Abstract
Disclosed is a method and device for the mass spectrometric quantification of two or more known target ion species, in which a mass spectrometric signal, composed of signal components of the two or more target ion species, which are not mass resolved in the mass spectrum, is weighted with quantification functions and summed, and a quantification parameter is determined for the two or more target ion species from the weighted sums.
Claims
1. A method for mass spectrometric quantification of two or more known target ion species, of which at least two differ in their masses, comprising the steps: providing quantification functions whose number is at least as large as a 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, which are not mass resolved in the mass spectrum, summing the signal weighted with the quantification functions, and determining a quantification parameter for the two or more target ion species from the weighted sums, where (a) the weighted sums form an inhomogeneous part of a system of equations, and the quantification parameter provides 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 prespecified lookup tables, which contain the quantification parameter as output value.
2. The method according to claim 1, wherein the system of equations is a system of linear equations.
3. The method according to claim 1, wherein the quantification parameter is a ratio of the signal components of two target ion species, or the signal component from a single target ion species of the signal.
4. The method according to claim 1, wherein the quantification functions are (a) polynomials of different order, (b) delta functions or rectangular functions, which are all preferably centered near the respective maximum position of the target ion species, (c) step functions, where each step position is preferably near the respective maximum position of the target ion species, (d) harmonic functions of different periodicity, or (e) functions used for a wavelet transform.
5. The method according to claim 1, wherein the signal is composed of the signal components of two target ion species.
6. The method according to claim 5, wherein the signal is weighted with a constant function of value one and the mass axis of the acquired mass spectrum, and the centroid of the signal is calculated from the two weighted sums, where the centroid is used as the input value of a lookup table to determine the ratio of the two signal components of the two target ion species.
7. The method according to claim 5, wherein the signal at the known masses of the two target ion species is interpolated, from whose ratio the ratio of the two signal components of the two target ion species is determined by means of a lookup table.
8. The method according to claim 1, wherein the target ion species are isobaric fragment ion species, which are produced by fragmentation of precursor ion species, where the precursor species all have a same species of biomolecule and are each labeled with different isobaric mass tags, each of which has a reporter group and a mass balance group, and where the precursor ion species are isolated from other precursor ion species regarding mass, and optionally isolated regarding mobility in addition, before the fragmentation.
9. The method according to claim 8, wherein the isobaric fragment ion species have isobaric reporter groups.
10. The method according to claim 1, wherein several mass spectra with the signal are provided, where the weighted sums are calculated separately in each case for individual mass spectra or for partially summed mass spectra before being summed separately according to the quantification function to determine quantification parameters therefrom.
11. The method according to claim 10, wherein the weighted sums are each calculated separately for each mass spectrum and afterwards summed separately, according to quantification function, to determine the quantification parameters therefrom.
12. The method according to claim 1, wherein several mass spectra with the signal are provided, where the weighted sums are calculated separately in each case for individual mass spectra or for partially summed mass spectra, and quantification parameters for the individual mass spectra or partially summed mass spectra are determined therefrom.
13. The 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 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 a second partially summed mass spectrum, where the first and second quantification functions are different.
14. A device for the quantification of two or more target ion species comprising a data processing unit which is designed and configured to execute a method according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] The disclosure can be better understood by referring to the following illustrations. The elements in the illustrations are not necessarily to scale, but are primarily intended to illustrate the principles of the disclosure (mostly schematically).
[0047] FIG. 1 is a schematic representation of a hybrid mass spectrometric system (100) known from the Prior Art, which is suitable for quantification by means of isobaric mass tags. The hybrid mass spectrometric system (100) contains 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) to record and process mass spectrometric data.
[0048] FIG. 2 shows a flow chart of a method according to the invention for the quantification of target ion species labeled with isobaric mass tags, said target ion species being isolated according to retention time, mobility, and mass before their fragmentation, and quantified by means of signals of reporter ion species or complementary fragment ion species.
[0049] FIGS. 3A to 3E show in one embodiment how, for a signal (30) composed of two signal components (31, 32), the intensity ratio of the two signal components (31, 32) can be determined, by means of weighted sums and a prespecified lookup table (36).
[0050] FIG. 3A shows a section from a simulated mass spectrum of a time-of-flight mass analyzer with the signal (30), composed of the two signal components (31, 32) of target ion species, which are not mass resolved in the mass spectrum.
[0051] FIG. 3B shows one of the two quantification functions (33, 34) together with the composite signal (30).
[0052] FIG. 3C shows the other of the two quantification functions (33, 34) together with the composite signal (30).
[0053] FIG. 3D shows the composite signal (30) together with the centroid (35) of the signal (30), said centroid resulting from weighted sums of the signal (30) as a derived quantity.
[0054] FIG. 3E is a logarithmic graphic representation of the prespecified lookup table (36), which is used to determine the intensity ratio of the two signal components (31, 32).
[0055] FIGS. 4A to 4E show in a second embodiment how, for a signal (40), composed of two signal components (41, 42), the intensity ratio of the two signal components (41, 42) can be determined, by means of weighted sums and a prespecified lookup table (47).
[0056] FIG. 4A shows a section from a simulated MS2 spectrum of a time-of-flight mass analyzer with the signal (40), composed of two signal components (41, 42), of isobaric fragment ion species, which are not mass resolved in the MS2 spectrum.
[0057] FIG. 4B shows one of the two quantification functions (43, 44) together with the composite signal (40).
[0058] FIG. 4C shows the other of the two quantification functions (43, 44) together with the composite signal (40).
[0059] 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 maximum position of each of the two signal components (41, 42).
[0060] FIG. 4E is a logarithmic graphic representation of the prespecified lookup table (47), which is used to determine the intensity ratio of the two signal components (41, 42).
[0061] FIG. 5A shows a section from 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.
[0062] FIG. 5B shows three quantification functions (54, 55, 56), each together with the composite signal (50), with which the signal (50) is weighted to calculate three weighted sums, which are used as the inhomogeneous part of a system of linear equations. The intensities of the three signal components (51, 52, 53) provide the direct solution of the system of linear equations.
DETAILED DESCRIPTION
[0063] FIG. 2 shows a flow chart for a method according to the invention for the quantification of precursor ion species labeled with isobaric mass tags, said precursor ion species being isolated according to LC retention time, mobility, and mass before their fragmentation, and quantified by means of signals of reporter ion species or complementary fragment ion species.
[0064] The precursor species, from which the precursor ion species are produced in an ion source, can be digest peptides from several proteome samples, for example, where the digest peptides in each proteome sample are labeled with one of the different isobaric mass tags and combined afterwards. The method according to the invention can be carried out with the mass spectrometric system (100) depicted in FIG. 1, for example, whose detection unit 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 parameters of the two or more isobaric fragment ion species are determined.
[0065] The digest peptides, which are labeled and combined differently, are pre-separated in an LC separation run according to their retention time. After the LC separation run has started, the precursor ion species, which are produced from the labeled digest peptides in the ESI ion source (121), are separated in an IMS scan of the mobility separator (144) according to mobility in the gaseous phase, while MS1 spectra are continuously acquired with the time-of-flight mass analyzer (170), with both mass filter (150) and fragmentation cell (160) switched off. The MS1 spectra acquired during the IMS scan provide an IMS-MS overview, which is examined to establish whether specified precursor ion species are present at the time the IMS scan is acquired. The precursor ion species, whose retention time, mobility, and mass are known, can be found by a comparison with the mobility and the mass of signals in the IMS-MS overview and via the acquisition time of the IMS-MS overview during the LC separation run (retention time). The acquisition of IMS-MS overviews is repeated until at least one of the specified precursor ion species is present in an IMS-MS overview.
[0066] After a precursor ion species has been found, an IMS scan (separation) is started, where the mass filter (150) is switched during the period in which the precursor ion species leaves the mobility separator (144) such that during this period, only the precursor ion species can pass through the mass filter (150), as far as possible. The precursor ion species isolated in this way 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 weighted sums are calculated therefrom. The signals here are preferably those of reporter ion species with isobaric labels in the lower mass range of the MS2 spectrum. As a rule, a precursor ion species separated according to mobility has a substance batch with a duration of around one millisecond at the time-of-flight mass analyzer (170), which means that a plurality of MS2 spectra can be acquired for one precursor ion species at an acquisition rate of 10 kHz. In the modified detection unit (181), the weighted sums are typically determined in real time for each individual MS2 spectrum, and passed to the data storage device (183) via the local bus (184). The central processor (182) or further decentralized processors (not shown in FIG. 1) can determine quantification parameters for the fragment ion species, such as the intensity ratio of two isobaric reporter ion species. The intensity ratio and the intensity of the signal, which is composed of the isobaric reporter ion species, can be used to determine the intensity of each of the two isobaric reporter ion species and compare (quantify) them with the intensities of other reporter ion species present in the MS2 spectra. It is possible to determine the quantification parameters for each individual MS2 spectrum, or to first sum the weighted sums for all MS2 spectra of a fragment ion species and determine the quantification parameters therefrom. During an IMS scan, MS2 spectra of different precursor ion species, which can be separated according to mobility, can be acquired.
[0067] An IMS scan typically takes between 10 and 100 milliseconds. Therefore, several precursor ion species, separable according to mass and mobility, can be quantified during an IMS scan. Further IMS scans can follow to acquire MS2 spectra of other precursor ion species or acquire MS2 spectra of precursor ion species in two or more IMS scans before a new IMS-MS overview is acquired.
[0068] FIGS. 3A to 3E show in a first embodiment how, for a signal (30) composed of two signal components (31, 32), the intensity ratio S1/S2 of the two signal components (31, 32) can be determined by means of weighted sums and a prespecified lookup table (36).
[0069] FIG. 3A shows a section from 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 with a noise component is scanned with a typical sampling rate of 5 GS/s (giga-samples per second) and digitized with a 10-bit resolution. 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, where the scanning time points (bins) are given as whole numbers. The two signal components (31, 32) belong to two target ion species, for which a mass of around 128 daltons and a mass difference of three millidaltons is used. The simulated mass spectrum has a mass resolution of around 12,000, whereas a mass resolution of 42,000 would be required in order to resolve the two signal components (31, 32) as separate signals. The signal, which extends over a finite number of mass channels (bins), here around 25, of which for example 11 bins are used for the summation, can have been determined and extracted from a mass spectrum with a much higher number of bins, for example by using a peak picking algorithm. It is possible that a mass spectrum has a plurality of superimposed signals, which can be found by appropriate algorithms.
[0070] FIG. 3B shows a first quantification function (33) together with the composite signal (30). The first quantification function (33) is a constant function of value one. The summation of the signal (30), weighted with the first quantification function (33), gives a first weighted sum g1.
[0071] FIG. 3C shows a second quantification function (34) together with the composite signal (30). The second quantification function (34) is the (linear) time-of-flight axis. The summation of the signal (30), weighted with the second quantification function (34), gives a second weighted sum g2.
[0072] FIG. 3D shows the composite signal (30) together with the centroid (35) of the signal (30), which is shown as a dashed line and occurs as a derived quantity from the weighted sums: to =g2/g1.
[0073] FIG. 3E is a logarithmic graphic representation of the prespecified lookup table (36), on whose abscissa (input values of the lookup table) the centroid of the composite signal is plotted, and on whose ordinate (output value of the lookup table) the intensity ratio of the signal components (31, 32) is plotted. In FIG. 3E, the centroid (35) calculated from the weighted sums g1 and g2 and the corresponding intensity ratio S2/S1 of the two signal components (31, 32) are represented by dashed lines. The intensity ratio is 1.1, and in this case is determined with a mean relative error of less than 0.5%.
[0074] FIGS. 4A to 4E show in a second embodiment how, for a signal (40), composed of two signal components (41, 42), the intensity ratio of the two signal components (41, 42) can be determined, by means of weighted sums and a prespecified lookup table (47).
[0075] FIG. 4A shows a section from a simulated MS2 spectrum of a time-of-flight mass analyzer with the signal (40), which is composed of two signal components (41, 42) of two isobaric reporter ion species, which are not mass resolved in the MS2 spectrum. An analog detector signal with a noise component is scanned with a typical sampling rate of 5 GS/s (giga-samples per second) and digitized with a resolution of 10 bit. 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, where the scanning time points (bins) are given as whole numbers. The two isobaric reporter ion species have a mass of around 128 daltons and a mass difference of 6.3 millidaltons. The simulated MS2 spectrum has a mass resolution of around 12,000, whereas a mass resolution of 20,000 would be required in order to resolve the two signal components (41, 42) as separate signals.
[0076] FIG. 4B shows a first quantification function (43) together with the composite signal (40). The first quantification function (43) is a delta function, which is centered at the maximum position of the first signal component (41). The summation of the signal (40), weighted with the first quantification function (43), gives a first weighted sum g1 and corresponds to an interpolation of the signal (40) at the position of the first delta function.
[0077] FIG. 4C shows a second quantification function (44) together with the composite signal (40). The second quantification function (44) is a delta function, which is centered at the maximum position of the second signal component (42). The summation of the signal (40), weighted with the second quantification function (44), gives a second weighted sum g2 and corresponds to an interpolation of the signal (40) at the position of the second delta function.
[0078] FIG. 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 shown as two cruciform symbols at the end of the dashed lines.
[0079] FIG. 4E is a logarithmic graphic representation of a prespecified lookup table (47), on whose abscissa (input values of the lookup table) the ratio of the weighted sum g2/g1 is plotted, and on whose ordinate (output value of the lookup table) the intensity ratio S2/S1 of the signal components (41, 42) is plotted. In FIG. 4E, the ratio g2/g1 (48) and the corresponding intensity ratio S2/S1 (49) of the two signal components (41, 42) are represented by dashed lines. The intensity ratio is 0.2 and is determined here with a mean 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 margin of the lookup table, and the intensity of the composite signal (40) from the example of FIGS. 4A-E is considerably lower than the intensity of the composite signal (30) from the example of the FIGS. 3A-E.
[0080] FIG. 5A shows a section from 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 with a noise component is scanned with a typical sampling rate of 5 GS/s (giga-samples per second) and digitized with a 10-bit resolution. 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, where the scanning time points (bins) are given as whole numbers. The three target ion species have a mass of around 128 daltons. The mass difference between the center and the left-hand signal component (52, 51) is 3 millidaltons. The mass difference between the center and the right-hand signal component (52, 53) is 5 millidaltons.
[0081] FIG. 5B shows three quantification functions (54, 55, 56), each together with the composite signal (50).
[0082] The first quantification function (54) is a constant function of value one. The summation of the signal (50), weighted with the first quantification function (54), gives a first weighted sum g1. The second quantification 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 summation of the signal (50), weighted with the second quantification function (55), gives 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), gives a third weighted sum g3.
[0083] The relative intensities li (i=1 . . . 3) of the three signal components (51, 52, 53) result as the solution of the system of linear equations:
[00004]
where Qi(x) is the quantification functions and Si(x) the individual normalized signal components and the weighted sums gi form the inhomogeneous part of the system of equations. The individual signal components Si(x) can be acquired in a prior measurement. The average relative error of the intensity ratios between the signal components is less than 1% in this example.
[0084] The invention has been described above with reference to different, specific example embodiments. It is to be understood, however, that various aspects or details of the example embodiments described can be modified without deviating from the scope of the invention.
