using a mass spectrometer to perform absolute quantification of a target analyte

Abstract

A targeted quantitation system for mass spectrometry analysis can acquire an observation mode mass spectrum when operating in an observation mode, the observation mode mass spectrum including mass peaks of ions generated from a plurality of internal standard variants added to a sample including a target analyte. Each internal standard variant included in the plurality of internal standard variants includes a unique isotopologue of the target analyte and is added to the sample in a unique amount. The targeted quantitation system can generate a calibration curve based on the observation mode mass spectrum. The targeted quantitation system can acquire a quantitation mode mass spectrum when operating in a quantitation mode, the quantitation mode mass spectrum including mass peaks of ions generated from the target analyte included in the sample. The targeted quantitation system can determine a concentration of the target analyte included in the sample based on the calibration curve and the second mass spectrum.

Description

[0001] Priority Information

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 031,188, filed May 25, 2020, and entitled “SYSTEMS AND METHODS FOR ABSOLUTE QUANTITATION OF A TARGET ANALYTE,” the contents of which are hereby incorporated by reference in their entirety. BACKGROUND

[0003] A mass spectrometer is a sensitive instrument that can be used to detect, identify, and / or quantify molecules based on their mass-to-charge (m / z) ratios. A mass spectrometer (MS) generally comprises an ion source for generating ions from a sample, a mass analyzer for separating the ions based on their mass-to-charge ratios, and an ion detector for detecting the separated ions. The mass spectrometer can be connected to a computer-based software platform that uses data from the ion detector to construct a mass spectrum showing the relative abundance of each of the detected ions as a function of their mass-to-charge ratios. The measured ion mass-to-charge ratios can be used to detect and quantify molecules in simple and complex mixtures.

[0004] However, selective detection and quantification of a particular target molecule of interest in a complex mixture is often very difficult, even with targeted acquisition. For example, in proteomics studies, a peptide target of interest can be contained in a complex biological matrix composed of a mixture of tens of thousands of peptides, with the abundances of the peptides spanning many orders of magnitude. Tandem mass spectrometry can be used to quantify target molecules in complex mixtures. For example, in a refined targeted acquisition technique known as internal standard-triggered parallel reaction monitoring (IS-PRM), a sample containing a peptide target can be spiked with a known amount of a corresponding internal standard (IS) (e.g., a synthetic peptide with the same amino acid sequence but containing one or more heavy stable isotopes) and scanned by the instrument in a dual mode combining single-stage and tandem mass spectrometry. Detection of the internal standard triggers the mass spectrometer to monitor the particular peptide target of interest.

[0005] However, this refined targeted acquisition technique can suffer from several limitations. For example, the technique suffers from false-positive triggers due to the presence of near-isobaric co-eluting interferences in the complex sample that mimic the accurate mass of the internal standard during the scan phase. Additionally, absolute quantification of the target analyte requires extrapolation or interpolation from a calibration curve established from independent analyses of the internal standard collected from multiple additional mass spectrometry experiments. SUMMARY

[0006] The following description presents a simplified summary of one or more aspects of the methods and systems described herein in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, and is intended to neither identify key or critical elements of all aspects nor delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects of the methods and systems described herein in a simplified form as a prelude to the more detailed description that is presented later.

[0007] In some example embodiments, a system includes a memory storing instructions and a processor communicatively coupled to the memory and configured to execute the instructions to: acquire an observation mode mass spectrum when operating in an observation mode, the observation mode mass spectrum including mass peaks of ions produced from a plurality of internal standard variants added to a sample including a target analyte, each internal standard variant included in the plurality of internal standard variants including a unique isotopologue of the target analyte and added to the sample in a unique amount; generate a calibration curve based on the observation mode mass spectrum; acquire a quantitation mode mass spectrum when operating in a quantitation mode, the quantitation mode mass spectrum including mass peaks of ions produced from the target analyte included in the sample; and determine a concentration of the target analyte included in the sample based on the calibration curve and the quantitation mode mass spectrum.

[0008] In some example embodiments, the processor is further configured to execute the instructions to, when operating in the observation mode, acquire mass spectra during an analytical run of the sample, the mass spectra including the observation mode mass spectrum; analyze the mass spectra based on a set of criteria associated with the plurality of internal standard variants; and determine, based on the analysis, that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standard variants added to the sample.

[0009] In some example embodiments, acquiring the quantitation mode mass spectrum is performed in response to determining that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standard variants added to the sample.

[0010] In some example embodiments, generating the calibration curve is performed in response to determining that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standard variants added to the sample.

[0011] In some example embodiments, determining that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standard variants added to the sample includes determining that a set of mass peaks included in the observation mode mass spectrum satisfies the set of criteria, the set of mass peaks including the mass peaks of the ions produced from the plurality of internal standard variants.

[0012] In some example embodiments, the set of criteria includes one or more of a mass-to-charge ratio, a signal intensity, or a peptide sequence of each of the plurality of internal standard variants added to the sample.

[0013] In some example embodiments, the observation mode includes a basic observation mode and an enhanced observation mode, the observation mode mass spectrum includes an enhanced observation mode mass spectrum, and the processor is further configured to execute the instructions to acquire a basic observation mode mass spectrum when operating in the basic observation mode, the basic observation mode mass spectrum containing mass peaks of precursor ions produced from the plurality of internal standard variants; in response to acquiring the basic observation mode mass spectrum, direct the mass spectrometry analysis system to selectively fragment the precursor ions into a plurality of fragment ions, the plurality of fragment ions including the ions produced from the plurality of internal standard variants; and acquire the enhanced observation mode mass spectrum from a scan of the plurality of fragment ions when operating in the enhanced observation mode.

[0014] In some example embodiments, the processor is further configured to execute the instructions to direct the mass spectrometry analysis system to scan the ions produced from the plurality of internal standard variants added to the sample according to a first mode of operation such that the observation mode mass spectrum is generated when the mass spectrometry analysis system operates in the first mode of operation; and direct the mass spectrometry analysis system to scan the ions produced from the target analyte contained in the sample according to a second mode of operation such that the quantitation mode mass spectrum is generated when the mass spectrometry analysis system operates in the second mode of operation.

[0015] In some example embodiments, the method includes acquiring, by a targeted quantitation system when operating in an observation mode, an observation mode mass spectrum containing mass peaks of ions produced from a plurality of internal standard variants added to a sample including a target analyte, each of the plurality of internal standard variants including a unique isotopologue of the target analyte and added to the sample in a unique amount; generating, by the targeted quantitation system, a calibration curve based on the observation mode mass spectrum; acquiring, by the targeted quantitation system when operating in a quantitation mode, a quantitation mode mass spectrum containing mass peaks of ions produced from the target analyte contained in the sample; and determining, by the targeted quantitation system, a concentration of the target analyte contained in the sample based on the calibration curve and the quantitation mode mass spectrum.

[0016] In some example embodiments, a non-transitory computer readable medium stores instructions that, when executed, direct at least one processor of a computing device of a mass spectrometry analysis system to: acquire an observation mode mass spectrum when operating in an observation mode, the observation mode mass spectrum including mass peaks of ions produced from a plurality of internal standard variants added to a sample including a target analyte, each internal standard variant included in the plurality of internal standard variants including a unique isotopologue of the target analyte and added to the sample in a unique amount; generate a calibration curve based on the observation mode mass spectrum; acquire a quantitation mode mass spectrum when operating in a quantitation mode, the quantitation mode mass spectrum including mass peaks of ions produced from the target analyte included in the sample; and determine a concentration of the target analyte included in the sample based on the calibration curve and the quantitation mode mass spectrum. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings illustrate various embodiments and are a part of the specification. The illustrated embodiments are merely examples and do not limit the scope of the disclosure. Throughout the drawings, the same or like reference numerals designate the same or like elements. Furthermore, the drawings are not necessarily to scale as one or more of the elements shown in the drawings can be expanded or sized for ease of discussion and illustration.

[0018] Figure 1A Functional components of an example mass spectrometer are illustrated.

[0019] Figure 1B Functional components of an example tandem mass spectrometer are illustrated.

[0020] Figure 2 An example combined separation and mass spectrometer system is illustrated.

[0021] Figure 3 An example targeted quantitation system configured to quantify a target analyte included in a sample is illustrated.

[0022] Figure 4 An example implementation of a targeted quantitation system of Figure 3 is illustrated.

[0023] Figure 5 An example flowchart of operations performed by a targeted quantitation system of Figure 3 during a targeted quantitation analysis of a target analyte included in a sample is illustrated.

[0024] Figures 6-8 Various example flowcharts of operations performed by a targeted quantitation system of Figure 3 during a targeted quantitation analysis of a target analyte are illustrated.

[0025] Figure 9An exemplary workflow depicting operations associated with targeted quantitative analysis of a sample is shown, as performed by a targeted quantitation system and a combined liquid chromatography-mass spectrometry analysis system of Figure 3

[0026] An exemplary mass spectrum is shown that includes a predetermined mass peak pattern comprising a plurality of mass peaks of ions to be produced from a set of five internal standard variants added to a sample. Figure 10A

[0027] An exemplary mass spectrum acquired during targeted quantitative analysis is shown. Figure 10B

[0028] An exemplary method of quantifying a target analyte contained in a sample is shown. Figure 11

[0029] Another exemplary method of quantifying a target analyte contained in a sample is shown. Figure 12

[0030] An exemplary computing device is shown that can be specifically configured to perform one or more of the processes described herein. Figure 13 DETAILED DESCRIPTION

[0031] Systems and methods for absolute quantification of a target analyte are described herein. In some embodiments, a plurality of different isotopomer variants of an internal standard variant corresponding to the target analyte are added to a sample that includes the target analyte. Each internal standard variant can include a unique isotopomer of the target analyte. Further, each internal standard variant is added to the sample in a unique amount (e.g., at a different concentration in the sample). A targeted quantitation system can acquire an observation mode mass spectrum when operating in an observation mode, the observation mode mass spectrum containing mass peaks of ions produced from the plurality of internal standard variants. The targeted quantitation system can generate a calibration curve based on the observation mode mass spectrum. When operating in a quantification mode, the targeted quantitation system can also acquire a quantification mode mass spectrum, the quantification mode mass spectrum containing mass peaks of ions produced from the target analyte contained in the sample. The targeted quantitation system can determine a concentration of the target analyte contained in the sample based on the calibration curve and the quantification mode mass spectrum.

[0032] ​The systems and methods described herein can provide various benefits, which can include one or more advantages over conventional targeted quantitation systems and methods. For example, a single sample can be used to generate a calibration curve and to quantify target analytes contained in the sample. This eliminates the need to perform separate experiments to generate a calibration curve. Furthermore, detection and quantitation of target analytes can depend on detection of multiple different internal standard variants, thereby reducing the occurrence of false positive triggers as compared to existing internal standard variant-triggered techniques that rely on a single internal standard variant trigger per target analyte. Moreover, due to the high specificity provided by detection of multiple internal standard variants, the use of a signal intensity threshold for triggering targeted acquisition can be eliminated, thereby improving the efficiency of targeted quantitation analysis.

[0033] The targeted quantitation systems and methods described herein can be implemented in conjunction with a mass spectrometer. Figure 1A Functional components of an example mass spectrometer 100A are shown. The example mass spectrometer 100A is illustrative and not limiting. As shown, the mass spectrometer 100A includes an ion source 102, a mass analyzer 104, and a controller 106. The mass spectrometer 100A can further include any additional or alternative components (not shown) that can be suitable for a particular implementation (e.g., ion optics, filters, autosamplers, etc.).

[0034] The ion source 102 is configured to generate a plurality of ions 108 from a sample to be analyzed and to deliver the ions to the mass analyzer 104. The ion source 102 can use any suitable ionization technique, including but not limited to electron ionization, chemical ionization, matrix-assisted laser desorption / ionization, electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, inductively coupled plasma, etc. The ion source 102 can include various components for generating ions from a sample and delivering the generated ions 108 to the mass analyzer 104.

[0035] The mass analyzer 104 is configured to separate the ions 108 according to a mass-to-charge ratio (m / z) of each of the ions. The mass analyzer 104 can be implemented, for example, by a quadrupole mass filter, an ion trap (e.g., a three-dimensional quadrupole ion trap, a cylindrical ion trap, a linear quadrupole ion trap, a toroidal ion trap, etc.), a time-of-flight (TOF) mass analyzer, an electrostatic trap mass analyzer (e.g., an orbital electrostatic trap such as an Orbitrap mass analyzer, a Kingdon trap, etc.), a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, a sector mass analyzer, etc.

[0036] In some examples, the mass analyzer 104 can include one or more multipole rod assemblies having a plurality of rod electrodes (e.g., quadrupole rods, hexapole rods, octapole rods, etc.) for guiding, trapping, and / or filtering ions. In a quadrupole rod, opposite phases of an RF voltage can be applied to pairs of rod electrodes, thereby generating a quadrupole rod electric field that guides or traps ions within a central region of the quadrupole rod. In a quadrupole rod mass filter, a mass resolving direct current (DC) voltage can also be applied to pairs of rod electrodes, thereby superimposing a DC electric field on the quadrupole rod electric field and causing the trajectories of some ions to become unstable and cause the unstable ions to discharge against one of the rod electrodes. In such a quadrupole rod mass filter, only ions having a certain mass-to-charge ratio will maintain a stable trajectory and traverse the length of the quadrupole rod, where the ions are emitted from the mass filter and subsequently detected by an ion detector included in the mass analyzer 104. In some examples, the mass analyzer 104 can be coupled to an oscillating voltage power supply (not shown) configured to supply an RF voltage to the multipole rod assembly, and can be coupled to a DC voltage power supply configured to supply a mass resolving DC voltage to the multipole rod assembly.

[0037] The mass analyzer 104 includes an ion detector configured to detect ions of each of the various different mass-to-charge ratios separated by the mass analyzer 104 and responsively generate an electrical signal representative of the intensity of the ions (the number of ions) or the relative abundance of the ions. The electrical signal is transmitted to the controller 106 for processing in order to construct a mass spectrum of the sample. For example, the mass analyzer 104 can emit a beam of the separated ions to an ion detector configured to detect the ions in the beam and generate or provide data that can be used by the controller 106 to construct a mass spectrum of the sample. The ion detector can be implemented by any suitable detection means, including but not limited to an electron multiplier, a Faraday cup, etc.

[0038] The controller 106 can be communicatively coupled with the ion source 102 and / or the mass analyzer 104 and configured to control the operation of the ion source and / or the mass analyzer. For example, the controller 106 can be configured to control the operation of various hardware components included in the ion source 102 and / or the mass analyzer 104. To illustrate, the controller 106 can be configured to control the fill time of the ion source 102 and / or the mass analyzer 104. The controller 106 can be further configured to control an oscillating voltage power supply and / or a DC power supply for supplying an RF voltage and / or a DC voltage to the mass analyzer 104 (e.g., to the multipole rod assembly included in the mass analyzer 104), and adjust the values of the RF voltage and the DC voltage to select an effective range of mass-to-charge ratios of ions to be detected. The controller 106 can also adjust the sensitivity of the ion detector, e.g., by adjusting a detector gain.

[0039] The controller 106 can also include and / or provide a user interface configured to enable interaction between a user of the mass spectrometer 100A and the controller 106. The user can interact with the controller 106 via the user interface through tactile, visual, audible, and / or other sensory types of communication. For example, the user interface can include a display device (e.g., a liquid crystal display (LCD) display screen, a touch screen, etc.) for displaying information (e.g., mass spectra, notifications, etc.) to the user. The user interface can also include an input device (e.g., a keyboard, a mouse, a touch screen device, etc.) that allows the user to provide input to the controller 106. In other examples, the display device and / or the input device can be separate from but communicatively coupled to the controller 106. For example, the display device and the input device can be included in a computer (e.g., a desktop computer, a laptop computer, etc.) that is communicatively connected to the controller 106 through a wired connection (e.g., through one or more electrical cables) and / or a wireless connection.

[0040] The controller 106 can include hardware (e.g., a processor, circuitry, etc.) and / or software configured to control the operation of the various components of the mass spectrometer 100A. Although FIG. 1 shows the controller 106 included in the mass spectrometer 100A, the controller 106 can alternatively be implemented in a manner that is completely or partially separate from the mass spectrometer 100A, such as through a computing device that is communicatively coupled to the mass spectrometer 100A through a wired connection (e.g., an electrical cable) and / or a network (e.g., a local area network, a wireless network (e.g., Wi-Fi), a wide area network, the Internet, a cellular data network, etc.).

[0041] Operation of the mass spectrometer 100A will now be described. During mass analysis of a sample, the controller 106 directs the ion source 102 to generate ions 108 from the sample and to deliver the ions 108 to the mass analyzer 104. The controller 106 directs the mass analyzer 104 to scan across a range of mass-to-charge ratios to selectively filter the generated ions according to their mass-to-charge ratios. At any given point in time during the analysis scan, the ions detected in the mass analyzer 104 have the selected mass-to-charge ratio. The mass spectrometer 100A can operate in a mode in which the selected mass-to-charge ratio gradually increases (or gradually decreases) during the scan. Alternatively, the mass spectrometer 100A can operate in a mode in which the selected mass-to-charge ratio does not gradually increase (or decrease), but is constant or discontinuous, such as in a selected ion monitoring (SIM) mode or a selected reaction monitoring (SRM) mode.

[0042] The detector detects the ion intensity (number) of ions of each mass-to-charge ratio scanned in the mass analyzer 104. The detector generates an electrical signal (ion signal) corresponding to the detected ion intensity and transmits the ion signal to the controller 106, which can save the data, process the data, and / or generate a mass spectrum based on the data.

[0043] In some embodiments, the mass spectrometer can include a tandem mass spectrometer. Figure 1B Functional components of an example tandem mass spectrometer 100B are shown. The example tandem mass spectrometer 100B is illustrative and not limiting. The mass spectrometer 100B is the same as the mass spectrometer 100A except that the mass analyzer 104 is replaced with a first mass analyzer 104-1, a collision cell 104-2, and a second mass analyzer 104-3. The first mass analyzer 104-1 is configured to receive the ions 108 produced by the ion source 102 and produce precursor ions 110. The collision cell 104-2 is configured to receive the precursor ions 110 and produce product ions 112 (e.g., fragment ions) from the precursor ions 110. The second mass analyzer 104-3 is configured to scan and / or filter the product ions 112 from the collision cell 104-2. The mass analyzers 104-1 and 104-3 can be implemented by any suitable mass analyzer and need not be implemented by the same type of mass analyzer. The collision cell 104-2 can be implemented by any suitable collision cell. As used herein, the term “collision cell” can encompass any structure arranged to produce product ions by a controlled dissociation process and is not limited to devices for collision-activated dissociation. For example, the collision cell 104-2 can be configured to fragment the precursor 110 ions using collision-induced dissociation, electron transfer dissociation, electron capture dissociation, photo-induced dissociation, surface-induced dissociation, ion / molecule reactions, etc.

[0044] Although the mass spectrometer 100B is shown as a spatial tandem mass spectrometer, the mass spectrometer 100B can alternatively be implemented as a temporal tandem mass spectrometer.

[0045] In some embodiments, the mass spectrometer 100 (e.g., the mass spectrometer 100A or 100B, each of which is referred to herein as the mass spectrometer 100) can be coupled with a separation system configured to separate components of a sample to be analyzed by the mass spectrometer 100. Figure 2 An example combined separation and mass spectrometer system 200 (“combined system 200”) is shown. As shown, the combined system 200 includes a separation system 202, a mass spectrometer 204, and a controller 206. The combined system 200 can include additional or alternative components that can be suitable for a particular implementation.

[0046] In an analytical run performed by the combined system 200, the separation system 202 is configured to receive a sample to be analyzed and separate certain components within the sample. In some examples, the separation system 202 can also detect the relative abundance of the separated components, such as by generating a chromatogram representing the components within the sample. The separation system 202 can be implemented by any device configured to separate components contained in a sample, such as a liquid chromatograph (LC) (e.g., a high performance liquid chromatograph (HPLC)), a gas chromatograph (GC), an ion chromatograph, a capillary electrophoresis system, etc. The components 208 separated by the separation system 202 are delivered to the mass spectrometer 204 for mass analysis by the mass spectrometer 204.

[0047] For example, in an LC, a sample can be injected into a mobile phase (e.g., a solvent) that carries the sample through a column containing a stationary phase (e.g., a sorbent packing material). As the mobile phase passes through the column, components 208 within the sample elute from the column at different times based on, for example, their size, their affinity for the stationary phase, their polarity, and / or their hydrophobicity. The retention time of a component 208 can also be influenced by LC conditions, such as the flow rate and solvent composition of the mobile phase. A detector (e.g., a spectrophotometer) can measure the relative intensity of a signal modulated by each separated component (eluent) in the effluent from the column and represent the signal as a chromatogram. In some examples, the relative intensity can be correlated to or represent the relative abundance of the separated component. The data generated by the LC can be output to the controller 206.

[0048] The mass spectrometer 204 can be implemented by any suitable type of mass spectrometer (e.g., the mass spectrometer 100). Thus, the combined system 200 can include, for example, an LC-MS system, an LC-MS / MS system, a GC-MS system, a GC-MS / MS system, etc.

[0049] The controller 206 is communicatively coupled with the combined system 200 (e.g., the separation system 202 and / or the mass spectrometer 204) and is configured to control the operation of the combined system. The controller 206 can include hardware (e.g., a processor, circuitry, etc.) and / or software configured to control the operation of the various components of the combined system 200. Although the controller 206 is shown as a single device, the controller 206 can include multiple devices that are communicatively coupled with one another and / or with the various components of the combined system 200. Figure 2The controller 206 is shown as being included in the combined system 200, but the controller 206 can alternatively be implemented in a manner that is entirely or partially separate from the combined system 200, such as by a computing device that is communicatively coupled to the combined system 200 by a wired connection (e.g., a cable) and / or a network (e.g., a local area network, a wireless network (e.g., Wi-Fi), a wide area network, the Internet, a cellular data network, etc.). In instances in which the mass spectrometer 204 is implemented by the mass spectrometer 100, the controller 206 can be implemented entirely or partially by the controller 106.

[0050] Figure 3 An exemplary targeted quantification system 300 (“system 300”) is shown that is configured to quantify target analytes included in a sample analyzed by the mass spectrometer 100 or the combined system 200. As shown, the system 300 can include, without limitation, a storage facility 302 and a processing facility 304 that are selectively and communicatively coupled to one another. The system 300 (e.g., the facilities 302 and 304) can include or be implemented by hardware and / or software components (e.g., processors, memories, communication interfaces, instructions stored in a memory for execution by a processor, etc.). In some instances, the facilities 302 and 304 can be distributed among multiple devices and / or multiple locations that can serve a particular implementation. Figure 3

[0051] The storage facility 302 can maintain (e.g., store) executable data used by the processing facility 304 to perform any of the operations described herein. For example, the storage facility 302 can store instructions 306 that can be executed by the processing facility 304 to perform any of the operations described herein. The instructions 306 can be implemented by any suitable application, software, code, and / or other executable data instance. The storage facility 302 can also maintain any data received, generated, managed, used, and / or transmitted by the processing facility 304. For example, the storage facility 302 can maintain standard data, mass spectrometry data, and / or calibration data.

[0052] The processing facility 304 can be configured to perform (e.g., execute the instructions 306 stored in the storage facility 302 to perform) various processing operations associated with the targeted quantification of target analytes included in a sample. These and other operations that can be performed by the processing facility 304 are described herein. In the following description, any reference to an operation performed by the system 300 can be understood as being performed by the processing facility 304 of the system 300.

[0053] Figure 4 ​An exemplary embodiment 400 of the system 300 is shown. As shown, the system 300 is communicatively coupled with a liquid chromatography-mass spectrometry analysis system 402 ("LC-MS system 402"). The LC-MS system 402 includes a combined system (e.g., the combined system 200). As shown, the LC-MS system 402 includes a liquid chromatography system 404 ("LC 404") and a mass spectrometer 406 ("MS 406"). The LC 404 can be implemented by any suitable liquid chromatography system, and the MS 406 can be implemented by any suitable mass spectrometer (e.g., the mass spectrometer 100A or the tandem mass spectrometer 100B). In alternative embodiments, the LC-MS system 402 can be replaced with a GC-MS system or a standalone mass spectrometer (e.g., only the MS 406).

[0054] The system 300 is communicatively coupled to the LC-MS system 402 by a bidirectional communication link 408. The communication link 408 can be implemented by any suitable wired and / or wireless communication connection(s) and / or network(s) (e.g., local area network, wireless network (e.g., Wi-Fi), wide area network, the Internet, cellular data network, etc.) that can serve a particular embodiment. The system 300 can use the communication link 408 to control certain operations of the LC-MS system 402 and to receive signals and / or data from the LC-MS system 402. As shown, the system 300 is separate from the LC-MS system 402. In alternative embodiments, the system 300 is implemented entirely or partially as part of the LC-MS system 402. For example, the system 300 can be implemented entirely or partially by the controller 106 of the mass spectrometer 100 or by the controller 206 of the combined system 200.

[0055] The LC-MS system 402 can perform an analysis run on a sample 410 containing target analytes 412. The sample 410 can be any suitable sample, such as a sample collected from a body or from an environment, a synthetic composition, etc. The sample 410 can include, for example, nucleotides, peptides, steroids, hormones, dyes, alcohols, phenols, alkaloids, organic acids, fatty acids, amino acids, amines, polyamines, nucleosides, prostaglandins, carotenoids, eicosanoids, esters, fragrances, terpenes, waxes, volatiles, flavonoids, lipids, etc. The target analytes 412 can be any individual component included in the sample 410. For example, in a targeted proteomics analysis, the sample 410 can be collected from a cell, tissue, or other biological source, and the target analytes 412 can be particular peptides included in the sample 410 (e.g., peptides generated by proteolytic digestion of proteins included in the sample 410).

[0056] To facilitate the targeted quantitation operations performed by the system 300, the sample 410 is spiked with a known but unique (different) amount of a plurality of internal standard variants (“IS variants”) 414-1 through 414-3. For example, the amount of IS variant 414-1 is different than the amount of IS variant 414-2 and the amount of IS variant 414-3, and the amount of IS variant 414-2 is different than the amount of IS variant 414-3. Each IS variant corresponds to a target analyte 412. Although Figure 4 Three IS variants 414 are shown as added to the sample 410, but any other suitable number of IS variants 414 greater than one can be added to the sample 410.

[0057] The IS variants 414 can be implemented by any suitable compound. In some examples, the IS variants 414 are isotopologues of the target analytes 412 (e.g., different isotopologue forms of unique amino acid sequences, signature peptides, protein-type peptides, etc.). For example, when the target analytes 412 are protein-type peptides, the IS variants 414 can be stable isotope labeled (SIL) peptides (e.g., peptides labeled with stable, non-radioactive isotopes, such as 13 C (carbon-13), 15 N (nitrogen-15), and 2 H (deuterium)). The IS variants 414 co-elute with the corresponding target analytes 412 from the LC 404 due to their similar structures. Thus, as will be described in greater detail below, the system 300 can determine that the target analytes 412 are also present and available for detection and targeted quantitation based on the detection of the plurality of IS variants 414 in the mass spectra acquired from the LC-MS system 402.

[0058] The mass of each IS variant 414 is different than the mass of the other IS variants 414. For example, each IS variant 414 can include a different isotopologue of the target analyte 412. Thus, the mass of IS variant 414-1 is different than the mass of IS variants 414-2 and 414-3; the mass of IS variant 414-2 is different than the mass of IS variants 414-1 and 414-3; and the mass of IS variant 414-3 is different than the mass of IS variants 414-1 and 414-2. The mass of the target analyte 412 is also different than the mass of the IS variants 414. Thus, the target analyte 412 and each IS variant 414 will have independently detectable mass peaks in the mass spectra acquired during an analysis run of the sample 410.

[0059] In addition to the different masses, each IS variant 414 is added to the sample 410 in a known but unique amount. That is, the concentration of each IS variant 414 within the sample 410 is different from the concentration of the other IS variants 414 within the sample 410. For example, IS variant 414-1 can be added to the sample 410 in a first concentration (e.g., 100 femtomole per liter (fmol / L)), IS variant 414-2 can be added to the sample 410 in a second concentration (e.g., 10 fmol / L), and IS variant 414-3 can be added to the sample 410 in a third concentration (e.g., 0.1 fmol / L). Thus, in the mass spectra acquired during the analysis run, the mass peaks corresponding to each IS variant 414 will have different signal intensities.

[0060] The IS variants 414 can be added to the sample 410 in any suitable manner. For example, the IS variants 414 can be manually added to the sample 410 prior to injection of the sample 410 into the LC-MS system 402. Alternatively, the IS variants 414 can be automatically added to the sample 410 by an autosampler included in the LC-MS system 402.

[0061] In some examples, the IS variants 414 are included in a kit designed for targeted quantitative analysis. The kit can include a plurality of containers having predetermined but different concentrations of the IS variants 414. In some examples, the containers can also include labels that provide information about the particular IS variant 414 (e.g., the mass, quantity, and / or ID of the IS variant in the container). The IS variant containers and sample containers (for the sample 410) can be placed in an autosampler of the LC-MS system 402, which can automatically cause the sample 410 to be spiked with the IS variants 414. In some examples, the kit can also include hardware (e.g., the system 300) and / or software (e.g., executable instructions) that can be executed by the system 300 to perform the operations described herein.

[0062] The LC-MS system 402 can perform an analysis run (e.g., a chromatographic run and mass analysis) on the sample 410 containing the target analytes 412 and the IS variants 414 added to the sample 410. During the analysis run, the system 300 can perform various operations associated with the targeted quantification of the target analytes 412, as will now be described.

[0063] Figure 5 An example flowchart 500 illustrating operations performed by the system 300 during targeted quantitative analysis of the target analytes 412 is shown. Although Figure 5 Example operations in accordance with one embodiment are illustrated, but other embodiments can omit, add to, reorder, and / or modify any of the operations shown in Figure 5 Any of the operations illustrated in FIG. 5 can be performed by the system 300.

[0064] In step 502, the system 300 obtains a set of criteria 504 associated with the IS variants 414 and / or the target analyte 412. The criteria 504 include values for one or more parameters that can be detected by the LC-MS system 402 (e.g., by the MS 406) and that, when detected, can indicate in real-time the presence of the IS variants 414 and / or the target analyte 412 in the MS 406 (e.g., can indicate that the IS variants 414 and / or the target analyte 412 eluted from the LC 404 and entered the MS 406). As will be explained in greater detail below, real-time detection of the IS variants 414 can trigger a quantification phase in which the LC-MS system 402 can be configured for operation parameters for detection and quantification of the target analyte 412.

[0065] The parameters of the criteria 504 can include any suitable parameters that can be detected by the LC-MS system 402. In some examples, the parameters include a mass-to-charge ratio (m / z) of one or more ions produced from each IS variant 414, an expected signal intensity of one or more mass peaks of the ions produced from each IS variant 414, and / or a peptide sequence of the IS variant 414. The criteria can specify a particular value or range of values for each parameter. For example, the criteria 504 for targeted quantification of a particular peptide target can include specific values for the mass-to-charge ratio (e.g., 606.824 + / - 0.03), the minimum signal intensity (e.g., > 22,000), and / or the peptide sequence (e.g., LCDSGELVAIK) of the IS variant 414-1, as well as specific values for the mass-to-charge ratio, signal intensity, and peptide sequence of the IS variants 414-2 and 414-3.

[0066] The system 300 can obtain the criteria 504 in any suitable manner. In some examples, the system 300 can provide a graphical user interface (GUI) through which a user can input the parameter values. The system 300 can receive user input indicating each of the values for the parameters. In additional or alternative examples, the system 300 can obtain values for one or more of the parameters automatically (e.g., without user input of the parameter values). For example, certain parameter values can be provided or encoded (e.g., as a barcode, QR code, etc.) on a label attached to the sample container and / or the IS variant container. The parameter values can be obtained by the LC-MS system 402 (e.g., in an autosampler) or a scanner included in the system 300 scanning the label code. In yet additional examples, the system 300 can access one or more parameter values from a database (local or remote) based on a target analyte identifier (ID), IS variant ID, etc., which can be input by a user through the GUI or automatically identified, as described above.

[0067] When operating in the watch mode of targeted quantitative analysis, the system 300 monitors mass spectra generated during an analysis run to detect elution of the IS variant 414 and the target analyte 412 from the LC 404 in real-time. In the watch mode, the system 300 acquires a mass spectrum 508 (“watch mode mass spectrum”) generated during an analysis run of the sample 410 in step 506. The system 300 can acquire the mass spectrum 508 in any suitable manner. In some examples, the system 300 receives data and / or ion intensity signals from the LC-MS system 402 (e.g., from an ion detector) and generates the mass spectrum 508 based on the received data and / or signals. Alternatively, the LC-MS system 402 generates the mass spectrum 508 and transmits data representing the mass spectrum 508 to the system 300.

[0068] The mass spectrum 508 can be generated from scans by any mass analyzer included in the LC-MS system 402 when operating in the watch mode. For example, when the LC-MS system 402 includes the tandem mass spectrometer 100B, the mass spectrum 508 can be generated from scans by the first mass analyzer 104-1 (e.g., MS1 scans). Alternatively, the mass spectrum 508 can be generated from scans by the second mass analyzer 104-3 (e.g., MS2 scans).

[0069] In some examples, the system 300 acquires the mass spectrum 508 by directing the LC-MS system 402 to operate in a first mode of operation such that the mass spectrum 508 is generated and / or acquired while the LC-MS system 402 is operating in the first mode of operation. In the first mode of operation, one or more operating parameters of the LC-MS system 402 can be configured to quickly detect the IS variant 414. Operating parameters that can be adjusted can include, for example, resolution, ion injection time, ion fill time, m / z range, and / or any other suitable operating parameter(s).

[0070] To illustrate, the LC-MS system 402 can perform high resolution MS1 full range scans of the sample 410 when operating in the first mode of operation. As another illustration, the LC-MS system 402 can perform fast low resolution MS2 scans of fragment ions produced from precursor ions of the IS variant 414 when operating in the first mode of operation. In some examples, the first mode of operation can specify a particular type of scan (e.g., MS1 scans or MS2 scans). In further examples, the LC-MS system 402 can perform MS2 scans with data independent acquisition when operating in the first mode of operation.

[0071] In step 510, the system 300 analyzes at least the mass spectrum 508 to determine whether the IS variants 414 are detected. The system 300 can detect the IS variants 414 in any suitable manner. In some examples, the system 300 determines whether the mass spectrum 508 includes mass peaks corresponding to the IS variants 414 (e.g., mass peaks including at least one precursor ion for each IS variant 414 or mass peaks including at least one precursor ion for one IS variant 414). For example, the system 300 can determine whether the mass spectrum 508 includes mass peaks that satisfy the mass-to-charge ratio (m / z) and possibly intensity values in the criteria 504. Additionally or alternatively, the system 300 can compare the mass spectrum 508 to a library of known mass spectra for the IS variants 414. The system 300 can use any suitable comparison method that can be suitable for a particular implementation, such as spectral matching or fragmentation matching.

[0072] If the system 300 does not detect the IS variants 414 in the mass spectrum 508, the system 300 returns processing to step 506 and acquires the next mass spectrum 508. On the other hand, if the system 300 detects one or more (or all) of the IS variants 414 in the mass spectrum 508, the system 300 proceeds to step 512. In some examples, the system 300 proceeds to step 512 in response to detecting all of the IS variants 414 (e.g., IS variants 414-1, 414-2, and 414-3) added to the sample 410. In alternative examples, the system 300 can proceed to step 512 in response to detecting at least some of the IS variants (e.g., detecting both IS variants 414-1 and 414-2, but not IS variant 414-3) or at least one of the IS variants (e.g., IS variant 414-3) added to the sample 410.

[0073] In step 512, system 300 generates a calibration curve 514 based at least in part on mass spectrometry 508. Therefore, in some instances, the detection of IS variant 414 during observation mode can trigger the generation of calibration curve 514. Because each IS variant 414 has a different mass and a different but known concentration in sample 410, system 300 can generate calibration curve 514 based at least in part on the mass peaks in mass spectrometry 508 of the ions generated from IS variant 414. Calibration curve 514 plots the detected intensity against concentration. Calibration curve 514 can take any form, such as a table, a regression curve (e.g., a straight line generated based on linear regression analysis), a set of data, etc. Calibration curve 514 can be generated based on the detection of as few as two data points (e.g., based on two different IS variants 414 in sample 410). However, the accuracy and / or reliability of calibration curve 514 can increase with additional data points (e.g., additional IS variants 414). System 300 can store calibration data representing calibration curve 514 in storage facility 302.

[0074] The detection of IS variant 414 during observation mode can trigger the initiation of quantitative mode. In step 516, system 300 acquires mass spectrum 518 (“quantitative mode mass spectrometry”) generated during the analytical run of sample 410 when operating in quantitative mode. The acquisition of mass spectrum 518 is triggered by the detection of IS variant 414 and / or the generation of calibration curve 514. Because system 300 detects IS variant 414 in step 510, mass spectrum 518 contains mass peaks of product ions generated from target analyte 412. System 300 can acquire mass spectrum 518 in any suitable manner (including any manner described herein). Mass spectrum 518 can be acquired at any time after acquisition of mass spectrum 508. In some instances, mass spectrum 518 may represent an aggregation (e.g., average, etc.) of multiple mass spectra acquired after acquisition of mass spectrum 508.

[0075] Although Figure 5 Not shown, but system 300 may also include a verification step, wherein system 300 verifies that mass spectrometry 518 detects target analyte 412 and / or IS variant 414. This can be accomplished by any suitable means, such as by comparing mass spectrometry 518 with known mass spectra of target analyte 412 and / or IS variant 414 (e.g., tandem mass spectrometry). Exemplary methods for verifying the detection of IS variant 414 are described in more detail below.

[0076] Mass spectrum 518 can be generated from a scan by any mass analyzer included in LC-MS system 402. For example, when LC-MS system 402 includes tandem mass spectrometer 100B, mass spectrum 518 can be generated from a scan by first mass analyzer 104-1 (e.g., a MS1 scan). Alternatively, mass spectrum 508 can be generated from a scan by second mass analyzer 104-3 (e.g., a MS2 or MS / MS scan).

[0077] In some instances, system 300 can acquire mass spectrum 518 by also directing LC-MS system 402 to operate according to a second operating mode, such that mass spectrum 518 is generated while LC-MS system 402 is operating in the second operating mode. The second operating mode is different from the first operating mode. In the second operating mode, one or more operating parameters of LC-MS system 402 can be configured to identify and quantify target analyte 412. For example, in the second operating mode, LC-MS system 402 can be configured to perform scans for target analyte 412 with high mass and high accuracy to produce high quality data. For example, when LC-MS system 402 is implemented by an orbitrap-based tandem mass spectrometer, LC-MS system 402 can operate at high resolution and high ion fill times.

[0078] As shown in FIG. 5B, step 516 is performed after step 512. Alternatively, step 512 can be performed after step 516, or step 512 and step 516 can be performed concurrently. In some instances, step 512 and / or step 516 can be performed in response to determining that mass spectrum 508 includes a mass peak corresponding to IS variant 414 (step 510). In this way, as explained above, detection of IS variant 414 triggers generation of calibration curve 514 and triggers a quantification mode, such as a targeted scan for target analyte 412. Figure 5

[0079] In step 520, system 300 can quantify target analyte 412 based on calibration curve 514 and mass spectrum 518. System 300 can quantify target analyte 412 in any suitable manner. For example, system 300 can determine a concentration of target analyte 412 in sample 410 by extrapolating or interpolating from calibration curve 514 based on a detected signal intensity of target analyte 412 in mass spectrum 518. Based on the identified concentration of target analyte 412 and a known quantity of sample 410, system 300 can also determine an absolute quantification of target analyte 412.

[0080] ​In the above examples, the spectrum 508 and the mass spectrum 518 are described in the singular. However, the examples described herein are not limited to a single mass spectrum, as the spectrum 508 and / or the mass spectrum 518 can represent multiple mass spectra. Thus, a calibration curve 514 can be generated based on multiple mass spectra 508, and the target analyte 412 can be quantified based on the calibration curve 514 and multiple mass spectra 518.

[0081] Figure 6 Another example flowchart 600 of operations performed by the system 300 during targeted quantitative analysis of the target analyte 412 is shown. Although Figure 6 Exemplary operations are shown in accordance with one embodiment, other embodiments can omit, add to, reorder, and / or modify any of the operations shown in Figure 6 The flowchart 600 is similar to the flowchart 500, except that the observation mode is divided into a basic observation mode and an enhanced observation mode.

[0082] When operating in the basic observation mode, the system 300 can detect the IS variant 414 (step 510) and optionally generate the calibration curve 514 (step 512). Detection of the IS variant 414 and / or generation of the calibration curve 514 can trigger initiation of the enhanced observation mode, in which the system 300 can confirm detection of the IS variant 414. In the enhanced observation mode, the system 300 can acquire a mass spectrum 604 (“enhanced observation mode mass spectrum”) in step 602. The mass spectrum 604 can be acquired at any time after the acquisition of the mass spectrum 508, and can be acquired in any suitable manner, including any of the manners described herein. In some examples, the system 300 can acquire the mass spectrum 604 by directing the LC-MS system 402 to operate in a third mode of operation, which is different from the first mode of operation and the second mode of operation, such that the mass spectrum 604 is generated while the LC-MS system 402 is operating in the third mode of operation. In the third mode of operation, one or more operating parameters of the LC-MS system 402 can be configured to confirm the IS variant 414. In some examples, the third mode of operation specifies a particular type of scan (e.g., an MS1 scan or an MS2 scan).

[0083] To illustrate, if the LC-MS system 402 performs high resolution full range scans when operating in the first mode of operation, the LC-MS system 402 can perform low resolution fast scans when operating in the third mode of operation. Additionally or alternatively, the LC-MS system 402 can perform MS2 scans when operating in the third mode of operation. For example, the LC-MS system 402 can selectively isolate a precursor ion detected in step 510 and produced by the first mass analyzer 104-1 from the IS variants 414, fragment the precursor ion in the collision cell 104-2, and scan the fragment ions in the second mass analyzer 104-3. A mass spectrum 604 can be generated from the scan of the fragment ions in the second mass analyzer 104-3. Thus, the mass spectrum 604 can include mass peaks of fragment ions produced from the IS variants 414.

[0084] In step 606, the system 300 determines whether to confirm detection of the IS variants 414 based on the mass spectrum 604. The system 300 can confirm detection of the IS variants 414 in any suitable manner, such as by spectral matching (e.g., dot product spectral matching), fragmentation matching, or any other suitable method. The fragmentation matching can be performed based on one or more reference peptide sequences specified in the criteria 504. For example, the system 300 can obtain a reference mass spectrum of known fragment ions for a particular peptide sequence based on the criteria 504 (e.g., based on the peptide sequence information). The system 300 can confirm detection of the IS variants 414 when the system 300 detects at least a minimum number of fragment ions of the precursor ion for each IS variant 414 in the mass spectrum 604. The minimum number of fragment ions (e.g., 5) can be pre-set, either automatically (e.g., based on known reference data) or manually by a user.

[0085] If the system 300 does not confirm detection of the IS variants 414, the system 300 can return to step 602. Alternatively, the system 300 can return to step 506 or step 510. If the system 300 confirms detection of the IS variants 414, the system 300 can initiate and operate in the quantification mode (e.g., perform steps 516 and 520) in response to the confirmation. That is, confirmation of detection of the IS variants 414 triggers the start of the quantification mode.

[0086] As with the mass spectrum 508 and the mass spectrum 518, the mass spectrum 604 is not limited to a single mass spectrum, but can represent multiple mass spectra.

[0087] Figure 7 Another example flowchart 700 illustrating operations performed by the system 300 during targeted quantification analysis of the target analyte 412 is shown. Although Figure 7Exemplary operations are demonstrated in accordance with one embodiment, although other embodiments can omit, add to, reorder, and / or modify any of the operations shown Figure 7 The flowchart 700 is similar to the flowchart 600, except that the step 512 (generate the calibration curve 514) is performed in response to the step 606 (confirm that the IS variant 414 is detected) instead of in response to the step 510 (detect the IS variant 414). The calibration curve 514 can be generated based on the mass spectrum 508 and / or the mass spectrum 604.

[0088] Figure 8 Another exemplary flowchart 800 demonstrates operations performed by the system 300 during a targeted quantitative analysis of the target analyte 412. Although Figure 8 Exemplary operations are demonstrated in accordance with one embodiment, although other embodiments can omit, add to, reorder, and / or modify any of the operations shown Figure 8 The flowchart 800 is similar to the flowchart 600, except that the step 512 (generate the calibration curve 514) is performed in response to the step 516 (acquire the mass spectrum 518) instead of in response to the step 510 (detect the IS variant 414). Thus, the calibration curve 514 can be generated based on the mass spectrum 508, the mass spectrum 604, and / or the mass spectrum 518.

[0089] Exemplary operations of the system 300 and the LC-MS system 402 will now be described with reference to Figure 9 Exemplary operations of the system 300 and the LC-MS system 402 will now be described with reference to Figure 9 An exemplary workflow 900 is demonstrated that depicts operations associated with a targeted quantitative analysis of the sample 410 as performed by the system 300 and the LC-MS system 402.

[0090] As shown, the LC-MS system 402 (e.g., the MS 406) includes a first mass analyzer 104-1, a collision cell 104-2, and a second mass analyzer 104-3. The first mass analyzer 104-1 can be implemented by any suitable mass analyzer, such as a quadrupole mass filter. The collision cell 104-2 can be implemented by any suitable collision cell. The second mass analyzer 104-3 can be implemented by any suitable mass analyzer, such as an orbitrap mass analyzer or a time-of-flight mass analyzer. The system 300 is communicatively coupled with the LC-MS system 402 through the link 408.

[0091] In step 902, while operating in the basic observation mode, the system 300 directs the LC-MS system 402 to perform a detection scan (e.g., a MS1 scan) of the sample 410 with the mass analyzer 104-1. In step 904, the LC-MS system 402 runs the detection scan of the sample 410 with the first mass analyzer 104-1. In alternative embodiments, the LC-MS system 402 can run the detection scan (e.g., a MS2 or MS / MS scan) with the second mass analyzer 104-3. The detection scan can be configured to detect the IS variants 414 in real-time (e.g., to detect that the IS variants 414 elute from the LC 404 and into the MS 406). To do so, the system 300 can direct the LC-MS system 402 to operate in a first operating mode while performing the detection scan. In the first operating mode, the LC-MS system 402 is configured to enable detection of precursor ions generated from the IS variants 414. For example, the detection scan can include a high resolution full range scan of the eluent from the LC 404.

[0092] In step 906, the system 300 acquires a mass spectrum 908 generated from the detection scan in step 904 (“basic observation mode mass spectrum”). In step 910, the system 300 analyzes the mass spectrum 908 to determine whether two or more (or all) of the IS variants 414 are detected. The system 300 can analyze the mass spectrum 908 and detect the IS variants 414 in any suitable manner, including any of the manners described herein. For example, the system 300 can analyze the mass spectrum 908 to identify mass peaks having a mass-to-charge ratio and signal intensity level that match a set of criteria. If the system 300 does not detect the IS variants 414 in the mass spectrum 908 (e.g., if the mass peaks in the mass spectrum 908 do not match the criteria), the system 300 can return to step 906 and analyze another mass spectrum. Alternatively, the system 300 can return to step 902 and direct the LC-MS system 402 to perform another detection scan (step 904).

[0093] If the system 300 detects the IS variants 414 in the mass spectrum 908, the system 300 can transition to operating in the enhanced observation mode. While operating in the enhanced observation mode, the system 300 can direct the LC-MS system 402 to perform a confirmation scan 912 to confirm the presence of the IS variants 414. In Figure 9In embodiments, the confirmation scan 912 includes an MS / MS scan of product ions generated from the sample 410. For example, in step 914, the LC-MS system 402 selectively filters ions generated from the sample 410 in the first mass analyzer 104-1 to produce precursor ions. In step 916, the LC-MS system 402 selectively fragments the precursor ions in the collision cell 104-2. In step 918, the LC-MS system 402 scans the fragment ions in the second mass analyzer 104-3. The system 300 can direct the LC-MS system 402 to operate in a second operational mode when performing the confirmation scan 912. In some instances, the LC-MS system 402 can perform the confirmation scan 912 with low resolution and high speed (e.g., low fill time) when operating in the second operational mode.

[0094] In step 920, the system 300 acquires a mass spectrum 922 generated from the scan in step 918 (“enhanced observation mode mass spectrum”). The system 300 can acquire the mass spectrum 922 in any manner described herein. In step 924, the system 300 confirms whether the IS variant 414 is detected based on the mass spectrum 922. The system 300 can confirm detection of the IS variant 414 in any suitable manner, such as by spectral matching, fragmentation matching, and / or any other suitable technique. If the system 300 does not confirm detection of the IS variant 414, the system 300 can return to step 914 and perform another confirmation scan 912. In alternative embodiments, the system 300 can return to step 902 and / or step 906.

[0095] If the system 300 confirms detection of the IS variant 414 in step 924, the system 300 can transition to operating in a quantification mode. When operating in the quantification mode, the system 300 can direct the LC-MS system 402 to perform a targeted quantification scan 926. In some instances, the targeted quantification scan 926 includes an MS / MS scan. In the targeted quantification scan 926, the LC-MS system 402 is configured to acquire high quality data for quantifying the target analyte 412. Accordingly, in step 928, the LC-MS system 402 selectively filters precursor ions generated from the target analyte 412 in the first mass analyzer 104-1. In step 930, the LC-MS system 402 fragments the target analyte precursor ions in the collision cell 140-2. In step 932, the LC-MS system 402 scans the fragment ions in the second mass analyzer 104-3. The system 300 can direct the LC-MS system 402 to operate in a third operational mode when performing the targeted quantification scan 926. For example, the LC-MS system 402 can perform the targeted quantification scan 926 with high resolution and high ion fill time when operating in the third operational mode.

[0096] In step 934, the system 300 acquires a mass spectrum 936 generated from the scan in step 932 ("quantification mode mass spectrum"). The system 300 can acquire the mass spectrum 936 in any suitable manner, including any of those described herein. In step 938, the system 300 generates a calibration curve. The system 300 can generate the calibration curve in any suitable manner, including any of those described herein. In some examples, the calibration curve is generated based on the mass spectrum 936. Alternatively, the calibration curve can be generated based on the mass spectrum 908 and / or the mass spectrum 922. Further, although Figure 9 Although it is shown that the calibration curve is generated after the targeted quantification scan 926, the generation of the calibration curve (step 938) can be performed at any other suitable time, such as in response to detecting the IS variant 414 (step 910) or in response to confirming the detection of the IS variant 414 (step 924).

[0097] In step 940, the system 300 quantifies the target analyte 412 based on the calibration curve and the mass spectrum 936. The system 300 can quantify the target analyte 412 in any suitable manner, including any of those described herein. For example, the system 300 can determine a concentration of the target analyte 412 in the sample 410 and / or determine an absolute quantification of the target analyte 412 based on the determined concentration of the target analyte 412 and a known quantity of the sample 410. In some examples, the system 300 can also store (e.g., in the storage facility 302) and / or output the calibration curve and / or quantification data (e.g., data representing the determined concentration of the target analyte 412 and / or the absolute quantification of the target analyte 412). Additionally or alternatively, the calibration curve and / or the quantification data can be output to a display device for display of the calibration curve and / or the quantification data by the display device.

[0098] In the above examples, the mass spectrum 906, the mass spectrum 920, and / or the mass spectrum 934 are not limited to a single mass spectrum, but can each represent a plurality of mass spectra.

[0099] In some modifications of the above examples, the system 300 can additionally perform feedback control of the LC-MS system 402 based on detecting (or not detecting) the IS variant 414 and / or the target analyte 412. For instance, the system 300 can adjust one or more operational parameters of the LC 404 based on detecting (or not detecting) the IS variant 414. To illustrate, the system 300 can monitor elution of the IS variant 414 from the LC 404. Upon detecting the IS variant 414, the system 300 can modify the LC gradient to spread out elution of the IS variant 414 to help the mass spectrometer (e.g., the mass spectrometer 204) keep up with the large amount of IS variants eluting from the LC system. In other words, the system 300 can space out the LC gradient in response to detecting the IS variant 414 (e.g., in response to the step 510, the step 606, the step 910, or the step 924). Additionally or alternatively, upon not detecting the IS variant 414, the system 300 can shorten the gradient to facilitate faster processing. By adjusting or optimizing control of the LC gradient, the system 300 can shorten the overall processing time of the LC-MS system 402 on the sample 410 without sacrificing the quality or accuracy of the targeted quantitative analysis. Adjustment or optimization of the LC gradient is just one example, as any other operational parameter of the LC 404 can be adjusted based on detecting (or not detecting) the IS variant 414 and / or the target analyte 412.

[0100] As another example of feedback control, the system 300 can adjust one or more operational parameters of the MS 406 based on detecting (or not detecting) the target analyte 412. To illustrate, the system 300 can determine that data (e.g., the mass spectrum 518 or the mass spectrum 936) of the target analyte 412 is insufficient or of insufficient quality to perform quantification of the target analyte 412 during the quantification phase of the targeted quantitative analysis. In response to this determination, the system 300 can adjust operational parameters of the MS 406 to improve the data quality. For instance, the system 300 can increase ion fill times in the ion source and / or the collision cell 104-2 in real-time. Increasing the ion fill times can increase the sensitivity of the MS 406 and increase the probability of generating high quality data. Additionally or alternatively, the system 300 can adjust or optimize the resolution of the second mass analyzer 104-3. Adjustment or optimization of the ion fill times and / or the resolution is just one example, as any other operational parameter of the MS 406 can be adjusted based on detecting (or not detecting) the IS variant 414 and / or the target analyte 412.

[0101] Various modifications can be made to the above examples. For example, in the above examples, the sample 410 has been described as containing only one target analyte 412. However, the sample 410 can contain any number of target analytes 412 that can be suitable for a particular implementation, as in target multiplexing. In examples involving target multiplexing, a different set of IS variants 414 can be added to the sample 410 for each target analyte 412. Further, during a particular analysis run of the sample 410, the processes and operations described herein can be performed independently for each different target analyte 412.

[0102] In some examples, the observation mode (e.g., the enhanced observation mode) and the quantification mode can be run partially concurrently. For example, during the enhanced observation mode of operation, MS2 analysis of the target analyte 412 can be interleaved with MS1 and MS2 analysis of the IS variants 414. The analysis of the IS variants 414 can be used to monitor the progress of elution in real time. However, it will be appreciated that the specificity provided by detecting multiple IS variants 414 during the observation mode can eliminate the need to monitor the progress of elution in real time, thereby improving the efficiency of performing targeted analysis.

[0103] Further, in the examples described herein, the calibration curve generation and quantification of the target analyte are performed substantially in real time during the targeted quantification analysis (e.g., in close temporal proximity to or in response to the acquisition of the mass spectra upon which the calibration curve is based). However, the examples are not limited to real time, as the calibration curve and quantification of the target analyte can be performed at any suitable time that can be suitable for a particular implementation, such as after the targeted quantification analysis, after a different batch of targeted quantification analyses, etc.

[0104] In the above examples, the system 300 can detect the IS variants 414 by determining whether a mass spectrum (e.g., the mass spectrum 508 or the mass spectrum 908) contains mass peaks that satisfy mass-to-charge ratio (m / z) and / or signal intensity values in a set of criteria (e.g., the criteria 504) (e.g., steps 510 or 910). In some examples, the set of criteria also specifies different patterns that must be satisfied as a condition for detection of the IS variants 414. Assuming that each of the IS variants 414-1, 414-2, and 414-3 has a different mass and a different concentration, the combination of the mass peaks of the IS variants 414-1, 414-2, and 414-3 within a mass spectrum can have a unique pattern. Thus, the unique combination of the masses and concentrations of the IS variants 414 can encode a specific signature that, when detected by the system 300, can satisfy the condition for detection of the IS variants 414.

[0105] This is illustrated with reference to Figure 10A and 10B . Figure 10AAn exemplary mass spectrum 1000A is shown that includes a plurality of mass peaks 1002-1 to 1002-5 of ions to be generated from a set of five IS variants added to a sample. As shown, each mass peak 1002 has a different relative abundance and a different mass-to-charge ratio (m / z). Figure 10A The mass peak pattern (e.g., the arrangement of mass peaks 1002 relative to each other) shown in the middle can be included in a standard set as a condition that the system 300 must detect to determine that the plurality of IS variants added to the sample have been detected. Thus, when the system 300 acquires a mass spectrum that matches the mass peak pattern of the Figure 10A The system 300 can determine that the set of five IS variants are detected during the targeted quantitative analysis when the system 300 acquires a mass spectrum that includes mass peaks matching the mass peak pattern of

[0106] Figure 10B An exemplary mass spectrum 1000B acquired during the targeted quantitative analysis is shown. In the mass spectrum 1000B, the mass peaks 1002-1 to 1002-5 have the same m / z values as in the mass spectrum 1000A, but different relative abundances. That is, the IS variants have been added to the sample at concentrations different from the concentrations required to generate the mass peak pattern of the mass spectrum 1000A. Thus, when the system 300 acquires the mass spectrum 1000B, the system 300 does not determine that the set of five IS variants are detected. Thus, as can be seen, the predefined mass peak pattern included in the standard set can improve the accuracy of the detection of the plurality of IS variants and reduce or even eliminate the possibility of false positive detection of the plurality of IS variants.

[0107] In further or alternative examples, the standard set (e.g., the standard 504) for detecting the IS variants 414 can include a unique non-linear calibration curve, and the system 300 can determine or confirm detection of the IS variants 414 only when the calibration curve generated by the system 300 (e.g., the calibration curve 514 or the calibration curve generated in step 928) matches the non-linear calibration curve specified in the standard set. The non-linear calibration curve can be based on the IS variants added to the sample in a non-linear concentration range. The non-linear calibration curve can have a curve unique to a particular IS variant. A calibration curve that does not match the predefined calibration curve generated by the system 300 during the targeted quantitative analysis of the target analyte and associated with the IS variants of the target analyte will not trigger the start of the detection confirmation and / or quantification phase. Thus, the predefined calibration curve included in the standard set can improve the accuracy of the detection of the plurality of IS variants and reduce or even eliminate the possibility of false positive detection of the plurality of IS variants.

[0108] In some examples, a targeted quantification kit can include a unique set of IS variants in a unique pre-determined amount in a set of containers. In some examples, the pre-determined amount of IS variants in the kit is configured to produce a mass spectrum in a mass analysis that matches a pre-defined mass peak pattern of a standard set. Additionally or alternatively, the pre-determined amount of IS variants in the kit is configured to produce a pre-defined non-linear calibration curve. In some examples, the kit can also include standard data including data representing the pre-defined mass peak pattern and / or the pre-defined non-linear calibration curve associated with the set of IS variants. In further examples, the kit can also include hardware (e.g., system 300, storage facility 302, etc.) and / or software (e.g., executable instructions) that can be executed by system 300 to perform the targeted quantification operations described herein.

[0109] In the foregoing embodiments, the concentration and / or absolute quantification of a target analyte included in a sample can be determined from a single targeted quantification analysis of the sample. In cases where multiple different IS variants are spiked into the sample at different concentrations, system 300 can generate a calibration curve from the sample during the same targeted quantification analysis, thereby eliminating the need to acquire calibration curve data in a separate analysis. This allows for more efficient monitoring, detection, and quantification of the target analyte. Additionally, detection and / or confirmation of detection of the multiple IS variants triggers acquisition of high resolution, high quality data of the target analyte, which can then be used in conjunction with the calibration curve to quantify the target analyte. Furthermore, accurate detection and confirmation of the IS variants can eliminate or substantially reduce the occurrence of false positive triggers. Moreover, the specificity of the triggers provided by detection of the multiple IS variants can eliminate the need to monitor the detected signal intensity against a signal intensity threshold, thereby reducing necessary processing power and time and improving the efficiency with which system 300 can perform targeted quantification analysis.

[0110] Figure 11 An exemplary method 1100 of quantifying a target analyte included in a sample is demonstrated. Although Figure 11 An exemplary operation according to one embodiment is demonstrated, but other embodiments can omit, add to, reorder, and / or modify any of the operations shown in Figure 11 One or more of the operations shown in Figure 11 may be performed by system 300, any component included therein, and / or any implementation thereof.

[0111] In operation 1102, an observation mode mass spectrum (e.g., mass spectrum 508, mass spectrum 604, mass spectrum 908, or mass spectrum 922) is acquired when operating in an observation mode. The observation mode mass spectrum includes mass peaks of ions produced from a plurality of IS variants (e.g., IS variants 414-1 through 414-3) added to a sample (e.g., sample 411) that includes a target analyte (e.g., target analyte 412). Each IS variant included in the plurality of IS variants includes a unique isotopologue of the target analyte and is added to the sample in a unique amount. Operation 1102 can be performed in any of the ways described herein.

[0112] In operation 1104, a calibration curve (e.g., calibration curve 514 or calibration curve generated by step 938 of method 930) is generated based on the observation mode mass spectrum. Operation 1104 can be performed in any of the ways described herein. Figure 9

[0113] In operation 1106, a quantification mode mass spectrum (e.g., mass spectrum 518 or mass spectrum 936) is acquired when operating in a quantification mode. The quantification mode mass spectrum includes mass peaks of ions produced from a target analyte included in a sample. Operation 1106 can be performed in any of the ways described herein.

[0114] In operation 1108, a concentration of the target analyte included in the sample is determined based on the calibration curve and the quantification mode mass spectrum. Operation 1108 can be performed in any of the ways described herein.

[0115] Figure 12 Another example method 1200 of quantifying a target analyte included in a sample is demonstrated. Although Figure 12 Example operations according to one embodiment are demonstrated, other embodiments can omit, add to, reorder, and / or modify any of the operations shown in Figure 12 One or more of the operations shown in method 1200 can be performed by system 300, any component included therein, and / or any implementation thereof. Figure 12 One or more of the operations shown in method 1200 can be performed by system 300, any component included therein, and / or any implementation thereof.

[0116] In operation 1202, a base observation mode mass spectrum (e.g., mass spectrum 508 or mass spectrum 908) is acquired when operating in a base observation mode of an observation mode. The base observation mode mass spectrum includes mass peaks of precursor ions produced from a plurality of IS variants (e.g., IS variants 414-1 through 414-3) added to a sample (e.g., sample 410) that includes a target analyte (e.g., target analyate 412). Each IS variant included in the plurality of IS variants includes a unique isotopologue of the target analyte and is added to the sample in a unique amount. Operation 1202 can be performed in any of the ways described herein. In operation 1202, a base observation mode mass spectrum (e.g., mass spectrum 508 or mass spectrum 908) is acquired when operating in a base observation mode of an observation mode. The base observation mode mass spectrum includes mass peaks of precursor ions produced from a plurality of IS variants (e.g., IS variants 414-1 through 414-3) added to a sample (e.g., sample 410) that includes a target analyte (e.g., target analyate 412). Each IS variant included in the plurality of IS variants includes a unique isotopologue of the target analyte and is added to the sample in a unique amount. Operation 1202 can be performed in any of the ways described herein.

[0117] In operation 1204, in response to acquiring the base observation mode mass spectrum, the mass spectrometry analysis system (e.g., LC-MS system 402) is directed to selectively fragment the precursor ions into a plurality of fragment ions. Operation 1204 can be performed in any of the ways described herein.

[0118] In operation 1206, an enhanced observation mode mass spectrum (e.g., mass spectrum 604 or mass spectrum 922) is acquired while operating in an enhanced observation mode of the observation mode. The enhanced observation mode mass spectrum contains mass peaks for at least a subset of the plurality of fragment ions. Operation 1206 can be performed in any of the ways described herein. In some examples, a different enhanced observation mode mass spectrum is acquired from each MS2 scan of an IS variant.

[0119] In operation 1208, a calibration curve (e.g., calibration curve 514 or calibration curve generated by step 938 of method 900) is generated based on the base observation mode mass spectrum and / or the enhanced observation mode mass spectrum. Operation 1208 can be performed in any of the ways described herein. In some examples, operation 1208 is performed in response to operation 1206. Figure 9

[0120] In operation 1210, a quantification mode mass spectrum (e.g., mass spectrum 518 or mass spectrum 936) is acquired. The quantification mode mass spectrum contains mass peaks for ions produced from a target analyte contained in the sample. Operation 1210 can be performed in any of the ways described herein. In some examples, operation 1210 is performed in response to operation 1206 and / or operation 1208.

[0121] In operation 1212, a concentration of the target analyte contained in the sample is determined based on the calibration curve and the quantification mode mass spectrum. Operation 1212 can be performed in any of the ways described herein.

[0122] In some examples, a non-transitory computer-readable medium storing computer- readable instructions can be provided in accordance with the principles described herein. The instructions, when executed by a processor of a computing device, can direct the processor and / or computing device to perform one or more operations, including one or more of the operations described herein. Such instructions can be stored and / or transmitted using any of a variety of known computer-readable media.

[0123] ​As used herein, a non-transitory computer-readable medium can include any non-transitory storage medium that contributes to providing data (e.g., instructions) that can be read and / or executed by a computing device (e.g., by a processor of the computing device). For example, a non-transitory computer-readable medium can include, but is not limited to, any combination of non-volatile storage media and / or volatile storage media. Exemplary non-volatile storage media include, but are not limited to, read-only memory, flash memory, solid-state drives, magnetic storage devices (e.g., hard disks, floppy disks, magnetic tapes, etc.), ferroelectric random access memory (“RAM”), and optical discs (e.g., compact discs, digital video discs, Blu-ray discs, etc.). Exemplary volatile storage media include, but are not limited to, RAM (e.g., dynamic RAM).

[0124] Figure 13 An exemplary computing device 1300 is shown, which can be specifically configured to perform one or more processes described herein. Figure 13 As shown, computing device 1300 may include a communication interface 1302, a processor 1304, a storage device 1306, and an input / output (“I / O”) module 1308 that are communicatively connected to each other via communication infrastructure 1310. Although Figure 13 An exemplary computing device 1300 is shown, but Figure 13 The components shown are not intended to be limiting. Additional or alternative components may be used in other embodiments. These will now be described in further detail. Figure 13 The components of the computing device 1300 shown.

[0125] Communication interface 1302 can be configured to communicate with one or more computing devices. Examples of communication interface 1302 include, but are not limited to, wired network interfaces (such as network interface cards), wireless network interfaces (such as wireless network interface cards), modems, audio / video connections, and any other suitable interfaces.

[0126] Processor 1304 generally refers to any type or form of processing unit capable of processing data and / or interpreting, executing, and / or directing the execution of one or more of the instructions, procedures, and / or operations described herein. Processor 1304 may perform operations by executing computer-executable instructions 1312 (e.g., applications, software, code, and / or other executable data instances) stored in storage device 1306.

[0127] The storage 1306 can include one or more data storage media, devices, or configurations and can employ any type, form, and combination of data storage media and / or device. For example, the storage 1306 can include, but is not limited to, any combination of the non-volatile media and / or volatile media described herein. Electronic data, including the data described herein, can be temporarily and / or permanently stored in the storage 1306. For example, data representative of the computer-executable instructions 1312 configured to direct the processor 1304 to perform any of the operations described herein can be stored within the storage 1306. In some examples, the data can be arranged in one or more databases residing in the storage 1306.

[0128] The I / O module 1308 can include one or more I / O modules configured to receive user input and provide user output. The input for a single virtual experience can be received using one or more I / O modules. The I / O module 1308 can include any hardware, firmware, software, or combination thereof supportive of input and output capabilities. For example, the I / O module 1308 can include hardware and / or software for capturing user input, including, but not limited to, a keyboard or keypad, a touchscreen component (e.g., a touchscreen display), a receiver (e.g., an RF or IR receiver), a motion sensor, and / or one or more input buttons.

[0129] The I / O module 1308 can include one or more devices for presenting output to a user, including, but not limited to, a graphics engine, a display (e.g., a display screen), one or more output drivers (e.g., display drivers), one or more audio speakers, and one or more audio drivers. In certain embodiments, the I / O module 1308 is configured to provide graphical data to a display for presentation to a user. The graphical data can be representative of one or more graphical user interfaces and / or any other graphical content serving a particular implementation.

[0130] In some examples, any of the systems (e.g., system 300), computing devices, and / or other components described herein can be implemented by the computing device 1300. For example, the processing facility 304 can be implemented by the processor 1304, and the storage facility 302 can be implemented by the storage 1306.

[0131] In the foregoing description, various example embodiments have been described with reference to the accompanying drawings. It will, however, be evident that various modifications and changes can be made thereto without departing from the scope of the application as set forth in the claims above. For example, certain features of one embodiment described herein can be combined with or substituted for features of another embodiment described herein. Accordingly, the description and drawings are to be regarded in an illustrative rather than a restrictive sense.

Claims

1. A system for mass spectrometric analysis, the system comprising: a memory storing instructions; and a processor communicatively coupled to the memory and configured to execute the instructions to: acquire an observation mode mass spectrum when operating in an observation mode, the observation mode mass spectrum including mass peaks of ions generated from a plurality of internal standards added to a sample including a target analyte, each internal standard included in the plurality of internal standards including a unique isotopologue of the target analyte and added to the sample in a unique amount; generate a calibration curve based on the observation mode mass spectrum; acquire a quantitation mode mass spectrum when operating in a quantitation mode, the quantitation mode mass spectrum including mass peaks of ions generated from the target analyte included in the sample; and determine a concentration of the target analyte included in the sample based on the calibration curve and the quantitation mode mass spectrum.

2. The system of claim 1, wherein the processor is further configured to execute the instructions to: acquire mass spectra during an analysis run of the sample when operating in the observation mode, the mass spectra including the observation mode mass spectrum; analyze the mass spectra based on a set of criteria associated with the plurality of internal standards; and determine, based on the analysis, that the observation mode mass spectrum includes the mass peaks of the ions generated from the plurality of internal standards added to the sample.

3. The system of claim 2, wherein acquiring the quantitation mode mass spectrum is performed in response to determining that the observation mode mass spectrum includes the mass peaks of the ions generated from the plurality of internal standards added to the sample.

4. The system of claim 2, wherein generating the calibration curve is performed in response to determining that the observation mode mass spectrum includes the mass peaks of the ions generated from the plurality of internal standards added to the sample.

5. The system of claim 2, wherein determining that the observation mode mass spectrum includes the mass peaks of the ions generated from the plurality of internal standards added to the sample includes determining that a set of mass peaks included in the observation mode mass spectrum satisfies the set of criteria, the set of mass peaks including the mass peaks of the ions generated from the plurality of internal standards.

6. The system of claim 5, wherein the set of criteria includes one or more of: a mass-to-charge ratio, a signal intensity, or a peptide sequence of each internal standard in the plurality of internal standards added to the sample.

7. The system of claim 1 or 2, wherein: the observation mode includes a basic observation mode and an enhanced observation mode, the observation mode mass spectrum includes an enhanced observation mode mass spectrum, and the processor is further configured to execute the instructions to: acquire a basic observation mode mass spectrum when operating in the basic observation mode, the basic observation mode mass spectrum including mass peaks of precursor ions generated from the plurality of internal standards; directing a mass spectrometry analysis system to selectively fragment the precursor ions into a plurality of fragment ions, including the ions produced from the plurality of internal standards, in response to acquiring the basic observation mode mass spectrum; and acquiring the enhanced observation mode mass spectrum from a scan of the plurality of fragment ions when operating in the enhanced observation mode.

8. The system of any one of claims 1 to 7, wherein the processor is further configured to execute the instructions to: direct a mass spectrometry analysis system to scan the ions produced from the plurality of internal standards added to the sample according to a first mode of operation, such that the observation mode mass spectrum is generated when the mass spectrometry analysis system operates in the first mode of operation; and direct the mass spectrometry analysis system to scan the ions produced from the target analyte contained in the sample according to a second mode of operation, such that the quantitative mode mass spectrum is generated when the mass spectrometry analysis system operates in the second mode of operation.

9. A method for mass spectrometry analysis, the method comprising: acquiring, by a targeted quantification system when operating in an observation mode, an observation mode mass spectrum containing mass peaks of ions produced from a plurality of internal standards added to a sample including a target analyte, each internal standard contained in the plurality of internal standards including a unique isotopologue of the target analyte and added to the sample in a unique amount; generating, by the targeted quantification system, a calibration curve based on the observation mode mass spectrum; acquiring, by the targeted quantification system when operating in a quantitative mode, a quantitative mode mass spectrum containing mass peaks of ions produced from the target analyte contained in the sample; and determining, by the targeted quantification system, a concentration of the target analyte contained in the sample based on the calibration curve and the quantitative mode mass spectrum.

10. The method of claim 9, further comprising: acquiring, by the targeted quantification system when operating in the observation mode, mass spectra during an analysis run of the sample, the mass spectra containing the observation mode mass spectrum; analyzing, by the targeted quantification system, the mass spectra based on a standard set associated with the plurality of internal standards; and determining, by the targeted quantification system based on the analysis, that the observation mode mass spectrum contains the mass peaks of the ions produced from the plurality of internal standards added to the sample.

11. The method of claim 10, wherein acquiring the quantitative mode mass spectrum is performed in response to determining that the observation mode mass spectrum contains the mass peaks of the ions produced from the plurality of internal standards added to the sample.

12. The method of claim 10, wherein generating the calibration curve is performed in response to determining that the observation mode mass spectrum contains the mass peaks of the ions produced from the plurality of internal standards added to the sample.

13. The method of claim 10, wherein determining that the observed mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standards added to the sample comprises determining that a set of mass peaks included in the observed mode mass spectrum satisfies the standard set, the set of mass peaks including the mass peaks of the ions produced from the plurality of internal standards.

14. The method of claim 13, wherein the standard set includes one or more of a mass-to-charge ratio, a signal intensity, or a peptide sequence of each internal standard of the plurality of internal standards added to the sample.

15. The method of claim 9, wherein: the observation mode includes a basic observation mode and an enhanced observation mode, the observed mode mass spectrum includes an enhanced observed mode mass spectrum, and the method further comprises: acquiring, by the targeted quantitation system when operating in the basic observation mode, a basic observed mode mass spectrum, the basic observed mode mass spectrum including mass peaks of precursor ions produced from the plurality of internal standards; determining, by the targeted quantitation system, that the mass peaks of the precursor ions satisfy a standard set associated with the plurality of internal standards; directing, by the targeted quantitation system, a mass spectrometry analysis system to selectively fragment the precursor ions into a plurality of fragment ions including the ions produced from the plurality of internal standards in response to determining that the mass peaks of the precursor ions satisfy the predefined standard set; and acquiring the enhanced observed mode mass spectrum from a scan of the plurality of fragment ions when operating in the enhanced observation mode.

16. The method of claim 9, further comprising: directing, by the targeted quantitation system during acquisition of the observed mode mass spectrum, a mass spectrometry analysis system to scan the ions produced from the plurality of internal standards added to the sample according to a first mode of operation; and directing, by the targeted quantitation system during acquisition of the quantitation mode mass spectrum, the mass spectrometry analysis system to scan the ions produced from the target analyte included in the sample according to a second mode of operation.

17. A non-transitory computer-readable medium storing instructions that, when executed, direct at least one processor of a computing device for mass spectrometry analysis to: acquire, when operating in an observation mode, an observed mode mass spectrum, the observed mode mass spectrum including mass peaks of ions produced from a plurality of internal standards added to a sample including a target analyte, each internal standard included in the plurality of internal standards including a unique isotopologue of the target analyte and added to the sample in a unique amount; generate a calibration curve based on the observed mode mass spectrum; acquire, when operating in a quantitation mode, a quantitation mode mass spectrum, the quantitation mode mass spectrum including mass peaks of ions produced from the target analyte included in the sample; and determine a concentration of the target analyte included in the sample based on the calibration curve and the quantitation mode mass spectrum.

18. The computer-readable medium of claim 17, wherein the instructions, when executed, further direct the at least one processor to: acquiring a mass spectrum during an analysis run of the sample when operating in the observation mode, the mass spectrum including the observation mode mass spectrum; analyzing the mass spectrum based on a set of criteria associated with the plurality of internal standards; and determining, based on the analysis, that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standards added to the sample.

19. The computer-readable medium of claim 18, wherein acquiring the quantitative mode mass spectrum is performed in response to determining that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standards added to the sample.

20. The computer-readable medium of claim 18, wherein generating the calibration curve is performed in response to determining that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standards added to the sample.

21. The computer-readable medium of claim 18, wherein determining that the observation mode mass spectrum includes the mass peaks of the ions produced from the plurality of internal standards added to the sample includes determining that a set of mass peaks included in the observation mode mass spectrum satisfies the set of criteria, the set of mass peaks including the mass peaks of the ions produced from the plurality of internal standards.

22. The computer-readable medium of claim 21, wherein the set of criteria includes one or more of a mass-to-charge ratio, a signal intensity, or a peptide sequence of each of the plurality of internal standards added to the sample.

23. The computer-readable medium of claim 17, wherein: the observation mode includes a basic observation mode and an enhanced observation mode, the observation mode mass spectrum includes an enhanced observation mode mass spectrum, and the instructions, when executed, further direct the at least one processor to: acquire a basic observation mode mass spectrum when operating in the basic observation mode, the basic observation mode mass spectrum including mass peaks of precursor ions produced from the plurality of internal standards; determine that the mass peaks of the precursor ions satisfy a set of criteria associated with the plurality of internal standards; in response to determining that the mass peaks of the precursor ions satisfy the predefined set of criteria, direct a mass spectrometry analysis system to selectively fragment the precursor ions into a plurality of fragment ions, the plurality of fragment ions including the ions produced from the plurality of internal standards; and acquire the enhanced observation mode mass spectrum from scans of the plurality of fragment ions when operating in the enhanced observation mode.

24. The computer-readable medium of claim 17, wherein the instructions, when executed, further direct the at least one processor to: direct a mass spectrometry analysis system to scan the ions produced from the plurality of internal standards added to the sample according to a first mode of operation during acquisition of the observation mode mass spectrum; and direct the mass spectrometry analysis system to scan the ions produced from the target analytes included in the sample according to a second mode of operation during acquisition of a second mass spectrum.

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