate the tuning of the mass spectrometer and yield a match status.

[0073] In embodiments, the degree of consistency or a degree of mismatch may be presented along with or instead of a match or mismatch determination. The match status and/or the degree of mismatch may be presented to the user on an interface or other output device. In embodiments, the match status and/or the degree of mismatch are record and stored in association with mass data. For example, the match status and/or degree of mismatch are recorded into a log file, accessible to a user.

[0074] Consistency between the actual appearance period and the intended appearance period indicates proper tuning and that accurate results can be calculated according to the intended acquisition value. In embodiments, the system may be configured to respond automatically to a match or mismatch status. For example, in response to a mismatch, the system may generate a mismatch alert. In embodiments, the mismatch alert may be an audible or visual indicator that the data calculations may need to be checked or that the mass spectrometer is due for tuning. In a further example, the system may adjust, based on the degree of mismatch, the transmission window of the first mass separator. The adjustment may be a preset incremental adjustment or may be based on the particular degree of mismatch determined.

[0075] In embodiments, the match status includes a width of the actual transmission window. Using the comparison between the appearance period and the intended appearance period, the system can determine the width of the actual ion transmission window. [0076] In some embodiments, the match status includes a function of a shape of the transmission window. In cases where the ion transmission window is not close to square and therefore cannot be assumed to be close to square for calculation purposes without distorting the data, the match status may include determining the shape of the transmission window as a function. The function of the shape may also be compared with the expected transmission window, with a mismatch used to determine if the shape of the transmission window indicates tuning should be addressed.

[0077] At 512, the mass data is processed into a standardized file format. In embodiments, the match status is incorporated into the standardized file format. In embodiments, processing the mass data into the standardized file format is the final data processing step and the standardized file format incorporating the mass data is stored for further processing and user access. The standardized file format may be used to transmit the processed mass data to another device or system for further processing or access. In some embodiments, the processing of the mass spectrum data into the standardized format may be performed in conjunction with tuning processing and analysis by a common component or module, such tuner 210 of FIG. 2. In embodiments, the processing of the mass spectrum data into the standardized format may be performed by a separate component or module, such as file formatter 212 of FIG. 2.

[0078] Match status may be used to validate or correct tuning of the mass spectrometer. In embodiments, an alert may be generated indicating possible problems with the mass spectrum data calculation and/or indicating the mass spectrometer may be in need of tuning. In some cases, match status and/or degree of mismatch may be provided as further data output to a user. The user evaluates their data and/or the mass spectrometer according to the match status and/or degree of mismatch.

[0079] Automatic actions may be initiated in response to the match status. For example, flagging the mass data as being generated with an ion transmission window inconsistent with the acquisition value. The actual ion transmission window may be automatically calculated, and the difference between the actual ion transmission window and the acquisition value appended to the data, stored as an element of the mass data, such as in a log file, and/or reported to a user. In embodiments, processing and analysis of the mass spectrum data may be adjusted according to the measured ion transmission window. In some cases, processing of the mass spectrum data may be able to effectively account for the mismatch in the ion transmission window. For example, if, after acquisition, the ion transmission window measurement is not as expected, the actual transmission window that can be the measured start and stop of the transmission window (based on the actual appearance and disappearance of the known ion) is used to encode the mass spectrum data into the storage file or to perform precursor inference, including real-time precursor inference. In embodiments, a shape of the QI trace (e.g. actual transmission window) is used as an input parameter, instead or in addition to the measured start and stop times, for encoding the mass spectrum data. In these examples, corrected data that includes compensation for improper tuning can be encoded and/or stored.

[0080] In embodiments, responsive actions to the match status include automatic tuning adjustments by the mass spectrometer. For example, using the calculated actual ion transmission window, the ion transmission window may be automatically adjusted, such as to the intended acquisition value. In some cases, an additional run of the sample may automatically be performed following adjustment of the ion transmission window. The timing of the ion transmission window of the additional run may also be verified according to the method 500, with additional adjustments made to the ion transmission window according to the determined match status. Verification and adjustment may proceed iteratively to achieve a satisfactory match status.

[0081] FIG. 6 illustrates an example block diagram of a virtual or physical computing system 150. One or more aspects of the computing system 150 can be used to implement the systems and methods for automatic tuning of a mass spectrometer. In particular, the computing system 150 may be used to implement the data processing system 200 and underlying or integrated components, such as preprocessor 203, peak detector 206, peak identifier 108, tuner 210, and file formatter 212.

[0082] In the embodiment shown, the computing system 150 includes one or more processors 152, a system memory 158, and a system bus 172 that couples the system memory 158 to the one or more processors 152. The system memory 158 includes RAM (Random Access Memory) 160 and ROM (Read-Only Memory) 162. A basic input/output system that contains the basic routines that help to transfer information between elements within the computing system 150, such as during startup, is stored in the ROM 162. The computing system 150 further includes a mass storage device 164. The mass storage device 164 is able to store software instructions and data. The one or more processors 152 can be one or more central processing units or other processors.

[0083] The mass storage device 164 is connected to the one or more processors 152 through a mass storage controller (not shown) connected to the system bus 172. The mass storage device 164 and its associated computer-readable data storage media provide nonvolatile, non-transitory storage for the computing system 150. Although the description of computer-readable data storage media contained herein refers to a mass storage device, such as a hard disk or solid state disk, it should be appreciated by those skilled in the art that computer-readable data storage media can be any available non- transitory, physical device or article of manufacture from which the central display station can read data and/or instructions.

[0084] Computer-readable data storage media include volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable software instructions, data structures, program modules or other data. Example types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROMs, DVD (Digital Versatile Discs), other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the computing system 150.

[0085] According to various embodiments of the invention, the computing system 150 may operate in a networked environment using logical connections to remote network devices through the network 148. The network 148 is a computer network, such as an enterprise intranet and/or the Internet. The network 148 can include a LAN, a Wide Area Network (WAN), the Internet, wireless transmission mediums, wired transmission mediums, other networks, and combinations thereof. The computing system 150 may connect to the network 148 through a network interface unit 154 connected to the system bus 172. It should be appreciated that the network interface unit 154 may also be utilized to connect to other types of networks and remote computing systems. The computing system 150 also includes an input/output controller 156 for receiving and processing input from a number of other devices, including a touch user interface display screen, or another type of input device. Similarly, the input/output controller 156 may provide output to a touch user interface display screen or other type of output device.

[0086] As mentioned briefly above, the mass storage device 164 and the RAM 160 of the computing system 150 can store software instructions and data. The software instructions include an operating system 168 suitable for controlling the operation of the computing system 150. The mass storage device 164 and/or the RAM 160 also store software instructions, that when executed by the one or more processors 152, cause one or more of the systems, devices, or components described herein to provide functionality described herein. For example, the mass storage device 164 and/or the RAM 160 can store software instructions that, when executed by the one or more processors 152, cause the computing system 150 to receive and execute managing network access control and build system processes.

[0087] The software instructions further include one or more software applications 166. Software applications may include dedicated systems and algorithms for performing specific tasks or actions or providing specific interfaces. One or more of data processing system 200 and/or one or more component of data processing system 200 may be encompassed by software applications 166.

[0088] Referring to the figures and examples presented herein generally, the disclosed environment provides a physical environment with which aspects of the outcome prediction and trade-off analysis systems can be implemented.

[0089] Having described the preferred aspects and implementations of the present disclosure, modifications and equivalents of the disclosed concepts may readily occur to one skilled in the art. However, it is intended that such modifications and equivalents be included within the scope of the claims which are appended hereto.

Claims

What is claimed is:

1. A method for evaluating tuning of a mass measuring system including a first mass separator in series with a second mass separator, wherein the second mass separator is faster than the first mass separator, the method comprising: receiving unprocessed mass data of detection of a set of ions from the second mass separator; identifying a known ion in the unprocessed mass data; mapping an actual appearance and an actual disappearance of the known ion to determine an appearance period of the known ion; comparing the appearance period with an intended appearance period for the known ion; and determining, based on the comparing, a match status between the appearance period and the intended appearance period for the unfragmented mass.

2. The method of claim 1, further comprising: processing the unprocessed mass data into a standardized file format; and incorporating the match status into the standardized file format.

3. The method of claim 1 or 2, further comprising determining a mass of the known ion.

4. The method of claim 3, wherein the intended appearance period for the known ion is based on the mass of the known ion.

5. The method of any preceding claim, wherein the mapping characterizes an actual transmission window of the first mass separator.

6. The method of claim 5, wherein the match status comprises a width of the actual transmission window.

7. The method of claim 5, wherein the match status comprises a function of a shape of the transmission window.

8. The method of claim 5, further comprising moving the transmission window in a series of overlapping steps across a mass range, wherein the mapping characterizes a series of transmission windows of the series of overlapping steps across the mass range.

9. The method of any preceding claim, wherein the match status comprises a match between the appearance period and the intended appearance period for the known ion.

10. The method of any preceding claim, wherein the match status comprises a mismatch between the appearance period and the intended appearance period for the known ion.

11. The method of claim 10, further comprising generating, in response to the mismatch, a mismatch alert.

12. The method of claim 10, further comprising determining, in response to the mismatch, a degree of mismatch between the appearance period and the intended appearance period for the known ion.

13. The method of claim 12, further comprising adjusting, based on the degree of mismatch, the transmission window of the first mass separator.

14. The method of claim 10, further comprising encoding, in response to the mismatch, the mass spectrum data using a measured start and stop of the transmission window, based respectively on the actual appearance and disappearance of the known ion.

15. The method of claim 10, further comprising encoding, in response to the mismatch, the mass spectrum data using a shape of the QI trace is used as an input parameter.

16. A system for tuning a mass spectrometer comprising: a first mass separator; a second mass separator in series with the first mass separator, the second mass separator being faster than the first mass separator, wherein each of the first and second mass separator is configured to: receive a set of ions; perform a detection of the set of ions; and generate a set of detection signals corresponding to detection of the set of ions; and a controller configured to: receive unprocessed mass data from second mass separator including the set of detection signals; identify an known ion in the unprocessed mass data; map an actual appearance and an actual disappearance of the known ion to determine an appearance period of the known ion; compare the appearance period with an expected appearance period for the unfragmented mass; and determine, based on the comparing, a match status between the appearance period and the expected appearance period for the known ion.

17. The system of claim 16, wherein the controller is further configured to: process the mass data into a standardized file format; and incorporate the match status into the standardized file format.

18. The system of claim 17, wherein the controller is further configured to store the standardized file format.

19. The system of claim 17, wherein the controller is further configured to transmit the standardized file format.

20. The system of any of claims 16-19, wherein the second mass separator is a time of flight mass analyzer.

21. The system of claim 20, wherein the first mass separator is a quadrupole.

22. A system for tuning a first mass separator with a scanning transmission window, the system comprising: a second mass separator, in series with the first mass separator and configured to: receive a set of ions; perform a detection of the set of ions; and generate a set of detection signals corresponding to the detection of the set of ions; and a controller configured to: receive unprocessed mass data including the set of detection signals; identify an known ion in the unprocessed mass data; map an actual appearance and an actual disappearance of the known ion to determine an appearance period of the known ion; compare the appearance period with an expected appearance period for the known ion; and determine, based on the comparing, a match status between the appearance period and the expected appearance period for the known ion, wherein the match status characterizes the scanning transmission window.

23. A method for processing data of a mass measuring system including a first mass separator in series with a second mass separator, wherein the second mass separator is faster than the first mass separator, the method comprising: receiving unprocessed mass data of detection of a set of ions from the second mass separator; identifying a known ion in the unprocessed mass data; mapping an actual appearance and an actual disappearance of the known ion to determine an actual transmission window of the known ion.

24. The method of claim 23, further comprising encoding the mass data using the actual transmission window as an input parameter.

25. The method of claim 24, further comprising encoding the mass data using a shape of the actual transmission window as an input parameter.