The technical field of the invention is that of ion characterisation.
A first aspect of the present invention relates to a method for characterising ions; a second aspect of the present invention relates to a device for characterising ions for the implementation of a method of characterising ions according to the first aspect of the invention.
The present invention notably finds applications in the fields of analytical chemistry, pharmacology and the environment.
The field of mass spectrometry is currently in full expansion. Numerous industrial concerns propose instruments, notably used in the fields of analytical chemistry, pharmacology and the environment, in order to characterise systems. However, these instruments uniquely make it possible to characterise the mass/charge ratio, noted m/z, of the systems detected, and not, notably, the different isomers or tautomers of the systems detected.
Tandem mass spectrometry, noted MS/MS or MS2, is a mass spectrometry technique for the identification and the characterisation of ions. The MS/MS technique consists in:
The study of third-generation ions makes it possible to obtain information on second-generation ions, and thus makes it possible to obtain indirectly information on first-generation ions. The study of third-generation ions may for example be carried out by means of electron spectroscopy.
Optical excitation, or photo-fragmentation, is another ion fragmentation technique that may be used. The following are typically used:
Electron spectroscopy of an ion makes it possible to obtain a spectrum giving information on the overall structure of said ion, but it remains difficult to determine the tautomeric form of the ion, notably on account of a phenomenon of spectral congestion when the ion is hot. The phenomenon of spectral congestion reflects the fact that the spectrum obtained, which contains characteristic fine rays when the ion is cold, becomes a wide and non-characteristic band when the ion is hot. It should be noted that an ion at room temperature is typically a hot ion.
The inventors are the authors of the article “Communication: Identification of daughter ions through their electronic spectroscopy at low temperature”, The Journal of Chemical Physics (2014). This article proposes a method for characterising the isomeric structure of an ionic photo-product, according to which:
In the article, the characterisation method is applied to the specific case of protonated tyrosine TyrH+ and makes it possible to characterise structurally the different photo-products of TyrH+. The method having made it possible to characterise fragments derived from a protonated amino acid, it is envisaged in the conclusion of the article to test the method for characterising fragments derived from a protonated peptide, in a more complete manner than the characterisation obtained by mass spectrometry.
However, experiments carried out in order to apply said method to ions other than protonated tyrosine TyrH+ are not conclusive.
The invention offers a solution to the aforementioned problems, by proposing a method of characterising ions, and in particular a method for characterising the isomeric structure of ions, the method being applicable to all types of ions.
One aspect of the invention thus relates to a method for characterising ions comprising:
The first photo-fragmentation of the plurality of first-generation ions makes it possible to obtain the plurality of second-generation ions. The second-generation ions are photo-fragments of the first-generation ions, thus the second-generation ions are different to the first-generation ions. The second-generation ions may all be of a same type, or the second-generation ions may be of different types. “Ions of different types” is taken to mean ions that can be selected independently of each other. Moreover, first-generation ions may subsist at the end of the first photo-fragmentation. Step i) makes it possible to isolate a first type of second-generation ions in the ion trap. Thus, it is guaranteed that the second photo-fragmentation is carried out uniquely on this first type of second-generation ions. The third-generation ions are thus photo-fragments of this first type of second-generation ions. Isolating the first type of second-generation ions in the ion trap before carrying out the second photo-fragmentation enables a reliable characterisation of the first type of second-generation ions, by using the third-generation ions obtained. Indeed, the spectroscopic signature of the first type of second-generation ions is obtained by the detection of its photo-fragments, that is to say by the detection of third-generation ions. Yet ions identical to the third-generation ions may also be obtained, notably, by photo-fragmentation of residual first-generation ions or by photo-fragmentation of second-generation ions of a type different to the first type. It is also possible that ions of different generations absorb in the same spectral domain. For example, a first-generation ion and a second-generation ion may both absorb in the UV. In such cases, the reliable characterisation of the first type of second-generation ions is made impossible because the spectroscopic signature of the first type of second-generation ions is mixed with the spectroscopic signature of other ions such as first-generation ions and/or second-generation ions of a type different to the first type. Thus, the method described in the publication cited previously functions when it is applied to the specific case of protonated tyrosine TyrH+ because protonated tyrosine TyrH+ absorbs in the UV whereas second-generation ions, that is to say the photo-fragments of protonated tyrosine TyrH+, absorb in the visible.
More generally, isolating a unique type of Nth-generation ions before carrying out an Nth photo-fragmentation enables a reliable characterisation of the type of Nth-generation ions.
Apart from the characteristics that have just been mentioned in the preceding paragraph, the characterisation method according to one aspect of the invention may have one or more additional characteristics among the following, considered individually or according to all technically possible combinations thereof:
Another aspect of the invention relates to a device for characterising ions for the implementation of the method for characterising ions according to one aspect of the invention, the device being characterised in that it comprises:
The invention and its different applications will be better understood on reading the description that follows and by examining the figures that accompany it.
The figures are presented for indicative purposes and in no way limit the invention.
Unless stated otherwise, a same element appearing in the different figures has a single reference.
The plurality G1 of first-generation ions is typically obtained using an electrospray ionisation (ESI) technique.
The ion trap P is preferentially a quadrupole trap, or Paul trap, which enables good localisation of a cloud of ions there within. A good localisation of the cloud of ions within the ion trap enables efficient interaction between a photo-fragmentation laser and the cloud of ions. Alternatively, the ion trap P may also be a Penning trap, or a multipolar linear trap.
The method 100 according to the first embodiment of the invention may advantageously comprise a step (not represented) according to which the plurality G1 of first-generation ions is selected so as to obtain a plurality G1 of first-generation ions of a first type. The step of selecting a first type of first-generation ions is for example carried out by a mass spectrometry technique. The step of selection by mass spectrometry advantageously makes it possible to obtain a plurality G1 of first-generation ions all having the same mass/charge ratio, noted m/z. The step of selection by mass spectrometry may for example take place before the plurality G1 of first-generation ions is introduced and trapped in the ion trap P. Alternatively, the step of selection by mass spectrometry may also take place after the plurality G1 of first-generation ions has been introduced and trapped in the ion trap P. In the latter case, the step of selection consists in conserving, in the ion trap P, first-generation ions having the desired m/z ratio by ejecting, out of the ion trap P, first-generation ions not having the desired m/z ratio. The step of selection of a first type of first-generation ions may also be carried out by an ion mobility spectrometry (IMS) technique.
The buffer gas is for example helium He. The buffer gas source may thus be a compressed helium cylinder. Alternatively, the buffer gas source may be a helium cylinder at ambient pressure which is used with a compressor.
a,
2
a and 2b show a step 130 according to which the plurality G1 of cooled first-generation ions is photo-fragmented by means of a photo-fragmentation laser L emitting at a first wavelength λ1, to obtain a plurality of second-generation ions. The first wavelength λ1 is selected as a function of the plurality G1 of first-generation ions to photo-fragment. The plurality of second-generation ions is different to the plurality of first-generation ions, and the plurality of second-generation ions is at least of one first type. At the end of the photo-fragmentation of the first-generation ions, the particular example represented in
a,
2
b and 2c show a step i), which is referenced 140 in
The selection typically takes place by adding a radiofrequency voltage to an electrode of the ion trap P. This radiofrequency voltage is selected and adjusted to eject ions having a certain m/z ratio. In the particular example illustrated in
a,
2
c and 2d show a step iii), which is referenced 160 in
In the case that has just been described, the photo-fragmentation laser L emits at the first wavelength λ1 for the first photo-fragmentation of step 130, and emits at the second wavelength λ2 for the second photo-fragmentation of step 160. Alternatively, two photo-fragmentation lasers may be used: a first photo-fragmentation laser emitting at the first wavelength λ1 for the first photo-fragmentation of step 130, and a second photo-fragmentation laser emitting at the second wavelength λ2 for the second photo-fragmentation of step 160.
In the particular example that has just been described, a first type G2T1 of second-generation ions and a second type G2T2 of second-generation ions are obtained at the end of the photo-fragmentation of the first-generation ions G1. After having obtained the spectroscopic signature of the first type G2T1 of second-generation ions by carrying out a first cycle of steps 110 to 170, it is then possible to obtain the spectroscopic signature of the second type G2T2 of second-generation ions by carrying out a second cycle 100′, represented in
In the same way as described for step 140, the mass spectrometer Sp is used during step 140′. Whatever the content of the ion trap P before the selection step 140′, it is guaranteed that the ion trap P now only substantially contains the second type of second-generation ions G2T2 at the end of said selection step 140′. “Substantially” is taken to mean the fact that a small residual quantity ε of the ions to eject may subsist within the ion trap P after the selection step 140, as described previously for step 140.
In the same way as described for step 150, the cooling module Re is used during step 150′.
The photo-fragmentation laser L emitting at a third wavelength λ2′ is used during step 160′. The third wavelength λ2′ is selected as a function of the second-generation ions of the second type G2T2 to photo-fragment. The third wavelength λ2′ is typically different to the first wavelength λ1 and the second wavelength λ2.
In the case that has just been described, the photo-fragmentation laser L emits at the first wavelength λ1 for the first photo-fragmentation of step 130, emits at the second wavelength λ2 for the second photo-fragmentation of step 160 and emits at the third wavelength λ2′ for the third photo-fragmentation of step 160′. Alternatively, two photo-fragmentation lasers may be used: a first photo-fragmentation laser emitting at the first wavelength λ1 for the first photo-fragmentation of step 130, and a second photo-fragmentation laser emitting at the second wavelength λ2 for the second photo-fragmentation of step 160 and emitting at the third wavelength λ2′ for the third photo-fragmentation of step 160′. According to another alternative, three photo-fragmentation lasers may be used: a first photo-fragmentation laser emitting at the first wavelength λ1 for the first photo-fragmentation of step 130, a second photo-fragmentation laser emitting at the second wavelength λ2 for the second photo-fragmentation of step 160 and a third photo-fragmentation laser emitting at the third wavelength λ2′ for the third photo-fragmentation of step 160′.
In the same way as described for step 170, the detector De is used during step 170′.
Generally speaking, advantageously as many cycles may be carried out as different types of second-generation ions, so as to obtain the spectroscopic signature of each type of second-generation ion.
An exemplary embodiment of the method 200 according to the second embodiment will now be described, according to which the number N of times the sequence of steps i), ii) and iii) is carried out is equal to 2.
The first chain of steps 110, 120, 130, 140, 150 and 160 of the method 200 according to the second embodiment is identical to the chain of steps 110, 120, 130, 140, 150 and 160 of the method 100 according to the first embodiment, which has been described previously. Indeed, for N=1, the method 200 according to the second embodiment is identical to the method 100 according to the first embodiment. At this stage, the sequence of steps i), ii) and iii) has thus been carried out once. For N=2, the sequence of steps i), ii) and iii) is next carried out a second time:
Finally, the method 200 according to the second embodiment of the invention comprises step 170, according to which the plurality of last-generation ions, in this case the plurality of fourth-generation ions, is detected. The spectroscopic signature of the first type of third-generation ions is thereby obtained. Generally speaking, the spectroscopic signature of each type of third-generation ion is advantageously determined.
Number | Date | Country | Kind |
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FR1554093 | May 2015 | FR | national |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2016/060057 | 5/4/2016 | WO | 00 |