Method for detecting the labelling state of unknown species of molecules

Abstract

In one aspect, a method for detecting the labelling state of unknown species of molecules M contained in a sample exposed to a non-changing isotope labelling process using mass spectrometry is described.

Claims

1. Method for detecting the labelling state of unknown species of molecules M contained in a sample exposed to a non-changing isotope labelling process using mass spectrometry comprising: measuring at least one mass spectrum I.sub.ul(p.sub.ul) of a reference sample which has not been exposed to the non-changing isotope labelling process or has been only exposed to the non-changing isotope labelling process at the beginning of said process with a mass spectrometer; labelling the sample in the non-changing isotope labelling process; measuring, after the beginning of the non-changing isotope labelling process, mass spectra I.sub.l(p.sub.l) of the sample with a mass spectrometer; determining of one or more most likely elemental compositions C.sub.M,i(+0) of at least one unknown species of molecules M contained in the reference sample based on the at least one measured mass spectrum I.sub.ul(p.sub.ul) of the reference sample; detecting, for each determined one or more most likely elemental compositions C.sub.M,i(+0) of at least one unknown species of molecules M contained in the reference sample, the corresponding species of molecules M.sub.l contained in the sample in the measured mass spectra I.sub.l(p.sub.l) of the sample using for each identified one or more most likely elemental compositions C.sub.M,i(+0) of at least one unknown species of molecules M contained in the reference sample by: determining for the elemental compositions C.sub.M,i(+0) and at least one discrete labelled state C.sub.M,i(+n.sub.1, +n.sub.2, . . . , +n.sub.w) of said elemental compositions C.sub.M,i(+0) the isotope peak patterns I.sub.M(+u),l, determining for the elemental compositions C.sub.M,i(+0) detectable masses m.sub.Ml,m of the corresponding species of molecules M.sub.l using the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i of the elemental composition C.sub.M,i(+0) and the at least one discrete labelled state C.sub.M,i(+n.sub.1, +n.sub.2, . . . , +n.sub.w) of said elemental composition C.sub.M,i(+0), determining for the elemental composition C.sub.M,i(+0) the isotope peak pattern I.sub.Ml,m of the corresponding species of molecules M.sub.l from at least one measured mass spectrum I.sub.l(p.sub.l) of the sample at the determined detectable masses m.sub.Ml,m of the corresponding species of molecules M.sub.l, determining for each identified one or more most likely elemental compositions of at least one unknown species of molecules M contained in the reference sample a labelling state of the corresponding species of molecules M.sub.l in the sample from the measured mass spectra I.sub.l(p.sub.l) of the sample by: determining for the elemental composition C.sub.M,i(+0) the intensity contributions L.sub.M,i(n.sub.1, n.sub.2, . . . , n.sub.w) of the unlabelled state of the elemental composition C.sub.M,i(+0) and the discrete labelled states C.sub.M,i(+n.sub.1, +n.sub.2, . . . , +n.sub.w), for which the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i was determined, by comparing for the each determined detectable mass m.sub.Ml,m of the corresponding species of molecules M.sub.l the peak intensity at the determined detectable masses m.sub.Ml,k in the determined isotope peak pattern I.sub.Ml,m of the corresponding species of molecules M.sub.l with the peak intensity of the determined detectable masses m.sub.Ml,m in the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i of the elemental composition C.sub.M,i(+0) and the discrete labelled states C.sub.M,i(+n.sub.1, +n.sub.2, . . . , +n.sub.w), for which the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i was determined.

2. The method of claim 1, wherein in a time period after the beginning of the non-changing isotope labelling process mass spectra I.sub.l(p.sub.l) of the sample are measured and in the time period for each identified one or more most likely elemental compositions of at least one species of molecules M contained in reference sample the labelling state of the unknown species of molecules M in the sample is determined from the measured mass spectra I.sub.l(p.sub.l) of the sample.

3. The method of claim 1, wherein for all possible discrete labelled states the isotopic peak pattern are determined and used in the following steps of the method.

4. The method of claim 1, wherein for each identified one or more most likely elemental compositions of at least one unknown species of molecules M contained in the reference sample a labelling state of the corresponding species of molecules M.sub.l in the sample is determined from the measured mass spectra I.sub.l(p.sub.l) of the sample by comparison for the each determined detectable mass m.sub.Ml,m of the corresponding species of molecules M.sub.l the linear combination of the intensity contributions L.sub.M,i(n.sub.1, n.sub.2, . . . , n.sub.w) of the unlabelled state of the elemental composition C.sub.M,i(+0) and the discrete labelled states C.sub.M,i(+n.sub.1, +n.sub.2, . . . , +n.sub.w) and the peak intensity at the expected detectable masses m.sub.Ml,m in the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i of the elemental composition C.sub.M,i(+0) and the discrete labelled states C.sub.M,i(+n.sub.1, +n.sub.2, . . . , +n.sub.w), for which the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i was determined with the peak intensity of the determined detectable masses m.sub.Ml,m in the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i of the elemental composition C.sub.M,i(+0) and the discrete labelled states C.sub.M,i(+n.sub.1, +n.sub.2, . . . , +n.sub.w), for which the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i was determined.

5. The method of claim 1, wherein the sample is labelled by a non-changing single enriched isotope labelling process.

6. The method of claim 1, wherein detecting the labelling state of unknown species of molecules M contained in the sample is used in flux analysis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a flow chart to illustrate the inventive method.

(2) FIG. 2 shows the results of the inventive method applied to test samples having a defined labelling state

(3) FIG. 3 shows the defined labelling states of two specific molecules in the test samples

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(4) FIG. 1 shows in a flow diagram of one order how the processes of the inventive method can be executed. This is only an example of the inventive method.

(5) Now shall be described another example of the method using a different order of the processes.

(6) In this example as labelling process is used a single non-changing single enriched isotope labeling process using an isotopic marker which is labeled only with the specific isotope .sup.xE.

(7) In this example a homogeneous original sample, may be a bacteria culture, is divided at the beginning into to portions, a reference sample and a sample, which shall be labelled.

(8) In a first step of the method mass spectra I.sub.ul(p.sub.ul) was measured of a reference sample with a mass spectrometer.

(9) The mass spectra of the reference sample are measured with an LC/MS instrument. The reference sample is provided to the mass spectrometer, in particular to its ion source, via a liquid chromatography system, in particular a liquid chromatography column. Then a series of mass spectra of the effluent of the liquid chromatography system is measured by the mass spectrum. Then from the series of mass spectra mass traces can be derived. According to the chromatography process chromatographic peaks of can be detected at specific elution times, also called according to the chromatographic process retension times, for each species of molecules, which can be shown at this time as a mass trace. Typically, the mass traces are detected at the mass of an isotopologue of the species of molecules but to to coalescence effects the mass may deviate at some time periods of measuring the series of mass spectra. The detection of the such mass traces taking into account the coalescence effect is described in the unpublished European patent application 18170779.5 of the applicant, which is hereby incorporated with its complete disclosure.

(10) The at least one mass spectrum I.sub.ul(p.sub.ul) of the reference sample can be measured by a kind of mass spectrometer, independent on its resolving power. In particular it is preferred to use a mass spectrometer of high resolution like a mass spectrometer having an Orbitrap® mass analyser.

(11) In a further step of the inventive method the sample which shall be investigated is exposed to a non-changing single enriched isotope labelling process for a specific time period.

(12) In the next step of the method mass spectra I.sub.l(p.sub.l) of the sample are measured with the mass spectrometer, like a mass spectrometer having an Orbitrap® mass analyser after the beginning of the non-changing single enriched isotope labelling process of the sample.

(13) The mass spectra of the sample are measured with an LC/MS instrument. Then the sample is provided to the mass spectrometer, in particular to its ion source, via a liquid chromatography system, in particular a liquid chromatography column. Then a series of mass spectra of the effluent of the liquid chromatography system is measured by the mass spectrum. This means at different times of the elution of the effluent the effluent is provided to the mass spectrometer, in particular its ion source to measure a mass spectrum at a specific elution time. Then from the series of mass spectra mass traces can be derived. According to the chromatography process chromatographic peaks of can be detected at specific elution times, also called according to the chromatographic process retension times, for each species of molecules, which can be shown at this time as a mass trace. Typically, the mass traces are detected at the mass of an isotopologue of the species of molecules but to coalescence effects the mass may deviate at some time periods of measuring the series of mass spectra. The detection of such mass traces taking into account the coalescence effect is described in the unpublished European patent application 18170779.5 of the applicant, which is hereby incorporated with its complete disclosure.

(14) In general, after the beginning of the non-changing isotope labelling process mass spectra I.sub.l(p.sub.l) of the sample are measured a time period to observe the process of labelling the species of molecules M contained in the sample. In this time the time dependent enrichment of the specific isotope .sup.xE can be observed, which can be expressed by the time dependent relative abundance c.sub.M(n,t) of its n-times labelled molecules M.sub.l describing the labelling state of the species of molecule M at a specific time t, which may be time after the start of the non-changing single isotope labelling process of the sample.

(15) In the next step of the inventive method one or more most likely elemental compositions C.sub.M,i(+0) of at least one unknown species of molecules M contained in reference sample based on the at least one measured mass spectrum I.sub.ul(p.sub.ul) of the reference sample are determined.

(16) In the method the only the most likely elemental composition C.sub.M(+0) of at least one species of molecules M is determined with the process, which is disclosed in the unpublished European patent application 18156903.9 of the applicant.

(17) In the next step of the method for each determined most likely elemental compositions C.sub.M(+0) of at least one unknown species of molecules M contained in the reference sample the corresponding species of molecules M.sub.l contained in the sample is detected in the measured mass spectra I.sub.l(p.sub.l) of the sample.

(18) This process comprises the following three substeps, which are executed for each identified most likely elemental compositions C.sub.M,i(+0) of at least one unknown species of molecules M contained in the reference sample.

(19) In a first substep of this process to detect the corresponding species of molecules M.sub.l contained in the sample for the elemental composition C.sub.M(+0) and all possible discrete labelled state C.sub.M(+n) of said elemental compositions C.sub.M(+0) the isotope peak patterns I.sub.M(+u) are determined.

(20) Methods known to the skilled person are applied to determine the isotope peak patterns I.sub.M(+u1, +u2, . . . , +uw),i. The resolving power of the mass spectrometer measuring the mass spectra I.sub.l(p.sub.l) of the sample is taken into account to determine the isotope peak patterns I.sub.M(+u).

(21) Further on the labelling rates of the isotopic markers are taken into account in this determination.

(22) In second substep of the process to detect the corresponding species of molecules M.sub.l contained in the sample detectable masses m.sub.Ml,m of the corresponding species of molecules M.sub.l are determined for the elemental compositions C.sub.M(+0) using the isotope peak patterns I.sub.M(+u) of the elemental composition C.sub.M(+0) and all discrete labelled state C.sub.M(+n) of said elemental composition C.sub.M(+0).

(23) Hereby is taken account the resolving power and/or the mass tolerance of the used mass spectrometer.

(24) For peaks which cannot be resolved, it is defined a common center of mass m.sub.Ml,m by taking into account the mass value at the top value of the intensity peaks and the maximum intensity of intensity peaks, preferable to normalized maximum intensity of intensity peaks. The mass value at the top value of the intensity peaks are weighted by normalized maximum intensity of intensity peaks and then averaged to determine the common center of mass m.sub.Ml,m.

(25) In this third substep of the process to detect the corresponding species of molecules M.sub.l contained in the sample the isotope peak pattern I.sub.Ml,m of the corresponding species of molecules M.sub.l are determining for the elemental composition C.sub.M(+0) from the measured mass spectra I.sub.l(p.sub.l) of the sample at the determined detectable masses m.sub.Ml,m of the corresponding species of molecules M.sub.l.

(26) In the next step of the method for each identified most likely elemental compositions of at least one unknown species of molecules M contained in the reference sample a labelling state of the corresponding species of molecules M.sub.l in the sample is determined from the measured mass spectra I.sub.l(p.sub.l) of the sample.

(27) The labelling state of the corresponding species of molecules M.sub.l in the sample can be described for this method by its intensity contributions L.sub.M(n) or relative abundances c.sub.M(n) of the n-times labelled molecule M.sub.l and the unlabelled molecule M.

(28) In this process the labelling state of the corresponding species of molecules M.sub.l in the sample of the elemental composition C.sub.M(+0) is determined by determining the intensity contributions L.sub.M(n) of the unlabelled state of the elemental composition C.sub.M(+0) and all discrete labelled states C.sub.M(+n) by comparing the peak intensity at the determined detectable masses m.sub.Ml,m in the determined isotope peak pattern I.sub.Ml,m of the corresponding species of molecules M.sub.l with the linear combination of the intensity contributions L.sub.M(n) of the unlabelled state of the elemental composition C.sub.M(+0) and the discrete labelled states C.sub.M(+n) and the peak intensity at the expected detectable masses m.sub.Ml,m in the isotope peak patterns I.sub.M(+u) of the elemental composition C.sub.M(+0) and the all discrete labelled states C.sub.M(+n.sub.1).

(29) For the determination of the intensity contributions L.sub.M(n) of the unlabelled state of the elemental composition C.sub.M(+0) and the discrete labelled states C.sub.M(+n) the non-negative least square fitting method is used.

(30) In FIGS. 2 and 3 are shown test results proving the validity of the method. A test sample comprising several molecules is labelled with an isotope marker labelling with the specific isotope .sup.13C. As shown in the right column of the table at the bottom of FIG. 2 several labelling states (0%, 25%, 50%, 75%, 100%) are investigated to different test samples. As shown in the middle of the sample the test sample is comprising several species of molecules. One species is glucose. For a molecule having the molecular weight 88.1094 in the columns under the title “Status exchange rate” the result for the determination of the labelling state with the method are shown. The intensity contributions of the discrete labelling states of the molecule are shown as percentage values. In the left of the three columns is shown the percentage of the unlabeled molecules and in each neighbouring column the percentage is shown, when one more specific isotope .sup.13C is contained in the molecules of the molecular weight 88.1094.

(31) In FIG. 3 the same results are shown in the upper table for Glucose and in the lower table for another molecule with was found for the first time with the method, which identified the unknown molecule and determined its labelling state. The unknown molecule could be identified as an adduct of the Glucose with HCl.

(32) The results show clearly that the method is able to identify the labelling state of the labelled test samples for the shown molecules.