Quantitative method and apparatus for target ion species

Abstract

The invention relates to a method and a device for mass spectrometric quantification of two or more known target ion species, wherein mass spectrometric signals are weighted with a quantification function and summed up and from the weighted sum a quantification parameter of the two or more target ion species is determined, wherein the mass spectrometric signals consist of signal components of the two or more target ion species which are not mass resolved in the mass spectrum.

Description

Technical Field

[0001] This invention relates to mass spectrometry methods and apparatus for quantifying target ion species of known mass, particularly for quantifying peptide ion species labeled with isomass heterostomosing mass tags. Background Technology

[0002] Proteomics is a core technology in bioscience, aiming to elucidate the molecular mechanisms of disease at the protein level, identify biomarkers, and apply them to clinical diagnosis. This is typically achieved through mass spectrometry to identify the proteins present in a sample and their modifications. The field of proteomics has developed at the intersection of instrument design, biochemistry, and bioinformatics. Instruments used in proteomics often combine mass spectrometry with liquid chromatography (LC) or with liquid chromatography and ion mobility spectrometry (IMS).

[0003] Besides identifying proteins and their modifications, mass spectrometry plays an increasingly important role in elucidating the molecular mechanisms of disease and in future routine clinical diagnosis. Proteomics allows for the quantitative comparison of one type of proteome from different samples (e.g., from different test subjects or time series from a single test subject), or the quantitative comparison of different types of proteomes. Mass spectrometry quantification here can be performed label-free or with labeled reagents.

[0004] Various types of labels are known for use in mass spectrometry quantification, such as stable isotope labeling (SILAC) using amino acids in cell cultures or via isotopic mass tags (e.g., iTRAC). TM (Isotopic labels for relative and absolute quantification), TMT TM (Tandem mass tag) or EASI tag (easily extractable sulfoxide-based isotopic tag; published in: Virreira Winter et al., Nat. Methods 2018, 15, 527-530).

[0005] An isotopic mass tag consists of three components: a reactive group that couples the mass tag to the target species, a reporter group, and a mass balance group. The latter two groups are linked by a cleavable chemical unit, allowing the reporter group to separate from the complementary remainder (the target substance with the isotopic residual tag) during the fragmentation of the labeled target species in the gas phase. A key aspect of quantification using different isotopic mass tags is that the reporter group of the isotopic mass tag has a different mass, but the total mass of the isotopic mass tag is the same. The mass balance group balances the mass difference caused by the insertion of different isotopes into the reporter group. Therefore, in isotopic mass tags, the distribution of heavy isotopes differs between the reporter group and the mass balance group. The signal of the reporter ion species generated during fragmentation can be primarily distinguished by mass spectrometry. The relative ratio of the reporter group signal allows for the relative quantification of the analyzed target species.

[0006] Quantification of two or more proteomic samples using isotopic mass tags is typically performed as follows: The proteomic samples are digested enzymatically separately. The resulting digested peptides are aggregated together in the proteomic sample and labeled with one of the different isotopic mass tags before analysis. The digested peptides are typically separated by liquid chromatography (LC) and selectively also by ion mobility spectrometry (IMS) in the gas phase followed by mass spectrometry analysis. Peptides present in each enzymatically digested proteomic sample but labeled with different isotopic mass tags elute at the same retention time in LC separation and are not separated by selective mobility separation. Because the isotopic mass tags have the same total mass, the labeled ion species generated from the peptide species appear as a single signal in the acquired mass spectrometer (MS1). After the labeled ion species of the peptide species are separated according to retention time, mobility (selectively), and mass, and subsequently fragmented, the dominant distinguishable signal of the reporting ion species is measured in fragment mass spectrometry (MS2 or MS / MS) and used for quantification of peptide species in the proteomic sample. Protein types are quantified by one or more peptide types, which are produced by the digestion of proteins using enzymes.

[0007] The signal of the reporter ion species is located in the low-mass range of fragment mass spectrometry, where it typically does not superimpose with other fragment ion species. In addition to the reporter ion species, fragmentation also produces so-called complementary fragment ion species, which have labeled mass balance groups and peptide species, and can be distinguished in fragment mass spectrometry by the mass balance groups, and can be used for quantification.

[0008] The greatest advantage of quantification using isobaric tagging lies in its multiplexing capability, allowing for the simultaneous quantification of many samples (currently up to 16). Ogata et al.'s publication (Anal. Chem. 2020, 92, 8037-8040: "Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-Based Quantitation in Proteomic LC / MS / MS") investigated the isobaric interference problem that arises when more than one labeled ion is selected and broken during separation (LC / MS or LC / IMS / MS) when using isobaric tagging for quantification. This issue is addressed for samples with electrostatic ion traps. Different mass spectrometry systems, using time-of-flight separators as mass analyzers, were studied for isotropic and heterotropic interference.

[0009] Figure 1 is a schematic diagram of a known hybrid mass spectrometry system 100 in the prior art, preferably suitable for quantization via isoplethysmographic mass tags. The hybrid mass spectrometry system 100 includes an LC separation unit 110, an ion source 121, a mobility separator 144, a quadrupole mass filter 150, a fragmentation cell 160, a fly-by-flight mass analyzer 170, and a device 180 for recording and processing mass spectrometry data. Before fragmentation, target ion species are separated by time-separation based on retention time and mobility, and also by filtration based on the mass in the quadrupole mass filter 150.

[0010] The mobility separator 144 may be a TIMS (Trap Ion Mobility Spectroscopy) separator, preferably operating in a parallel accumulation mode. In this mode, ions accumulate in the upstream portion of the mobility separator 144 or in an upstream ion trap (not shown), while previously accumulated ions are simultaneously analyzed in the downstream portion of the mobility separator 144. The time-of-flight mass analyzer 170 is typically a time-of-flight mass analyzer with orthogonal ion implantation (OTOF) and at least one reflector.

[0011] The LC separation device 110 is coupled to an ion source 121, which is typically an electrospray ionization (ESI) source operating at atmospheric pressure. Ions generated in chamber 120 are fed into a first vacuum chamber 140 via a transport capillary 141 and then deflected into an RF ion funnel 142 by a repulsive DC potential applied to a deflection electrode. The RF ion funnel 143 guides the ions to a mobility separator 144. Ions released from the mobility separator 144 and separated according to mobility are guided to a quadrupole mass filter 150, which can transfer or filter ions according to mass. Ions separated in the quadrupole mass filter 150 are guided to a fragmentation cell 160, where fragment ion species can be generated from the ion species. Peptide ions labeled with isotopic mass tags are preferably fragmented by collision-induced dissociation (CID), but can also be fragmented by other types of fragmentation, such as electron transfer dissociation or photodissociation. Fragmentation can be turned on and off. Compared to the duration of the batch of material containing ion species downstream of the mobility separator 144, the time-of-flight mass analyzer 170 has such a short mass spectrometry acquisition time that multiple (fragment) mass spectra can be acquired for each ion species contained in the batch and separated according to mobility.

[0012] The mass spectrometry system 100 is controlled by device 180 via line 185. This device includes a detection unit 181, a central processing unit (CPU) 182, and a data storage device 183. The components of device 180 are interconnected via a local bus 184, such as via a Peripheral Component Interconnect Express (PCI express) bus. The detection unit 181 is connected to the ion detector 171 of the time-of-flight mass analyzer 170, which is located at the end of the flight path and generates a pulsed electron beam for impacting ion pulses. The ion detector 171 typically includes a secondary electron multiplier, such as a microchannel plate. The detection unit 181 has an analog-to-digital converter and a data processing unit for digitizing the electron current generated in the detector. For example, the data processing unit can determine the intensity and time of flight of individual signals in the time-of-flight mass spectrometer in real time.

[0013] As mentioned above, labeling with isotopic mass tags allows for the parallel quantification of multiple (currently up to 16) samples. However, isotopic reporter groups are also used for this purpose, some of which have mass differences of only a few millidaltons (Dalton = atomic mass unit) (isotopic reporter ion species), meaning that the mass analyzer used needs to have very high mass resolution. However, time-of-flight mass analyzers, in particular, have lower mass resolution in the lower mass range than in the higher mass range. In contrast, electrostatic ion traps, such as... Alternatively, magnetic ICR (ion cyclotron resonance) ion traps have higher mass resolution in the low mass range than in the high mass range, but require sufficient time to obtain adequate mass spectrometry mass resolution.

[0014] In a poster (67th ASMS Mass Spectrometry and Related Topics Conference, MP 735: "Improved identification, quantification accuracy, and workflow efficiency using a modified quadrupole orbitrap mass spectrometer and Tandem Mass Tags (TMT) approach"), Robitaille et al. disclosed that by using the "phased-array deconvolution" (ΦSDM) method to improve the mass resolution of isoplethysmographic reporter ion species in fragment mass spectrometry, it is possible to improve, for example... Acquisition rate of fragment mass spectrometry using an isostatic ion trap.

[0015] There remains a strong need to rapidly quantify as many biomolecules as possible from a sample simultaneously, such as peptides labeled with isotopic mass tags, especially in clinical diagnostics. Here, using isotopic mass tags presents a particular challenge because compact time-of-flight mass analyzers typically lack the mass resolution required to measure the signal of isotopic reporter ion species resolved by mass, while other mass analyzers require long measurement cycles to measure their signals at mass resolution. Summary of the Invention

[0016] This invention provides a method for mass spectrometry quantification of two or more known target ion species, wherein at least two of the target ion species have different masses. The method according to the invention includes the following steps:

[0017] - Provide a number of quantization functions that are at least as many as the number of target ion species of different masses;

[0018] - Provides a mass spectrum with a signal consisting of signal components of two or more target ion species that are not mass-resolved in the mass spectrum;

[0019] -Sum of signals weighted by a quantization function, and

[0020] - A weighted sum determines the quantization parameters of two or more target ion species, wherein (a) the weighted sum forms the non-homogeneous part of the system of equations and the quantization parameters provide or are derived from the solution of the system of equations, or (b) the weighted sum or the parameters derived therefrom are used as input values ​​to a pre-specified lookup table containing the quantization parameters as output values.

[0021] The weighted sum is generated by summing the signals weighted by a quantization function. The summation step also includes integrating the weighted signals using the quantization function, particularly integrating around the signal. It is preferable to perform the summation when the signal exceeds a certain (absolute or relative) threshold. The quantization functions are chosen such that at least some are linearly independent, i.e., they cannot be expressed as a linear combination of other quantization functions. The number of linearly independent quantization functions corresponds at least to the number of target ion species of different masses.

[0022] Quantization parameters can be, for example, the ratio of signal components of two target ion species, or the signal component of a single target ion species from the signal. The intensity of each target ion species can be determined by the signal components and intensity of each target ion species, and compared with the intensities of (isosigma) target ion species from other signals. Target ion species can also be quantified in absolute value by selectively using ion species whose concentrations are given or known (reference or standard species).

[0023] The mass position of the target ion species is preferably known. In addition to the quantization parameters of the target ion species, the solution to the equations may, for example, include the mass shift of the signal, which is caused by the joint shift of the signal components. According to the invention, it is therefore unnecessary to know the mass positions of individual signal components, but only the (relative or absolute) mass separation of the signal components.

[0024] The solution to the system of equations is preferably calculated directly from the weighted sum without any iterative approximation steps. Therefore, the method according to the invention differs significantly from time-consuming optimization methods (where curves for two or more target ion species are matched to the signal). In information technology and digital technology, lookup tables (transformation tables) are used to predefine information and avoid the need for complex calculations later. Quantization parameters are pre-determined as output values ​​(weighted sums or quantities derived therefrom) of certain input values ​​and stored as tables in a storage device. A lookup table can have one or more independent input values.

[0025] The fact that the signal components of two or more target ion species that make up the signal have no mass resolution in the mass spectrum may, for example, mean that the signal does not have a local maximum for each signal component, or that the full width at half maximum (FWHM) of the signal is greater than the resolution between the two target ion species, or that the ratio of the average mass of the target ion species to the minimum mass difference between the target ion species is greater than the mass resolution achieved in the mass spectrum provided around the signal. The FWHM of the provided signal may be 1.5, 2, or 5 times greater than the resolution between the two target ion species.

[0026] The provided mass spectrometer can be, for example, a time-of-flight mass analyzer or an electrostatic ion trap (ESI). Cassini ion traps or magnetic ICR (ion cyclotron resonance) ion traps are used for acquisition. In the case of electrostatic or magnetic ion traps, transient simulated mirror current signals are typically acquired by the detector, and this signal is a Fourier transform mirror current signal of the target ion species. The mass axis of the mass spectrometer can be, but is not necessarily, a calibrated mass axis or a calibrated m / z axis (mass-to-charge ratio); it can also be referenced to physical quantities (e.g., time of flight or frequency), from which a calibrated mass axis or m / z axis can be derived. For example, when a mass spectrometer is acquired using a time-of-flight mass analyzer, a time-of-flight axis is generated in an obvious way. For example, a frequency axis is generated after a Fourier transform of the measured transient mirror current signal (time signal), which is typically acquired using an electrostatic ion trap (Cassini ion trap). It can be obtained by either a Cassini trap or a magnetic ICR (ion cyclotron resonance) ion trap.

[0027] The target ion species can be, in particular, isotopic ion species or isotopic fragment ion species, wherein the sum of the number of protons and neutrons is the same, but the number of protons or neutrons differs, thus allowing for a mass difference of several millidaurates. Specifically, the target ion species is characterized by its individual mass-to-charge ratio m / z. Therefore, molecular ions composed of different isotopes must already be considered as a single target ion species. The difference between target ion species of different masses is particularly evident in that their mass difference is preferably less than 100 millidaurates, more preferably less than 10 millidaurates, and especially less than 1 millidaurate. The mass resolution m / Δm of the mass analyzer used for acquiring the mass spectrometer is preferably less than 15000, more preferably less than 10000, and especially less than 5000.

[0028] In a preferred embodiment, the target ion species is an isotopic fragment ion species, generated by fragmentation from a precursor ion species labeled with an isotopic mass tag. The precursor ion species all possess the same biomolecular species but different isotopic mass tags, and are separated from other precursor ion species based on mass and (selectively) mobility prior to fragmentation. Precursor species with isotopic tags from which precursor ion species are generated in the ion source of a mass spectrometry system are typically separated by liquid chromatography or liquid electrophoresis. The precursor ion species can be peptide species or other types of biomolecular species labeled with isotopic mass tags, such as lipid species, glycan species, sugar species, and analogues.

[0029] The preferred fragment ion species are isoplegic reporter groups, but complementary fragment ions and biomolecules with mass balance groups can also be distinguished in fragment mass spectrometry by these mass balance groups. For example, peptide species can be digestible peptides from proteins digested by enzymes, where the quantification of fragment ion species is used to quantify digestible peptides or proteins in different proteome samples.

[0030] The system of equations is preferably a linear system of equations:

[0031] Where i, j = 1...N

[0032] Where N is the number of target ion types, Q ij These are the matrix components of a system of linear equations, I j This is a quantification parameter for the target ion species. The signal S(x) consists of signal components representing the target ion species:

[0033] For example, where S j (x) is a single signal for the j-th target ion species, normalized to 1. The variable x can be (as described above) a mass-related physical quantity (e.g., time of flight or frequency). Weighted sum g i It is obtained by summing the signal S(x), which is then weighted by the quantization function Qi(x) to form the non-homogeneous part of the linear equation system:

[0034]

[0035] For example, the matrix components Q of a linear solution group ij It can be determined by measuring and normalizing the individual non-recombining signals for each target ion species. Matrix component Q ij It comes from the quantization function Q i (x) Weighted (normalized) individual signals S j The sum of (x). The quantization function is chosen such that the constant matrix components Q... ij The rank corresponds at least to the number of target ion species of different masses. If system overdeterminacy is acceptable, the number of linearly independent quantization functions can be greater than the number of target ion species of different masses. Direct numerical methods, such as determinant methods, are preferred for solving linear equation systems, rather than iterative methods that gradually improve the initial approximation.

[0036] The lookup table can also be determined from the measurement of the target ion species and the measurement of a normalized single or composite signal, the quantization parameters of which are known through appropriate sample preparation.

[0037] Quantization functions can be, for example, (a) polynomials of different orders, (b) delta functions or rectangular functions, preferably all concentrated near the corresponding maximum positions of the target ion species, (c) step functions, wherein each step position is preferably close to the corresponding maximum position of the target ion species, (d) harmonic functions of different periods, or (e) functions used for wavelet transform. The summation of the signal weighted by the delta function corresponds to signal interpolation at the position of the delta function, and is therefore preferably at the maximum position of one of the target ion species. For example, typically when the signal on the mass axis is given only for sampling points on the mass axis, interpolation can include using the signal value of the sampling point closest to the delta function position. If the delta function is centered between two sampling points, the signal can be interpolated, extrapolated, or approximated by regression from the signal values ​​of the two sampling points or other sampling points. The method according to the invention can also be used for saturated signals, i.e., when the signal exceeds a certain maximum value for some regions and is cut off there. A quantization function can be selected for the saturated signal such that it is equal to zero at least at the position where the signal is saturated.

[0038] In the first embodiment, the signal consists of signal components from two target ion species. The signal is weighted by a constant function of value one and the mass axis of the acquired mass spectrum. The sum of these two weights is used to calculate the centroid of the signal, which serves as the input value for a lookup table to determine the intensity ratio of the signal components from the two target ion species. As is known from Blom's publication (J. Am. Soc. Mass Spectrom., 1998, 9, 789-798: "Utility of Peak Shape Analyses in Determining Unresolved Interferences in Exact Mass Measurements at Low Resolution"), only the moments of the measured signal (e.g., the centroid) can be used to determine unresolved superpositions in the measured mass spectrum signal.

[0039] In the second embodiment, the signal also consists of signal components from two target ion species. The signal is weighted using two delta functions centered at the two maximum positions of the two target ion species. The weighted sum corresponds to the interpolated signal values ​​at the maximum positions of the two target ion species, and the ratio of these values ​​is used as input to a lookup table to determine the intensity ratio of the signal components of the two target ion species. Another lookup table may also take the signal component or intensity of one of the two target ion species as its output value.

[0040] In the third embodiment, multiple mass spectra with a signal are provided, and weighted sums are calculated separately for each individual mass spectrum or for partially summed mass spectra. These sums are then calculated separately according to a quantization function to determine the quantization parameters. Partial summation means faithfully adding a certain number of mass spectra to their mass values, thereby improving the signal-to-noise ratio in particular. Weighted sums can be calculated separately for each individual spectrum before calculating the quantization parameters according to the quantization function.

[0041] In the fourth embodiment, several mass spectra with a signal are provided, and a weighted sum is calculated separately for each individual mass spectrum or partially summed mass spectrum, from which quantization parameters for the individual mass spectrum or partially summed mass spectrum are determined. A representative quantization parameter is derived as a (weighted) average from the quantization parameters calculated separately for each individual spectrum or partially summed spectrum. A first quantization function can be used to separately calculate the weighted sum of a first individual mass spectrum or a first partially summed mass spectrum, and a second quantization function can be used to separately calculate the weighted sum of a second individual mass spectrum or a second partially summed mass spectrum. The first and second quantization functions are different.

[0042] The separate calculation of the weighted sum or quantization parameters allows for checking whether the values ​​of these parameters change during the acquisition of a single mass spectrum. If a target ion species is separated from other ion species based on mobility and mass, but overlaps with other ion species in terms of mobility, the resulting isoplegic interference can be read by the change in the weighted sum or quantization parameters and corrected if necessary.

[0043] Furthermore, the present invention provides an apparatus for quantifying two or more target ion species, the apparatus comprising a data processing unit designed and configured to perform the method according to the invention, for example, programmed accordingly. The apparatus according to the invention is preferably connected to a detector of a mass analyzer. In the case of a time-of-flight mass analyzer, the apparatus preferably has a detection unit in which an analog-to-digital converter and a data processing unit are integrated. The data processing unit here is preferably designed such that a weighted sum is determined in real time for each individual mass spectrum.

[0044] The method according to the invention is particularly applicable to mass spectrometry systems where the mass position of the signal component of the target ion species is stable over a relatively long measurement period (e.g., during LC separation runs). However, signal shifts occurring during measurement can be compensated for by additionally measuring the position of the signal component of a single target ion species during measurement and using it to correct a lookup table or a constant matrix component of a system of linear equations. Furthermore, signal shifts caused by joint shifts of signal components can be determined as partial solutions to (nonlinear) equations.

[0045] A key advantage of this invention is that the compact time-of-flight mass analyzer can be used to quantify isoplethysmotic reporter ion species, where the mass resolution of these analyzers within the mass range of isoplethysmotic reporter ion species is typically insufficient to measure the signal components of the reporter ion species at mass resolution. Another advantage of this invention is that existing data processing units can be adapted to allow for real-time determination of the weighted sum of individual spectra, even when acquiring individual spectra at acquisition rates greater than 1 kHz, 5 kHz, or 10 kHz.

[0046] Furthermore, signals can be acquired using electrostatic or magnetic ion traps in a shorter measurement time because a mass-resolved signal is not required when quantifying the target ion species according to the method of the invention. Conversely, this means that the acquisition rate of the mass spectrometer, and therefore the number of target ion species quantified per unit time, can be significantly increased. Attached Figure Description

[0047] The invention can be better understood by referring to the following figures. The elements in the figures are not necessarily drawn to scale, but are mainly intended to illustrate the principles of this disclosure (primarily schematic).

[0048] Figure 1 shows a schematic diagram of a known hybrid mass spectrometry system 100, suitable for quantization via isoplethysmographic mass tags. The hybrid mass spectrometry system 100 includes an LC or other separation device 110, an ion source 121, a mobility separator 144, a quadrupole mass filter 150, a fragmentation cell 160, a time-of-flight mass analyzer 170, and a device 180 for recording and processing mass spectrometry data.

[0049] Figure 2 A flowchart of a method according to the present invention for quantifying target ion species marked with isoplethysmographic mass tags is shown, wherein the target ion species are separated according to their retention time, mobility and mass before fragmentation and are quantified by a signal reporting the ion species or complementary fragment ion species.

[0050] Figures 3A to 3E In one embodiment, it is shown how the intensity ratio of the two signal components 31 and 32 can be determined by a weighted sum and a pre-given lookup table 36 for a signal 30 consisting of two signal components 31 and 32.

[0051] Figure 3A The image shows a portion of a simulated mass spectrum from a time-of-flight mass analyzer, where signal 30 consists of two signal components 31 and 32 of the target ion species, which are not mass-resolved in the mass spectrum.

[0052] Figure 3B and 3C Each of the two quantization functions 33 and 34 and the composite signal 30 is shown.

[0053] Figure 3D The composite signal 30 is shown together with its centroid 35, which is derived from the weighted sum of the signals 30.

[0054] Figure 3E It is a logarithmic graphical representation of a pre-given lookup table 36, which is used to determine the intensity ratio of the two signal components 31 and 32.

[0055] Figures 4A to 4E The second embodiment shows how the intensity ratio of the two signal components 41 and 42 can be determined by weighted sum and a pre-given lookup table 47 for a signal 40 composed of two signal components 41 and 42.

[0056] Figure 4A A portion of a simulated MS2 spectrum from a time-of-flight mass analyzer is shown, which has a signal 40 consisting of two signal components 41 and 42 of the same mass of exosite fragment ions, which is not mass-resolved in the MS2 spectrum.

[0057] Figure 4B and 4C One of the two quantization functions 43 and 44 and the composite signal 40 are shown respectively.

[0058] Figure 4D A composite signal 40 with a weighted sum of signals 45 and 46 is shown, which corresponds to the interpolated value of signal 40 at the maximum position of each of the two signal components 41 and 42.

[0059] Figure 4E A logarithmic graphical representation of a pre-given lookup table 47 is shown, which is used to determine the intensity ratio of the two signal components 41 and 42.

[0060] Figure 5A A portion of a simulated mass spectrum from a time-of-flight mass analyzer is shown, which has a signal 50 consisting of three signal components 51, 52, and 53 of the target ion species, which are not mass-resolved in the mass spectrum.

[0061] Figure 5B Three quantization functions 54, 55, and 56 are shown, each used in conjunction with a composite signal 50. The signal 50 is weighted by these functions to compute three weighted sums, which are used as the non-homogeneous part of the linear equation system. The strengths of the three signal components 51, 52, and 53 provide a direct solution to the linear equation system. Detailed Implementation

[0062] Figure 2A flowchart of a method according to the present invention for quantifying precursor ion species labeled with isotopic mass tags is shown, wherein the precursor ion species are separated according to their LC retention time, mobility and mass before fragmentation, and are quantified by a signal reporting the ion species or complementary fragment ion species.

[0063] The precursor substances from which precursor ion species are generated in the ion source can be digested peptides from several proteome samples, for example, wherein the digested peptides in each proteome sample are labeled with one of different isotopic mass tags and subsequently merged. The method according to the invention can be implemented, for example, with the mass spectrometry system 100 depicted in FIG. 1, whose detection unit has a modified data processing unit that determines the weighted sum of the composite signals of two or more isotopic fragment ion species, thereby determining the quantitative parameters of the two or more isotopic fragment ion species.

[0064] Digestible peptides labeled and combined in different ways are pre-separated based on their retention times during LC separation runs. After the start of the LC separation run, precursor ions generated from the labeled digestible peptides in ESI ion source 121 are separated based on their migration in the gas phase during IMS scans in a mobility separator 144, while MS1 spectra are continuously acquired using a time-of-flight mass analyzer 170, with mass filter 150 and fragmentation cell 160 turned off. The MS1 spectra acquired during the IMS scans provide an IMS-MS overview, which is examined to determine if a given precursor ion species is present at the time of IMS scan acquisition. Precursor ion species with known retention times, migrations, and masses can be identified by comparing their migration and signal quality with those in the IMS-MS overview and by the acquisition time (retention time) of the IMS-MS overview during the LC separation run. IMS-MS overviews are acquired repeatedly until at least one specified precursor ion species is present in the IMS-MS overview.

[0065] After the precursor ion species is identified, an IMS scan (separation) is initiated, during which the mass filter 150 is switched while the precursor ion species is leaving the mobility separator 144, such that only the precursor ion species can pass through the mass filter 150 as far as possible during this period. The precursor ion species separated from other ion species in this way based on mobility and mass is fragmented in the fragmentation cell 160, and an MS2 spectrum (fragment mass spectrometry) is obtained. In the modified detection unit 181, the signals of the fragment ion species (target ion species) are weighted by a quantization function, and a weighted sum is calculated thereby. Here, the signals are preferably those of reporter ion species with isoplethysmographic labels in the lower mass range of the MS2 spectrum. Typically, precursor ion species separated based on mobility have a batch of material in the time-of-flight mass analyzer 170 with a duration of approximately one millisecond, meaning that multiple MS2 spectrum ions can be obtained for one precursor species at an acquisition rate of 10 kHz. In the modified detection unit 181, the weighted sum is typically determined in real time for each individual MS2 spectrum and transmitted to the data storage device 183 via the local bus 184. A central processing unit 182 or further distributed processors (not shown in Figure 1) can determine quantization parameters for fragment ion species, such as the intensity ratio of two isoplethysmotic reporter ion species. The signal intensity ratio and intensity of the isoplethysmotic reporter ion species can be used to determine the intensity of each of the two isoplethysmotic reporter ion species and compare it with the intensities of other reporter ion species present in the MS2 spectrum (quantization). The quantization parameters can be determined for each individual MS2 spectrum, or by first summing the weighted sums of all MS2 spectra of the fragment ion species and determining the quantization parameters from there. During the IMS scan, MS2 spectra of different precursor ion species that can be separated according to mobility can be obtained.

[0066] IMS scans typically take 10 to 100 milliseconds. Therefore, several precursor ion species that can be separated based on mass and mobility can be quantified during an IMS scan. Further IMS scans can be performed to obtain MS2 spectra of other precursor ion species, or MS2 spectra of precursor ion species can be obtained in two or more IMS scans, before a new IMS-MS overview can be obtained.

[0067] Figures 3A to 3E The first embodiment shows how the intensity ratio S1 / S2 of the two signal components 31 and 32 can be determined by weighted sum and a pre-given lookup table 36 for a signal 30 composed of two signal components 31 and 32.

[0068] Figure 3AA portion of a simulated mass spectrum with signal 30 from a time-of-flight mass analyzer is shown. This signal consists of two signal components 31 and 32 from two target ion species, which are not mass-resolved in the mass spectrum. The simulated detector signal with noise components is scanned at a typical sampling rate of 5 GS / s (gigasamples per second) and digitized at 10-bit resolution. Circular symbols correspond to the digitized value pairs (time of flight, intensity) of the composite signal 30. The mass axis is the time-of-flight axis, where scan time points (bins) are given as integers. The two signal components 31 and 32 belong to two target ion species with masses of approximately 128 Daltons and a mass difference of three millidaltons. The simulated mass spectrum has a mass resolution of approximately 12,000, while a mass resolution of 42,000 is required to resolve the two signal components 31 and 32 into separate signals. The signal is determined and extracted from a mass spectrum with a larger number of bins, for example, by using a peak-picking algorithm, where the signal is extended over a finite number of mass channels (bins), here approximately 25, with, for example, 11 bins used for summation. A mass spectrum may contain multiple superimposed signals, which can be identified using appropriate algorithms.

[0069] Figure 3B The first quantization function 33 and the composite signal 30 are shown. The first quantization function 33 is a constant function with a value of one. The first weighted sum g1 is given by the sum of the signals 30 weighted by the first quantization function 33.

[0070] Figure 3C The second quantization function 34 and the composite signal 30 are shown. The second quantization function 34 is a (linear) time-of-flight axis. The second weighted sum g2 is given by the sum of the signals 30 weighted by the second quantization function 34.

[0071] Figure 3D The composite signal 30 is shown along with its centroid 35, which is shown as a dashed line and is generated as g2 / g1 as a quantity derived from the weighted sum.

[0072] Figure 3E It is a logarithmic graphical representation of a pre-given lookup table 36, plotting the centroid of the composite signal on its horizontal axis (the input value of the lookup table) and the intensity ratio of signal components 31 and 32 on its vertical axis (the output value of the lookup table). Figure 3E In the diagram, the centroid 35 calculated from the weighted sums g1 and g2, and the corresponding intensity ratio S2 / S1 of the two signal components 31 and 32, are represented by dashed lines. The intensity ratio is 1.1 and is determined in this case with an average relative error of less than 0.5%.

[0073] Figures 4A to 4EThe second embodiment shows how the intensity ratio of the two signal components 41 and 42 can be determined by weighted sum and a pre-specified lookup table 47 for a signal 40 composed of two signal components 41 and 42.

[0074] Figure 4A A portion of a simulated MS2 spectrum with signal 40 from a time-of-flight mass analyzer is shown. This signal consists of two signal components 41 and 42 from two isoplethysmotic reporter ion species, which are not mass-resolved in the MS2 spectrum. The simulated detector signal with noise components is scanned at a typical sampling rate of 5 GS / s (gigasamples per second) and digitized at 10-bit resolution. Circular symbols correspond to the digitized value pairs (time of flight, intensity) of the composite signal 40. The mass axis is the time-of-flight axis, where scan time points (bins) are given as integers. The masses of the two isoplethysmotic reporter ion species are approximately 128 Daltons, with a mass difference of 6.3 millidaltons. The simulated MS2 spectrum has a mass resolution of approximately 12,000, while a mass resolution of 20,000 is required to resolve the two signal components 41 and 42 into separate signals.

[0075] Figure 4B The first quantization function 43 and the composite signal 40 are shown. The first quantization function 43 is a delta function centered at the maximum position of the first signal component 41. The sum of the signals 40 weighted by the first quantization function 43 gives the first weighted sum g1 and corresponds to the interpolation of the signals 40 at the position of the first delta function.

[0076] Figure 4C The second quantization function 44 and the composite signal 40 are shown. The second quantization function 43 is a delta function centered at the maximum position of the second signal component 42. The second weighted sum g2 of the signal 40 weighted by the second quantization function 43 is given and corresponds to the interpolation of the signal 40 at the position of the second delta function.

[0077] Figure 4D A composite signal 40 is shown with interpolated signal values ​​g1(45) and g2(46) at the positions of two delta functions, which are shown as two cross symbols at the end of the dashed line.

[0078] Figure 4E It is a logarithmic graphical representation of a pre-given lookup table 47, plotting the weighted sum g2 / gl ratio on its horizontal axis (the input values ​​of the lookup table) and the intensity ratio S2 / S1 of signal components 41 and 42 on its vertical axis (the output values ​​of the lookup table). Figure 4EIn the diagram, the ratio g2 / g1(48) of the two signal components 41 and 42 and the corresponding intensity ratio S2 / S1(49) are represented by dashed lines. The intensity ratio is 0.2 and is determined here with an average relative error of less than 7.5%. The average relative error is larger than in the previous embodiment because the intensity ratio is closer to the edge of the lookup table and comes from... Figure 4A The intensity of the composite signal 40 in the -E example is significantly lower than that from... Figure 3A -E Example: The intensity of the composite signal 30.

[0079] Figure 5A A portion of a simulated mass spectrum from a time-of-flight mass analyzer is shown, featuring signal 50 composed of three signal components 51, 52, and 53 for the target ion species, which are not mass-resolved in the mass spectrum. The simulated detector signal with noise components is scanned at a typical sampling rate of 5 GS / s (gigasamples per second) and digitized at 10-bit resolution. Circular symbols correspond to digitized value pairs (time of flight, intensity) of the composite signal 50. The mass axis is the time-of-flight axis, where scan time points (bins) are given as integers. The masses of the three target ion species are approximately 128 Daltons. The mass difference between the center and left-hand signal components 52 and 51 is 3 millidaltons. The mass difference between the center and right-hand signal components 52 and 53 is 5 millidaltons.

[0080] Figure 5B Three quantization functions 54, 55, and 56 are shown, each associated with the composite signal 50.

[0081] The first quantization function 54 is a constant function with a value of one. The sum of signals 50 weighted by the first quantization function 54 gives the first weighted sum g1. The second quantization function 55 is a (linear) time-of-flight axis whose zero-crossing is shifted to the maximum position of the center signal component 52. The sum of signals 50 weighted by the second quantization function 55 gives the second weighted sum g2. The third quantization function 56 is the square of the second quantization function 55. The sum of signals 50 weighted by the third quantization function 56 gives the third weighted sum g3.

[0082] The relative intensity I of the three signal components 51, 52, and 53 i (i=1…3) is obtained as a solution to the system of linear equations:

[0083]

[0084] Q i (x) is the quantization function, S i (x) represents the individual normalized signal components, and the weighted sum g i The non-homogeneous part of the system of equations is formed. A single signal component S i(x) can be obtained from previous measurements. In this example, the average relative error of the intensity ratio between the signal components is less than 1%.

[0085] The invention has been described above with reference to various specific exemplary embodiments. However, it should be understood that various aspects or details of the described exemplary embodiments may be modified without departing from the scope of the invention.

Claims

1. A method for mass spectrometry quantification of two or more known target ion species, wherein at least two of the target ion species have different masses, the method comprising the following steps: - Provide a number of linearly independent quantization functions that are at least as many as the number of target ion species of different masses; - Provide a mass spectrum having a signal consisting of signal components of two or more target ion species, which are not mass-resolved in the mass spectrum, wherein the signal does not have a local maximum for each signal component, or the full width at half maximum of the signal is greater than the resolution between the two target ion species, or the ratio of the average mass of the target ion species to the minimum mass difference between the target ion species is greater than the mass resolution achieved in the mass spectrum provided around the signal. - Summing of signals weighted by a quantization function, and - A weighted sum is used to determine quantization parameters for two or more target ion species, wherein (a) the weighted sum forms the non-homogeneous part of a system of equations, and the quantization parameters provide a solution to the system of equations, or are derived from the solution, or (b) the weighted sum or the parameters derived therefrom are used as input values ​​to a pre-given lookup table containing the quantization parameters as output values. - The quantization parameter is the ratio of the signal components of two target ion species, or the signal component of a single target ion species from the signal.

2. The method according to claim 1, wherein the system of equations is a linear system of equations.

3. The method according to claim 1, wherein the quantization function is (a) a polynomial of different orders, (b) a delta function or a rectangular function, all of which are concentrated near the corresponding maximum position of the target ion species, (c) a step function, wherein each step position is close to the corresponding maximum position of the target ion species, (d) a harmonic function of different periods, or (e) a function used for wavelet transform.

4. The method of claim 1, wherein the signal consists of signal components of two target ion species.

5. The method of claim 4, wherein the signal is weighted by a constant function of value one and the mass axis of the acquired mass spectrum, and the centroid of the signal is calculated by the sum of the two weights, wherein the centroid is used as the input value of a lookup table to determine the intensity ratio of the two signal components of the two target ion species.

6. The method according to claim 4, wherein, The signal is interpolated at the known mass of the two target ion species, and the ratio of the two signal components of the two target ion species is determined by a lookup table based on the ratio of the interpolated signal values ​​at the known mass of the two target ion species.

7. The method according to claim 1, wherein, The target ion species are isomass-heterotopic fragment ion species, which are generated by the fragmentation of precursor ion species. The precursor ion species all have the same biomolecule species and are each labeled with different isomass-heterotopic mass tags. Each of them has a reporter group and a mass balance group. Furthermore, the precursor ion species are separated from other precursor ion species in terms of mass and are selectively further separated according to mobility before fragmentation.

8. The method according to claim 7, wherein, Iso-depositional fragment ions possess iso-depositional reporter groups.

9. The method according to claim 1, wherein, Multiple mass spectra with a signal are provided, where a weighted sum is calculated separately for each mass spectrum, either individually or as a partial sum, and then summed separately according to a quantization function to determine the quantization parameters.

10. The method according to claim 9, wherein, The weighted sums are calculated separately for each individual mass spectrometer, and then summed separately according to the quantization function to determine the quantization parameters.

11. The method according to claim 1, wherein, Several mass spectra with signals are provided, wherein a weighted sum is calculated for each individual mass spectrum or partially summed mass spectrum, and quantization parameters for the individual mass spectra or partially summed mass spectra are determined, wherein representative quantization parameters are derived as averages from multiple individually determined quantization parameters.

12. The method according to claim 11, wherein, The representative quantization parameter is derived as a weighted average from multiple individually determined quantization parameters.

13. The method according to claim 11, wherein, The first quantization function is used to calculate the weighted sum of the first individual mass spectrum or the first partial summation mass spectrum separately, and the second quantization function is used to calculate the weighted sum of the second individual mass spectrum or the second partial summation mass spectrum separately, wherein the first and second quantization functions are different.

14. An apparatus for quantifying two or more target ion species, the apparatus comprising a data processing unit designed and configured to perform the method according to claim 1.

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