Method and device for quantifying target ion species

Abstract

A method for the mass spectrometric quantification of two or more known target ion species, at least two of which differ in their masses, comprising the steps of: - Providing quantification functions whose number is at least as large as the number of target ion species of different masses, - Providing a mass spectrum with a signal composed of signal components of the two or more target ion species that are not mass-resolved in the mass spectrum, - Weighting the signals with the respective quantification functions, - Calculating a weighted sum by integrating the signal weighted with the respective quantification function, and - Determining a quantification quantity for the two or more target ion species from the weighted sums, wherein (a) the weighted sums form the inhomogeneous part of a system of equations and the quantification quantity results as a solution of the system of equations or is derived therefrom or (b) the weighted sums or quantities derived therefrom serve as input values ​​for predetermined lookup tables which contain the quantification quantity as an output value.

Description

Field of the InventionThe invention relates to mass spectrometric methods and apparatus for quantifying target ion species, the masses of which are known, and more particularly to quantifying peptide ion species labeled with isobaric mass tags.BACKGROUND OF THE INVENTIONProteomics is a core technology in the biosciences, which is directed to clarifying molecular mechanisms of protein-level diseases and finding biomarkers and using these in clinical diagnostics. For this purpose, the proteins present in a sample and their modifications are typically identified by mass spectrometry. Proteomics has developed as a diszipine at the cleavage site of instrument construction, biochemistry and bioinformatics. The instruments used in proteomics typically couple mass spectrometry to liquid chromatography (LC) or to liquid chromatography and ion mobility spectrometry (IMS).In addition to identification, quantification of proteins and their modifications plays an ever greater role in clarifying the molecular mechanisms of diseases and in diagnosing them in the future in clinical routine. Proteomics allows quantitative comparison of a proteome type from different samples (e.g., from different subjects or a time series of a subject) or quantitative comparison of different proteome types. Mass spectrometric quantification can be carried out without labeling or by using labeling reagents.Various types of labels for mass spectrometric quantification are known, such as, for example, stable isotope labeling (SILAC) or labeling by means of isobaric mass tags (such as, for example, iTRAC™ (isobaric tags for relative and absolute quantification), TMT™ (tandem mass tag) or EASI tag (Eastiy Abstractable Sulfoxide-based Isobaric tag; published in: Virriera Winter et al., Nat. Methods 2018, 15, 527-530).An isobaric mass tag consists of three components, namely a reactive group for coupling the mass tag to the target species, a reporter group and a mass balance group. The two latter groups are connected via a cleavable chemical unit in such a way that, when the labeled target species is fragmented in the gas phase, the reporter group is separated from the complementary radical (target species with isobaric radical label). It is decisive in the quantification with different isobaric mass tags that the mass of the reporter groups of the isobaric mass tags is different, but the total mass of the isobaric mass tags is identical. The mass balance groups thereby ensure the balance of the mass differences which arise as a result of the incorporation of different isotopes into the reporter groups. The heavy isotopes are thus distributed differently in the isobaric mass tags to the reporter and mass-balancing groups. The signals of the reporter ion species formed during the fragmentation can be distinguished in principle in the mass spectrum. The relative ratios of the signals of the reporter groups can be used to quantify the target species to be analyzed relative to one another.The quantification of two or more proteome samples by means of isobaric mass tags typically takes place as follows. The proteome samples are subjected separately to enzymatic digestion. The digestion peptides produced in this way are labeled with one of the different isobaric mass tags before all digested and differently labeled digestion peptides of the proteome samples are combined and analyzed. The digest peptides are generally separated by liquid chromatography (LC) and optionally additionally by ion mobility spectrometry (IMS) in the gas phase and analyzed by mass spectrometry. A peptide species present in each of the enzymatically digested proteome samples, but labeled with different isobaric mass tags, elutes in the LC separation at the same retention time and is also not separated by the optional mobility separation. Due to the same total mass of isobaric mass tags, the labeled ion species generated from a peptide species appear as a single signal in a recorded mass spectrum (MS1). After isolation of the labeled ion species of a peptide species according to retention time, mobility (optional) and mass and subsequent fragmentation, the principally distinguishable signals of the reporter ion species are measured in a fragment mass spectrum (MS2 or MS / MS) and used for quantification of the peptide species in the proteome samples. Quantification of a protein species is effected via one or more quantified peptide species which are generated from the protein species by the enzymatic digestion.The signals of the reporter ion species are in the low mass range of the fragment mass spectra and are generally not overlaid there by other fragment ion species. In addition to the reporter ion species, fragmentation also gives rise to so-called complementary fragment ion species which have the labeled mass balance group and the peptide species and which are likewise distinguishable in the fragment mass spectrum by the mass balance group and can be used for quantification.The greatest advantage of quantification using isobaric mass tags is the multiplex capability, i.e. that many (currently up to 16) samples can be quantified simultaneously. In the publication by Ogata et al. (Anal. Chem. 2020, 92, 8037-8040: "Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-Based Quantification in Proteomic LC / MS / MS"), the problem of isobaric interference which arises in the quantification by means of isobaric mass tags is investigated when more than one labeled ion species is selected and fragmented in the isolation (LC / MS or LC / IMS / MS). The isobaric interference is investigated here for different mass spectrometric systems which have an electrostatic ion trap (Orbitrap®) or a time-of-flight separator as mass analyzer.FIG. 1 shows a hybrid mass spectrometric system ( 100) known from the prior art in schematic representation, which is preferably suitable for quantification by means of isobaric mass tags. The hybrid mass spectrometric system (100) comprises an LC 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 acquiring and processing mass spectrometric data. Isolation of a target ion species before fragmentation thereof is effected by a temporal separation according to retention time and mobility and by filtering according to mass in the quadrupole mass filter ( 150).The mobility separator (144) may be of the TIMS type (TIMS= Tr Ion Mobility Spectrometry), which is preferably operated in a parallel accumulation mode. In this mode, ions are accumulated in an upstream portion of the mobility separator (144) or an upstream ion trap (not shown), while previously accumulated ions are analyzed in parallel in time in a downstream portion of the mobility separator (144). The time-of-flight mass analyzer (170) is typically an orthogonal ion injection (OTOF) time-of-flight mass analyzer and at least one reflector.The LC separator (110) is coupled to the ion source (121), which is typically an atmospheric pressure powered electrospray ion source (ESI). The ions generated in the chamber (120) are introduced into a first vacuum chamber (140) via a transfer capillary (141), and thereafter deflected into an RF ion funnel (143) by a repulsive DC electric potential applied to a deflection electrode (142). The RF ion funnel (143) directs the ions to the mobility separator (144). The ion species released from the mobility separator (144) and separated for mobility are directed to the quadrupole mass filter (150), which can transmit ions or filter for mass. An ion species isolated in the quadrupole mass filter (150) is directed to the fragmentation cell (160), in which fragment ion species can be generated from the ion species. The fragmentation of the peptide ion species labelled with isobaric mass tags is preferably effected by collision-induced dissociation (CID), but can also be effected by other types of fragmentation, such as e.g. electron transfer dissociation or photodissociation. Fragmentation can be switched on and off. The time-of-flight mass analyzer ( 170) has, in comparison with the time duration of a mass push of an ion species after the mobility separator ( 144), such a short recording time for a mass spectrum that a plurality of (fragment) mass spectra can be recorded for each mobility-separated ion species contained in the mass push.The mass spectrometric system (100) is controlled by the device (180), which has a detection unit (181), a central processor (182) (CPU) and a data memory (183), via the line (185). The components of the device (180) are interconnected via a local bus (184), e.g., via a peripheral component interconnect express (PCI express) bus. The detection unit ( 181) is connected to an ion detector ( 171) of the time-of-flight mass analyzer ( 170), which is located at the end of the flight path and which generates a pulsed electron current for impinging ion pulses. Typically, the ion detector (171) comprises a photomultiplier such as a microchannel plate. The detection unit ( 181) has an analog-to-digital converter with which the electron current generated in the detector is digitized, and a data processing unit. The data processing unit can ascertain, for example, in real time the intensities and the flight times of individual signals in the flight time mass spectrum.As described above, labeling with isobaric mass tags allows parallel quantification of multiple (currently up to 16) samples. However, isobaric reporter groups are also used for this purpose, some of which have a mass difference of only a few millidaltons (daltons=atomic mass unit) (isobaric reporter ion species), which presupposes a high requirement for the mass resolution of the mass analyzers used. However, especially time-of-flight mass analyzers have a lower mass resolution than in the higher mass range, especially in the lower mass range. In contrast, electrostatic ion traps such as the Orbitrap® or ICR magnetic ion trap (ICR) have a higher mass resolution in the low mass range than in the higher mass range, but require a sufficient time period for recording a mass spectrum with sufficient mass resolution.In a poster (67th ASMS Conference on Mass Spectrometry and Allied Topics, MP 735: "Improved identification, quantification accuracy, and workflow efficiency using a modified quadrupole Orbitrap mass spectrometer and Tandem Mass Tags (TMT) approach"), it is disclosed that, for an electrostatic ion trap such as the Orbitrap® the recording rate of fragment mass spectra can be increased if the mass resolution for isobaric reporter ion species in the fragment mass spectra is improved by using a "Phased Spectrum Deconvolution" method (ΦSDM).US 2013 / 0 309 774 A1, for example, discloses a method for quantifying peptides by mass spectrometry, wherein the peptides are each labeled with isobaric tags of different tag types in order to form a tag-labeled analyte in each case. Each of the tags contains a mass labeling group and a mass normalizing group, each of which is linked to the peptide via a bond. The tags labeled analytes are exposed to a dissociative energy level using a mass spectrometer. The bonds of the labeled analytes to the mass normalizing groups are cleaved to form a neutral first mass normalizing group and a first charged mass labeled analyte, wherein the mass normalizing groups are each configured to form a neutral molecule when the bond is cleaved. For both charged mass-labeled analytes, a mass-to-charge ratio is measured, wherein the mass-to-charge ratio of the first charged mass-labeled analyte is different from that of the second charged mass-labeled analyte. The concentrations of the two analytes are determined based on the frequency values of the respective mass-to-charge ratios of the first and second charged mass-labeled analytes.A method for quantitative multiplex analysis of proteins is disclosed, for example, in WO 2018 / 042 454 A2. The method described therein combines an MS1 and an MS20 labeling technique (hyperplexing) in order to enable efficient quantification with reduced technical variability and at the same time increased multiplexing capacity. In the method described therein, the peptides are labeled with isobaric tags and subsequently identified by means of LC mass spectrometry analysis. Quantification of the labeled peptide is performed with a specific tool for quantification based on the isobaric tag method in conjunction with software.There is still a great need to quantify the biomolecule species, e.g. peptide species, labeled with isobaric mass tags from as many samples as possible simultaneously and in a short time, in particular in clinical diagnostics. The use of isobaric mass tags represents a particular challenge in this case because compact time-of-flight mass analyzers often do not have the necessary mass resolution in order to measure the signals of isobaric reporter ion species in a mass-resolved manner, or other mass analyzers require a long measurement duration in order to measure their signals in a mass-resolved manner.SUMMARY OF THE INVENTIONThe present invention provides a method for mass spectrometric quantification of two or more known target ion species, at least two of which differ in mass. The method according to the invention comprises the following steps:providing quantification functions whose number is at least as great as the number of target ion species of different masses;providing a mass spectrum having a signal composed of signal components of the two or more target ion species that are not mass resolved in the mass spectrum;weighting the signals with the respective quantification functions; calculating a weighted sum in each case by integrating the signal weighted with the respective quantification function, anddetermining a quantification variable for the two or more target ion species from the weighted sums, wherein (a) the weighted sums form the inhomogeneous component of a system of equations and the quantification variable results as a solution of the system of equations or is derived therefrom or (b) the weighted sums or variables derived therefrom serve as input values for predetermined look-up tables which contain the quantification variable as output value.The weighted sums result from summing the signal weighted with the quantification functions. The summing step also comprises an integration of the signal weighted with the quantification functions, in particular the integration in the region of the signal. The summation preferably takes place where the signal exceeds a specific (absolute or relative) threshold value. The quantification functions are selected such that at least some are linearly independent, i.e. cannot be represented as a linear combination of the other quantification functions. The number of linearly independent quantification functions corresponds at least to the number of target ion species of different masses.The quantification variable can be, for example, a ratio of the signal components of two target ion species or the signal component of a single target ion species on the signal. From the signal component of the individual target ion species and the intensity of the signal, the intensity of the individual target ion species can be determined and compared with the intensities of (isobaric) target ion species of further signals. By optionally used ion species whose concentrations are predetermined or known (reference or standard species), the target ion species can also be quantified absolutely.Preferably, the mass positions of the target ion species are known. In addition to the quantification variables for the target ion species, the solution of the equation system can also comprise, for example, a mass shift of the signal which results from a joint shift of the signal components. According to the invention, therefore, the mass positions of the individual signal components do not have to be known, but only the (relative or absolute) mass distances of the signal components.The solution of the equation system is preferably calculated directly from the weighted sums without iterative approximation steps. The method according to the invention thus differs in particular from time-consuming optimization methods in which curve shapes of two or more target ion species are adapted to the signal. Look-up tables (conversion tables) are used in information and digital technology to define information in advance and later to use it to avoid complicated calculations. The quantification variable is previously determined as output values for specific input values (weighted sums or variables derived therefrom) and stored in a memory as a table. A lookup table may have one or more independent input values.The fact that the signal components of the two or more target ion species from which the signal is composed are not mass-resolved in the mass spectrum can mean, for example, that the signal does not have a local maximum for each signal component, or that the full full full half width of the signal is greater than the distance of two target ion species, or that the ratio between the average mass of the target ion species and the smallest mass difference between the target ion species is greater than the mass resolution which is achieved in the provided mass spectrum in the region of the signal. The full width at half maximum of the signal provided can be greater than the distance of two target ion species by more than a factor of 1.5, 2 or 5.The mass spectrum provided can be recorded, for example, with a time-of-flight mass analyzer or with an electrostatic ion trap (Orbitrap® Cassini ion trap) or a magnetic ion cyclotron resonance (ICR ion trap). In the case of electrostatic or magnetic ion traps, a detector typically captures a transient analog image current signal, the signal being the Fourier transformed image current signal of the target ion species. The mass axis of the mass spectrum may be, but need not be, a calibrated mass axis or calibrated m / z axis (mass to charge ratio), but may be related to a physical quantity (e.g., time of flight or frequency) from which a calibrated mass axis or m / z axis may be derived. A time-of-flight axis is obtained, for example, in an obvious manner if the mass spectrum is recorded with a time-of-flight mass analyzer. A frequency axis is obtained, for example, after a Fourier transformation of a measured transient image current signal (time signal), which is typically recorded with an electrostatic ion trap (Orbitrap® or Cassini trap) or a magnetic ICR ion trap (ion cyclotron resonance).The target ion species can be, in particular, isobaric ion species or isobaric fragment ion species in which the sum of the proton number and the neutron number is the same, but which differ in the proton number or neutron number and which can therefore have a mass difference of a few millidaltons. In particular, target ion species are distinguished by an individual mass-to-charge ratio m / z. Thus, molecular ions of different isotope composition are already to be regarded as individual target ion species. The target ion species of different masses differ in particular in that their masses are preferably spaced apart by less than 100 millidaltons, preferably less than 10 millidaltons and in particular less than 1 millidalton. The mass resolution m / Δm of the mass analyzer used for recording the mass spectra is preferably less than 15000, preferably less than 10000 and in particular less than 5000.In a preferred embodiment, the target ion species are isobaric fragment ion species resulting from fragmentation from precursor ion species labeled with isobaric mass tags. The precursor ion species all have the same biomolecule species and different isobaric mass tags and are isolated from other precursor ion species prior to fragmentation for mass and (optionally) mobility. The isobarmarked precursor species from which the precursor ion species are generated in an ion source of a mass spectrometric system are typically separated by liquid chromatography or liquid phase electrophoresis. The precursor ion species may comprise peptide species or other types of biomolecule species labeled with isobaric mass tags, e.g., lipid species, glycan species, saccharide species, and the like.The isobaric fragment ion species are preferably isobaric reporter groups, but may also be complementary fragment ion species that have the mass balance groups and the biomolecule species and that are also distinguishable in the fragment mass spectrum by the mass balance groups. A peptide species can be, for example, a digest peptide of an enzymatically digested protein, wherein the quantification of the fragment ion species is used for quantifying the digest peptide or the protein in different proteome samples.The equation system is preferably a linear equation system: wherein N is the number of target ion species, Q are ij matrix components of the linear equation system, and I are j quantification variables of the target ion species. The signal S(x) is composed of signal components of the target ion species: wherein S j( x) is, for example, the single signal of the jthtarget ion species normalized to one. The variable x may be a mass-related physical variable (e.g. time of flight or frequency) (as already described above). The weighted sums g i result from summing the signal S(x) weighted with the quantification functions Q i( x) and form the inhomogeneous component of the linear equation system:The matrix components Q ij of the linear solution system can be determined, for example, by measuring and normalizing an uncombusted single signal for each target ion species. The matrix components Q ij are obtained by summing the individual signals S j( x) weighted (normalized) with the quantification functions Q i( x). The quantification functions are selected such that the rank of the constant matrix components Q ij corresponds at least to the number of target ion species of different masses. The number of linearly independent quantification functions can be greater than the number of target ion species of different masses if one is wanting to accept a certain overdetermination. To solve the linear equation system, direct numerical methods are preferably used, such as the determinant method, without an initial approximation being improved stepwise, as in the case of iterative methods.The look-up tables can likewise be determined from measured and normalized individual signals of the target ion species or by measurements of composite signals for which the quantification variables are known by corresponding sample preparation.The quantification functions can be, for example, (a) polynomials of different order, (b) delta functions or rectangular functions, which are preferably each centered in the vicinity of the respective maximum position of the target ion species, (c) step functions, wherein the step positions are preferably each located in the vicinity of the respective maximum position of the target ion species, (d) harmonic functions of different periodicity, or (e) functions used for a wavelet transformation. The summing of the signal weighted with a delta function corresponds to an interpolation of the signal at the position of the delta function, i.e., preferably at the maximum position of one of the target ion species. If the signal on the mass axis is given only for sampling points on the mass axis as usual, the interpolation can consist, for example, in using the signal value from the sampling point which is closest to the position of the delta function. If the delta function is centered between two sampling points, the signal can be interpolated, extrapolated or approximated by regression there from the signal values of the two sampling points or further sampling points. The method according to the invention can also be applied to a saturated signal, i.e. if the signal goes beyond a specific maximum value for specific ranges and is cut off there. The quantification functions can be chosen for saturated signals to be at least zero where the signal is in saturation.In a first embodiment, the signal is composed of the signal components of two target ion species. The signal is weighted with a constant function and the mass axis of the recorded mass spectrum. From the two weighted sums, the centroid of the signal is calculated, which is used as an input value for a look-up table in order to determine the intensity ratio of the signal components of the two target ion species. From the publication by Blom (J. Am. Soc. Mass Spectrum., 1998, 9, 789-798: "Utility of Peak Shape Analyses in Determining Resolved Interferences in Exact Mass Measurements at Low Resolution") it is only known that moments of a measured signal (such as the centroid) are used to determine non-resolved overlays in a measured mass spectrometric signal.In a second embodiment, the signal is also composed of the signal components of two target ion species. The signal is weighted with two delta functions centered at the two maximum positions of the two target ion species. The weighted sums correspond to the interpolated signal values at the maximum positions of the two target ion species, the ratio of which is used as an input value for a look-up table in order to determine the intensity ratio of the signal components of the two target ion species. Another look-up table can also have the signal component or the intensity of one of the two target ion species as output value.In a third embodiment, a plurality of mass spectra are provided with the signal, wherein the weighted sums are respectively calculated separately for individual mass spectra or for partially summed mass spectra and are then summed separately according to a quantification function in order to determine the quantification variables therefrom. Partial summing means that a certain number of mass spectra is summed up with true mass in order in particular to improve the signal-to-noise ratio. The weighted sums can be calculated separately for each individual spectrum and then added separately according to the quantification function in order to determine the quantification variables therefrom.In a fourth embodiment, a plurality of mass spectra are provided with the signal, wherein the weighted sums are respectively calculated separately for individual mass spectra or for partially summed mass spectra and quantification variables for the individual mass spectra or partially summed mass spectra are determined therefrom. Representative quantification variables result as (weighted) averages from the quantification variables calculated separately for individual spectra or partially summed spectra. In this case, first quantification functions can be used in the separate calculation of the weighted sums of a first individual mass spectrum or of a first partially summed mass spectrum and second quantification functions can be used in the separate calculation of the weighted sums of a second individual mass spectrum or of a second partially summed mass spectrum, wherein the first and second quantification functions are different.A separate calculation of the weighted sums or the quantification variables enables a check as to whether the values of the weighted sums or the quantification variables change during the recording of the individual mass spectra. If the target ion species are isolated from other ion species by mobility and mass, but overlap with other ion species in terms of mobility, isobar interference resulting therefrom can be read and optionally corrected based on changes in the weighted sums or quantification variables.The present invention further provides an apparatus for quantifying two or more target ion species, comprising a data processing unit which is designed and configured, e.g. suitably programmed, for carrying out the method according to the invention. The device 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 device preferably has a detection unit in which an analog-to-digital converter and the data processing unit are integrated. The data processing unit is preferably designed such that the weighted sums are determined in real time for each individual mass spectrum.The method according to the invention is suitable in particular for mass spectrometric systems in which the mass positions of the signal components of the target ion species are stable over a longer measurement duration (e.g. during an LC separation). However, a shift in the signal that occurs during a measurement can be compensated for by additionally measuring the positions of the signal components of individual target ion species during the measurement and using these for correcting the look-up tables or the constant matrix components of the linear equation system. Furthermore, a shift of the signal, which results from a common shift of the signal components, can be determined as a partial solution of a (nonlinear) equation system.An important advantage of the present invention is in particular that compact time-of-flight mass analyzers can be used for quantifying isobaric reporter ion species, the mass resolution of which in the mass range of the isobaric reporter ion species is typically not sufficient to measure the signal components of the reporter ion species in a mass-resolved manner. A further advantage of the present invention is that existing data processing units can be adapted in such a way that the determination of the weighted sums of individual spectra is also possible in real time if these are recorded at a recording rate of more than 1 kHz, 5 kHz or 10 kHz.Furthermore, signals with electrostatic or magnetic ion traps can be recorded with a shortened measurement duration, since no mass-resolved signals are necessary for a quantification of the target ion species using the method according to the invention. Conversely, this means that the recording rate of the mass spectra and thus the number of quantified target ion species per time can be significantly increased.BRIEF DESCRIPTION OF THE DRAWINGSFor a better understanding of the disclosure, reference is made to the following figures. The elements in the figures are not necessarily to scale, and are intended to primarily illustrate the principles of the disclosure (for the most part, schematically).FIG. 1 shows a hybrid mass spectrometric system ( 100) known from the prior art in schematic representation, which is suitable for quantification by means of isobaric mass tags. The hybrid mass spectrometric system (100) comprises 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 acquiring and processing mass spectrometric data.FIG. 2 shows a flow chart for a method according to the invention for quantifying target ion species labeled with isobaric mass tags, which are isolated before their fragmentation according to retention time, mobility and mass and which are quantified by means of signals from reporter ion species or from complementary fragment ion species.FIGS. 3A to 3E show, in one embodiment, how the intensity ratio of the two signal components (31, 32) can be determined for a signal (30) which is composed of two signal components (31, 32) by means of weighted sums and a predefined look-up table (36).FIG. 3A shows a section of a simulated mass spectrum of a time-of-flight mass analyzer with the signal (30), which is composed of the two signal components (31, 32) of target ion species, which are not mass-resolved in the mass spectrum.Figures 3B and 3C each show one of the two quantification functions (33, 34) together with the composite signal (30).FIG. 3D shows the composite signal ( 30) with the center of gravity ( 35) of the signal ( 30), which results as a derived variable from weighted sums of the signal ( 30).FIG. 3E shows a logarithmic graphic representation of the predefined look-up table (36) which is used for determining the intensity ratio of the two signal components (31, 32).FIGS. 4A to 4E show, in a second embodiment, how the intensity ratio of the two signal components (41, 42) can be determined for a signal (40) which is composed of two signal components (41, 42) by means of weighted sums and a predefined look-up table (47).FIG. 4A shows a section of a simulated MS2 spectrum of a time-of-flight mass analyzer with the signal (40), which is composed of the two signal components (41, 42) of isobaric fragment ion species, which are not mass-resolved in the MS2 spectrum.Figures 4B and 4C each show one of the two quantification functions (43, 44) together with the composite signal (40).FIG. 4D shows the composite signal (40) with the weighted sums of the signal (45, 46) which correspond to the interpolated values of the signal (40) at the respective maximum positions of the two signal components (41, 42).FIG. 4E shows a logarithmic graphic representation of the predefined look-up table (47) which is used for determining the intensity ratio of the two signal components (41, 42).FIG. 5A shows a section of a simulated mass spectrum of a time-of-flight mass analyzer with a signal (50) which is composed of three signal components (51, 52, 53) of target ion species which are not mass-resolved in the mass spectrum.FIG. 5B shows three quantification functions ( 54, 55, 56), each together with the composite signal ( 50), with which the signal ( 50) is weighted in order to calculate three weighted sums which are used as inhomogeneous component of a linear equation system. The intensities of the three signal components ( 51, 52, 53) result as a direct solution of the linear equation system.DETAILED DESCRIPTION OF THE INVENTIONFIG. 2 shows a flow chart for a method according to the invention for quantifying precursor ion species marked with isobaric mass tags, which are isolated before their fragmentation according to LC retention time, mobility and mass and which are quantified by means of signals from reporter ion species or from complementary fragment ion species.The precursor species from which the precursor ion species are generated in an ion source may be, for example, digest peptides from multiple proteome samples, the digest peptides in each proteome sample being labeled with one of the different isobaric mass tags and then merged. The method according to the invention can be carried out, for example, with the mass spectrometric system ( 100) shown in FIG. 1, the detection unit of which has a modified data processing unit, with which weighted sums of a composite signal of two or more isobaric fragment ion species are determined, from which quantification variables of the two or more isobaric fragment ion species are determined.The digest peptides differently labelled and joined are pre-separated in an LC separation run according to their retention time. After the start of the LC separation run, the precursor ion species generated in the ESI ion source (121) from the labeled digest peptides are separated in an IMS scan of the mobility separator (144) for mobility in the gas phase, while with the mass filter (150) switched off and the fragmentation cell (160) switched off, MS1 spectra are continuously recorded with the time-of-flight mass analyzer (170). The MS1 spectra recorded during the IMS scan produce an IMS-MS overview which is examined to determine whether predefined precursor ion species are present at the recording time of the IMS scan. The precursor ion species whose retention time, mobility and mass are known can be found by comparison with the mobility and mass of signals in the IMS-MS map and on the acquisition time of the IMS-MS map during the LC separation (retention time). The acquisition of IMS-MS reviews is repeated until at least one of the predetermined precursor ion species is present in an IMS-MS review.After finding a precursor ion species, an IMS scan (separation) is started, in which the mass filter (150) is switched for the time period in which the precursor ion species leaves the mobility separator (144), such that only the precursor ion species can pass the mass filter (150) as far as possible during this time period. The precursor ion species thus isolated from other ion species according to mobility and mass is fragmented in the fragmentation cell (160) and an MS2 spectrum (fragment mass spectrum) is acquired. In the modified detection unit ( 181), signals of the fragment ion species (target ion species) are weighted with quantification functions and sums weighted therefrom are calculated. The signals are preferably the reporter ion species labeled isobar in the lower mass range of the MS2 spectrum. As a rule, a precursor ion species separated according to mobility has a mass transfer of a duration of approximately one millisecond at the time-of-flight mass analyzer ( 170), so that a plurality of MS2 spectra can be recorded for a precursor ion species at a recording rate of 10 kHz. The weighted sums are typically determined in real time in the modified detection unit ( 181) for each individual MS2 spectrum and are transferred to the data memory ( 183) via the local bus ( 184). The central processor (182) or other remote processors (not shown in Figure 1) may determine quantification magnitudes for the fragment ion species, such as the intensity ratio of two isobaric reporter ion species. From the intensity ratio and the intensity of the signal composed of the isobaric reporter ion species, the intensity of the two isobaric reporter ion species can be determined in each case and compared (quantified) with the intensities of other reporter ion species present in the MS2 spectra. In this case, there are the possibilities of determining the quantification variables for each individual MS2 spectrum or first summing the weighted sums for all MS2 spectra of a fragment ion species and determining the quantification variables therefrom. During an IMS scan, MS2 spectra can be recorded from various mobility-separable precursor ion species.An IMS scan typically takes between 10 and 100 milliseconds, so that during an IMS scan a plurality of precursor ion species separable by mass and mobility can be quantified. Additional IMS scans may follow to acquire MS2 spectra from other precursor ion species or acquire MS2 spectra from precursor ion species in two or more IMS scans before a new IMS MS map is acquired.FIGS. 3A to 3E show, in a first embodiment, how the intensity ratio S 1 / S 2 of the two signal components ( 31, 32) can be determined for a signal ( 30) composed of two signal components ( 31, 32) by means of weighted sums and a predefined look-up table ( 36).FIG. 3A shows a section of a simulated mass spectrum of a time-of-flight mass analyzer with the signal (30), which is composed of the two signal components (31, 32) of two target ion species, which are not mass-resolved in the mass spectrum. An analog detector signal having a noise component is detected at a typical sampling rate of 5 GS / s (engl. Gigasamples per second) are sampled and digitized with a resolution of 10 bits. The circular symbols correspond to the digitized value pairs (time of flight, intensity) of the composite signal (30). The mass axis is a time-of-flight axis, with the sampling instants (bins) being indicated as integers. The two signal components (31, 32) belong to two target ion species for which a mass of approximately 128 daltons and a mass difference of three millidaltons is used. The simulated mass spectrum has a mass resolution of approximately 12000, while a mass resolution of 42000 would be necessary in order to be able to resolve the two signal components ( 31, 32) as separate signals. The signal extending over a finite number of mass channels (bins), here somewhat 25, of which, for example, 11 bins are used for the summation, can have been determined and extracted from a mass spectrum with a significantly higher bin number, for example using a peak search algorithm (peak picking). It is possible that a mass spectrum has a plurality of overlapping signals, which are found by corresponding algorithms.FIG. 3B shows a first quantification function ( 33) together with the composite signal ( 30). The first quantification function ( 33) is a constant unifunction. The summation of the signal (30) weighted with the first quantification function (33) yields a first weighted sum g1.FIG. 3C shows a second quantification function ( 34) together with the composite signal ( 30). The second quantification function ( 34) is the (linear) flight time axis. The summation of the signal (30) weighted with the second quantification function (34) yields a second weighted sum g2.FIG. 3D shows the composite signal ( 30) with the center of gravity to ( 35) of the signal ( 30), which is shown as a dashed line and results as a derived variable from the weighted sums: to=g2 / g1FIG. 3E shows a logarithmic graphic representation of the predefined lookup table ( 36), on whose abscissa (input values of the lookup table) the center of gravity of the composite signal is plotted, and on whose ordinate (output values of the lookup table) the intensity ratio of the signal components ( 31, 32) is plotted. In FIG. 3E, the center of gravity ( 35) calculated from the weighted sums g1and g2and the corresponding intensity ratio S 1 / S 2 of the two signal components ( 31, 32) are shown as dashed lines. The intensity ratio is 1.1 and is correctly determined in this case with an average relative error of less than 0.5%.FIGS. 4A to 4E show, in a second embodiment, how the intensity ratio of the two signal components (41, 42) can be determined for a signal (40) which is composed of two signal components (41, 42) by means of weighted sums and a predefined look-up table (47).FIG. 4A shows a section of a simulated MS2 spectrum of a time-of-flight mass analyzer with the signal (40), which is composed of the two signal components (41, 42) of two isobaric reporter ion species, which are not mass-resolved in the MS2 spectrum. An analog detector signal having a noise component is detected at a typical sampling rate of 5 GS / s (engl. Gigasamples per second) are sampled and digitized with a resolution of 10 bits. The circular symbols correspond to the digitized value pairs (time of flight, intensity) of the composite signal (40). The mass axis is a time-of-flight axis, with the sampling instants (bins) being indicated as integers. The two isobaric reporter ion species have a mass of about 128 daltons and have a mass difference of 6.3 millidaltons. The simulated MS2 spectrum has a mass resolution of approximately 12000, while a mass resolution of 20000 would be necessary in order to be able to resolve the two signal components (41, 42) as separate signals.FIG. 4B shows a first quantification function ( 43) together with the composite signal ( 40). The first quantification function (43) is a delta function centered at the maximum position of the first signal portion (41). The summation of the signal (40) weighted with the first quantification function (43) yields a first weighted sum g1and corresponds to an interpolation of the signal (40) at the position of the first delta function.FIG. 4C shows a second quantification function ( 44) together with the composite signal ( 40). The second quantification function ( 43) is a delta function centered at the maximum position of the second signal portion ( 42). The summation of the signal (40) weighted with the second quantification function (43) yields a second weighted sum g2and corresponds to an interpolation of the signal (40) at the position of the second delta function.Figure 4D shows the composite signal (40) with the interpolated signal values g1 (45) and g2 (46) at the positions of the two delta functions, which are represented as two cross-shaped symbols at the end of the dashed lines.FIG. 4E shows a logarithmic graphic representation of a predetermined look-up table ( 47), on whose abscissa (input values of the look-up table) the ratio of the weighted sum g2 / g1is plotted and on whose ordinate (output values of the look-up table) the intensity ratio S2 / S1of the signal components ( 41, 42) is plotted. FIG. 4E shows the ratio g2 / g1 (48) and the corresponding intensity ratio S2 / S1 (49) of the two signal components (41, 42) as dashed lines. The intensity ratio is 0.2 and is correctly determined here with an average relative error of less than 7.5%. The mean relative error is greater than in the previous embodiment because the intensity ratio is closer to the edge of the look-up table and the intensity of the composite signal (40) from the example of FIGS. 4A-E is significantly less than the intensity of the composite signal (30) from the example of FIGS. 3A-E.FIG. 5A shows a section of a simulated mass spectrum of a time-of-flight mass analyzer with a signal (50) which is composed of three signal components (51, 52, 53) of target ion species which are not mass-resolved in the mass spectrum. An analog detector signal having a noise component is detected at a typical sampling rate of 5 GS / s (engl. Gigasamples per second) are sampled and digitized with a resolution of 10 bits. The circular symbols correspond to the digitized value pairs (time of flight, intensity) of the composite signal (50). The mass axis is a time-of-flight axis, with the sampling instants (bins) being indicated as integers. The three target ion species have a mass of about 128 daltons. The mass difference between the medium and left signal components (52, 51) is 3 millidaltons. The mass difference between the medium and the right signal components (52, 53) is 5 millidaltons.FIG. 5B shows three quantification functions ( 54, 55, 56), each together with the composite signal ( 50).The first quantification function (54) is a constant unifunction. The summation of the signal (50) weighted with the first quantification function (54) yields a first weighted sum g1. The second quantification function ( 55) is a (linear) time-of-flight axis, the zero crossing of which is shifted to the maximum position of the mean signal component ( 52). The summation of the signal (50) weighted with the second quantification function (55) yields a second weighted sum g2. The third quantification function ( 56) is the squared second quantification function ( 55). The summation of the signal (50) weighted with the third quantification function (56) yields a third weighted sum g3.The relative intensities I i( i=1... 3) of the three signal components (51, 52, 53) result as a solution of the linear equation system: where Q i( x) are the quantification functions and S i( x) are the individual normalized signal components and the weighted sums g i form the inhomogeneous part of the equation system. The individual signal components S i( x) can be recorded in a measurement upstream. The mean relative error of the intensity ratios between the signal components is less than 1% in this example.The invention has been described above with reference to various specific embodiments. It should be understood, however, that various aspects or details of the described embodiments may be changed without departing from the scope of the invention.

Claims

Method for mass spectrometric quantification of two or more known target ion species, at least two of which differ in their masses, comprising the steps of: - providing quantification functions, the number of which is at least as great as the number of target ion species of different masses, - providing a mass spectrum with a signal which is composed of signal components of the two or more target ion species which are not mass-resolved in the mass spectrum, - weighting the signals with the respective quantification functions, - calculating in each case a weighted sum by integration of the signal weighted with the respective quantification function, and - determining a quantification variable for the two or more target ion species from the weighted sums, wherein (a) the weighted sums form the inhomogeneous portion of an equation system and the quantification variable results as a solution of the equation system or is derived therefrom or (b) the weighted sums or variables derived therefrom serve as input values for predetermined look-up tables which contain the quantification variable as output value.The method of claim 1, wherein the equation system is a linear equation system.The method of claim 1 or 2, wherein the quantification quantity is a ratio of the signal components of two target ion species or the signal component of a single target ion species on the signal.Method according to one of Claims 1 to 3, in which the quantification functions are (a) polynomials of different order, (b) delta functions or rectangular functions which are preferably each centred in the vicinity of the respective maximum position of the target ion species, (c) step functions, wherein the step positions are preferably each located in the vicinity of the respective maximum position of the target ion species, (d) harmonic functions of different periodicity, or (e) functions used for a wavelet transformation.The method of any one of claims 1 to 4, wherein the signal is composed of the signal portions of two target ion species.Method according to Claim 5, in which the signal is weighted with a constant function and the mass axis of the recorded mass spectrum and the centre of gravity of the signal is calculated from the two weighted sums, which centre of gravity is used as input value of a look-up table in order to determine the ratio of the two signal components of the two target ion species.Method according to claim 5, in which the signal is interpolated at the known masses of the two target ion species, from the ratio of which the ratio of the two signal components of the two target ion species is determined by means of a look-up table.The method of any one of claims 1 to 7, wherein the target ion species are isobaric fragment ion species generated by fragmentation of precursor ion species, wherein the precursor species all have a same biomolecule species and are each labeled with different isobaric mass tags each having a reporter group and a mass balancing group, and wherein the precursor ion species are isolated from other precursor ion species for mass or for mass and mobility prior to fragmentation.The method of claim 8, wherein the isobaric fragment ion species have isobaric reporter groups.Method according to one of Claims 1 to 9, in which a plurality of mass spectra are provided with the signal, the weighted sums being calculated separately in each case for individual mass spectra or for partially summed mass spectra and then being summed separately according to a quantification function in order to determine the quantification variables therefrom.Method according to Claim 10, in which the weighted sums are respectively calculated separately for each of the mass spectra and are then summed separately after a quantification function in order to determine the quantification variables therefrom.Method according to one of Claims 1 to 9, in which a plurality of mass spectra are provided with the signal, the weighted sums being calculated separately in each case for individual mass spectra or for partially summed mass spectra and quantification variables for the individual mass spectra or partially summed mass spectra being determined therefrom.Method according to claim 12, wherein first quantification functions are used for the separate calculation of the weighted sums of a first individual mass spectrum or of a first partially summed mass spectrum and second quantification functions are used for the separate calculation of the weighted sums of a second individual mass spectrum or of a second partially summed mass spectrum, wherein the first and second quantification functions are different.An apparatus for quantifying two or more target ion species comprising a data processing unit, wherein the data processing unit is adapted and configured to perform a method according to any one of the preceding claims.

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