WO2008142170A1

WO2008142170A1 - Method and system of tandem mass spectrometry without primary mass selection with secondary ionization of dissociated neutral fragments.

Info

Publication number: WO2008142170A1
Application number: PCT/EP2008/056428
Authority: WO
Inventors: David Scigocki
Original assignee: Physikron
Priority date: 2007-05-24
Filing date: 2008-05-26
Publication date: 2008-11-27

Abstract

The invention proposes a method of tandem mass spectrometry for use in a mass spectrometer having a known characteristic function of mass-to-charge ratios of ions to be analysed, wherein it comprises the following steps: - providing a primary charged ions source to be analysed, - acquiring different spectra, one as primary ion spectrum, one as dissociation spectrum without ionized neutral fragments and one as dissociation spectrum with ionized neutral fragments, - determining potential pairs, - computing to identify from amongst said potential pairs, using a proximity criterion, each real pair of charged fragment peaks corresponding to primary ions by comparing which one of the potential pairs of values corresponds to a real dissociation mass peak pair in the dissociation mass spectrum produced with ionized neutral fragments. - generating dissociation mass spectra corresponding respectively to said parent primary ions of interest, comprising said real pairs of dissociation mass peaks identified.

Description

Method and system of tandem mass spectrometry without primary mass selection with secondary ionization of dissociated neutral fragments

Field of the invention

The invention relates to the general field of mass spectrometry.

State of the art

By way of a reminder, mass spectrometry (MS), whatever its type, generally includes steps used to identify the molecules present in a sample by measuring the mass of these molecules after they have been ionised, accelerated and injected into a mass spectrometer. A mass spectrometer generates a mass spectrum of the various molecules contained in the analysed sample, as a function of the value of the mass-to- charge ratio (M/Q) (M being the mass and Q the charge) of the ions generated, in the form of the current intensity of the ions detected by an ion detector in relation to a function of the mass-to-charge ratio F(M/Q) of the ions which is characteristic of the mass spectrum used, and is generally of the form:

where G is a function which depends on the type of spectrometer used, and which is independent from the mass-to-charge ratio of the ions. The main mass spectrometers used are time-of-flight (TOF) spectrometers, magnetic sector spectrometers, quadrupolar mass spectrometers, 3D ion traps, 2D ion traps, and FT-ICR mass spectrometers (for Fourier transform ion cyclotron resonance spectrometer) using a static magnetic field or a radial logarithmic electrical field to store the ions. The specific forms of operation, embodiments and the characteristic function corresponding to each of said mass spectrometers are known by the person skilled in the art. In particular, tandem mass spectrometry (MS-MS) is well known and used when the primary mass spectrum does not allow the identification of the analysed ions. It generally includes steps required to generate, by means of a first mass spectrometer, a primary mass spectrum (MS) of the ionised molecules present in the analysed sample, to perform a step for the selection of a primary mass, and then to fragment, i.e. to dissociate by means of a dissociation device, the primary ions of said selected primary mass, so as to generate a mass spectrum described as the dissociation mass spectrum of the charged fragments coming from the dissociation of said primary ions, by means of a second mass spectrometer.

The primary mass selection, generally implemented to realise each dissociation mass spectrum, limits the acquisition debit of the tandem mass spectrometer, as the mass spectra are generated one after the other.

It also limits the sensitivity of the tandem mass spectrometer, this sensitivity being defined as the amount of samples consumed to generate each mass dissociation spectrum, the remaining unselected ions provided by the ion source being actually eliminated for the generation of the mass spectrum of the selected primary ions.

The primary ions dissociation can be performed at high kinetic energy (about 0.8 to 20 keV) or low kinetic energy (about 10 to 200 eV).

Low kinetic energy dissociation can be used with any existing mass spectrometers, while high kinetic energy dissociation is generally used with tandem magnetic sector mass spectrometers or tandem time-of-flight mass spectrometers. The single charge of mono charged primary ions of small molecules dissociated in a pair of neutral/charged fragments is generally situated on the same fragment of the pair. For large molecule as peptides or proteins, the situation is different and the charge of the dissociated mono charged ion can be distributed alternatively on one or the other fragment of the pair (mobile proton theory), to finally produce a pair of fragment peaks in the dissociation spectrum produced by the tandem mass spectrometer used. Only large molecule can be multi charged (electric charge after ionization superior to one), and can be dissociated in pairs of charged fragments or in neutral/charged fragment pairs.

In the particular case of time-of-f light and ion trap mass spectrometry, in addition to the tandem mass spectrometers described previously with primary mass selection, tandem mass spectrometers without primary mass selection are also well known.

These can be used to generate several dissociation mass spectra simultaneously.

Methods of time-of-flight mass spectrometry without primary mass selection [1] [2] [3] [4] are also well known, which nevertheless necessitate several acquisitions in order to generate the different dissociation spectra, but with a lower number of successive acquisitions in relation to the appliances using primary mass selection.

In particular, two methods of tandem time-of-flight mass spectrometry employed to generate several dissociation mass spectra without primary mass selection in a single acquisition [5] [6] are known. The first method of time-of- flight mass spectrometry without primary mass selection is based on conversion of the times-of-flight into measured positions [5]. This method limits the range of primary masses simultaneously accessible. The second method of time-of-flight mass spectrometry without primary mass selection is based on the individual identification of dissociated neutral/charged fragment pairs [6].

The methods [1] [2] [3] [4] [5] and [6] are only compatible with the primary ions dissociation at high kinetic energy. A method of tandem mass spectrometry without primary mass selection compatible with low kinetic energy dissociation [7] for ion trap mass spectrometers is also known. It is a method based on the comparison of two dissociation mass spectra obtained with two different set of ion trapping conditions [7]. All these MS-MS methods without precursor mass selection are dependant of type of mass spectrometer used and of the dissociation mode used (low or high kinetic energy dissociation). WO-A-2008/003684 in the name of the Applicant describes an MS-MS method, which is the only existing universal MS-MS method without precursor mass selection compatible with all types of tandem mass spectrometers and all possible modes of dissociation. However this universal method using charged fragment pair correlations can be applied only to large molecules producing pairs or multiplets of charged fragments, and cannot be applied to dissociation channels producing neutral-charge fragment pairs.

Thus no technique of tandem mass spectrometry compatible with all existing types of mass spectrometers and both for large and small molecules is currently known to generate, simultaneously and in a single acquisition, a plurality of dissociation mass spectra without primary mass selection for charged precursors dissociated in neutral/charged fragment pairs.

Summary of the invention

One aim of the invention is therefore to overcome the drawbacks of the state of the art as presented above, in the case of charged primary ions dissociated in neutral/charged fragment pairs.

In particular, one aim of the invention is to propose a method of mass spectrometry without primary mass selection, compatible with known mass spectrometers, that is capable of simultaneously producing, in a single acquisition, dissociation spectra for a plurality of different primary masses present in a sample to be analysed.

To this end, the invention provides a method of mass spectrometry as defined in appended claim 1.

Other optional features of this method of mass spectrometry are defined in appended dependant claims 2 to 8.

The invention provides also a tandem mass spectrometer as defined in appended claim 9. Other optional features of this tandem mass spectrometer are defined in appended dependant claims 10 to 21. Finally, the invention provides a computer program as defined in appended claim 22.

Brief description of the figures Other aspects, aims and advantages of the invention will more clearly appear on reading the following description of the invention, which is provided by way of a non-limiting example and with reference to the appended drawings in which:

- figure 1 is a flow chart for a preferred embodiment of the spectrometry method of the invention,

- figure 2 illustrates components of a system designed to implement the method of mass spectrometry according to one example of the invention,

- figure 3 illustrates a tandem mass spectrometer according to an other embodiment of the invention, - figure 4 illustrates a primary mass spectrum of molecules to be identified, comprising three primary mass peaks,

- figure 5 illustrates a mass dissociation spectrum obtained without primary mass selection, comprising only three dissociation mass peaks produced by the dissociated charged fragments from the dissociation in neutral/charged fragment pairs of the primary ions of the three primary peaks of figure 4, each one corresponding to one of the three primary mass peaks of figure 4,

- figure 6 illustrates, a mass dissociation spectrum obtained without primary mass selection, comprising the three dissociation mass peaks of figure 5, and three additional dissociation mass peaks produced by the ionization of the neutral fragments of the dissociated neutral/charged fragment pairs of the primary ions of the three primary peaks of figure 4, each one corresponding to one of the three primary mass peaks of figure 4,

- figure 7 illustrates, an example of ionization device (3) with ionization of the neutral fragments inside the dissociation device (2), - figure 8 illustrates, an example of ionization device (3) with ionization of the neutral fragments outside the dissociation device (2). Description of preferred embodiments of the invention

First of all, It is recalled that what is meant by a "charged ion" is an ion that has a positive or negative electric charge whose absolute value is equal to Ze (where e is the electron charge value, and Z is the number of charges equal 1 for mono-charged ions and Z > 2 for multi-charged ions), and which, when dissociated, can generate a pair of neutral and charged fragments with a positive or negative electric charge.

Referring to figures 1 and 2 in particular, the method of the invention preferably aims at dissociation of the charged primary ions into neutral/charged fragment pairs.

In the present embodiment, implemented with whichever mass spectrometer having a known characteristic function of mass-to-charge ratio for ions to be analysed, a first step a and b comprises supplying a primary mass spectrum for charged ions obtained from molecules that are to be identified or studied.

This primary mass spectrum can be obtained from a database, such as a third-party database, in which it was previously saved.

It can also be obtained by implementing sub-steps a and b illustrated in figure 1. In sub-step a, molecules to be identified are ionized in a source 1 of charged ions, and accelerated with a substantially constant electric field, in order to provide a primary ion source of primary ions.

And then in sub-step b, the primary ions are injected into a mass spectrometer 4, in order to generate a primary mass spectrum of said primary ions, without dissociation, wherein said spectrum contains primary ions peaks of occurrence obtained following a measurements of characteristic function values.

According to the presentation graph conventionally used by the person skilled in the art (though in no way limiting) of mass spectrometry, the primary mass spectrum is generally shown with two perpendicular axes, with the characteristic function values on the abscissa axis, and the corresponding occurrences on the ordinate axis. This primary mass spectrum is then used to determine, in step c, the characteristic function values at maxima of occurrences F_max(M/Q) of the primary mass peaks, primary mass-to-charge ratios M/Q and primary electric charges Q of the ions, for each primary mass peak. The person skilled in the art will be able to determine the values of each primary mass-to-charge ratio M/Q and the primary electric charge Q of the primary ions corresponding to each primary mass peak with the F_max(M/Q) values obtained, by performing the usual prior primary calibration of the mass spectrometer used, with known molecules. Figure 4 shows an example of a primary mass spectrum containing three primary mass peaks of primary ions, having a mass-to-charge ratio of (M1/Q1),

(M2/Q2) and (M3/Q3) respectively, and the characteristic function values

F_max(M/Q) at maxima of occurrences for each of said primary mass peaks.

From the characteristic function values associated to said peaks, correlation laws are determined, considering every possible pair of characteristic function values and associated mass-to-charge ratio values corresponding to pairs of charged fragments liable to come from the dissociation of primary ions associated to said primary mass peaks, are determined. Here, only the case of the dissociation of charged primary ions of mass-to- charge ratio value (M/Q,), in neutral/charged fragment pairs, is considered with the charged fragment of mass-to-charge value (m/q,) of each pair keeping the primary charge (Q₁ = q,) and the ionization of the neutral fragment of each pair in a mono charged fragment of mass-to-charge value (m'/q¹,) (q¹, = e).

In this particular case, each correlation law of each of the primary mass peak associated to primary ions having the same mass-to-charge ratio (M,/Q,), dissociating into pairs of charged fragments, can always be described by the equation (with q', = e and Q₁ = q, = Ze):

Each correlation law corresponding to a primary mass peak of the primary mass spectrum can thus be determined by the method according to the invention, on the basis of the primary charge value and of the characteristic function value at maxima of occurrences of said primary mass peak given the mass-to-charge ratio value of the primary ions of the said primary mass peak.

It may happen that one of the two fragments of each dissociated neutral/charged fragment pairs loses another neutral fragment m_neutrai during the dissociation.

Generally, the different possible m_neutrai values are known by the professional using tandem mass spectrometers.

In this particular case of dissociation channels with additional neutral fragment loss of known mass, the equation of correlation (1 ) becomes:

d, charged primary ions are dissociated by a dissociation device 2 so as to obtain a pair of neutral/charged fragments for each of them.

In step e, the dissociated charged fragments are injected into the mass spectrometer 4, and characteristic function values for the occurrences (or ions current intensity) are measured for the dissociated charged fragments. A dissociation mass spectrum (MS-MS) is then generated, on the basis of said values, without primary mass selection, comprising all the mass dissociation peaks of each of dissociation spectra of the parent primary ions of the primary mass spectrum.

The mass dissociation peaks of the several mass dissociation spectra corresponding to the primary mass peaks of the primary mass spectrum are consequently mixed in the mass dissociation spectrum generated without primary mass selection.

According to the conventional graph presentation used by professionals in this field, although not limiting, each dissociation mass spectrum is generally shown with two perpendicular axes, with the identified characteristic function values on the abscissa axis, and the corresponding occurrences on the ordinate axis.

Figure 5 illustrates a mass dissociation spectrum (MS-MS) containing three dissociation peaks of fragment ions having mass-to-charge ratio values (rτii/qi), (m₂/q2), (rrVqs), corresponding for each of them to the dissociated charged fragments from the neutral/charged fragment pairs produced by the dissociation of the primary ions of each of the three primary mass peaks of figure 4.

In order to simplify the present description, the above example, illustrated on figure 5, only comprises one dissociation peak for each primary peak.

It will be understood that in reality, the dissociation spectrum, obtained without mass selection, contains generally several dissociation mass peaks obtained from the different possible dissociation channels of the charged primary ions of each primary mass peak. Then the characteristic function values at the maxima F_max(m/q) of each of the dissociation peaks of the dissociation mass spectrum are determined on the basis of the characteristic function values generated for the occurrences.

The person skilled in the art will be able to determine the values of each mass-to-charge ratio (m/q) of the charged fragments corresponding to each dissociation mass peak with the F_max(m/q) values obtained, by performing the usual prior primary calibration of the mass spectrometer used, with known molecules.

In step f, for each dissociation mass peak from the measured characteristic function values at maxima F_max(m/q) given the mass-to-charge ratio values (m/q) of the dissociated charged fragments, every potential pair {(m/q),(m',/q'i)} value corresponding to each primary peak of mass-to-charge ratio value (M/Q,) are formed using the possible pairs given by each correlation law corresponding to each primary mass peak of interest.

The number of potential pairs {(m/qj^m'/q¹,)} for each dissociation mass peak is equal to the number of primary mass peaks of interest. Each (m'i/q'i) value of each potential pair {(m/q),(m'Jq\)} is a solution of the equation (1 ) of the correlation law corresponding to each primary mass peak of interest.

In the example of figure 5, three potential pairs (not represented on figure 5) are associated to each one of the three dissociation mass peak corresponding to the three possible solutions of each equation (1 ) of correlation law corresponding to each one of the three primary mass peak of figure 4.

Step g begins as step d with adissociation of the charged primary ions by the dissociation device 2 so as to obtain a pair of neutral/charged fragments for each of them, following by a second step of ionization of the dissociated neutral fragments by an ionization device 3.

According to one preferred embodiment of the invention, the ionization device 3 is any ion source able to ionize dissociated neutral fragments in gaseous phase.

According to one preferred embodiment of the invention the dissociated neutral fragments can be ionized inside the dissociation device 2.

According to another preferred embodiment of the invention the dissociated neutral fragments can be ionized outside the dissociation device 2.

Then, in step h, the characteristic function values at the maxima Fm_3x(In₁Zq₁) and F_max(m'_l/q'_l) of each of the dissociation mass peak of the mass dissociation spectrum, and their corresponding mass-to-charge ratio values, are determined on the basis of the characteristic function values generated for the occurrences.

Figure 6 illustrates a mass dissociation spectrum (MS-MS) containing the three dissociation mass peaks of figure 5, and the three additional dissociation mass peaks forming for each of them by the ionized neutral fragments with mass-to-charge ratio values (mVq'i), (nrr₂/q'₂), (mVq'3), from the neutral/charged fragment pairs produced by the dissociation of the primary ions of each of the three primary mass peaks of figure 4. In step i, from amongst said potential pairs of values obtained for each dissociation mass peak obtained in step f, the real pair of dissociation mass peaks associated to dissociated neutral/charged fragment pairs (coming from the dissociation of identical primary ions having the same mass-to-charge ratio (IWQ₁) for each of the primary mass peaks), is identified using a proximity criterion by comparing which one of the potential pairs of values corresponds to a real dissociation mass peak pair in the dissociation mass spectrum produced with ionized neutral fragments.

This identification step i consists in comparing the dissociation mass spectrum obtained without using the ionization device 3 in steps d and e with the mass spectrum obtained using the ionization device 3 in the steps f and g.

Proximity criterion precision can be at least substantially equal to the precision of the characteristic function values at maxima Fm_3x(M₁ZQ₁) for the primary ions, or/and of the precision of the characteristic function values measurements at maxima F_max(m_l/q_l) for the dissociated charged fragments.

In the example of figure 6, the three parent primary peaks of the three dissociation mass peaks of figure 5 are identified with the three additional dissociation mass peaks produced by the ionized neutral fragments, by their characteristic function values at the maxima F_max(m'i/q'i), F_max(m'₂/q'2), F_max(m'₃/q'3) and their corresponding mass-to-charge ratio values (m'i/q'i),

Each one of these values is the only real value solution corresponding to a real dissociation mass peak, within the proximity criterion equal to the precision of these values, of the three potential pair solutions obtained for each dissociation mass peak of figure 5, corresponding to the three possible correlation laws of the three primary mass peaks of figure 4.

Each dissociation mass peak produced by ionized neutral fragments of the three real pairs of dissociation mass peaks, corresponding to the mass-to- charge ratio values {(mi/qi),(m'i/q'i)}, {(m₂/q2),(m'₂/q'2)}, {(ms/qsMmVq's)}, are positioned, in figure 6, at positions corresponding to said each one of the three potential solutions of the said corresponding correlation laws, and none dissociation mass peaks are produced at positions corresponding to the other potential solutions.

Finally, in step j, each dissociation mass spectrum, corresponding respectively to each of the primary mass peak of interest, and comprising the real pairs of dissociation mass peaks identified, is generated.

As indicated above, according to a preferred embodiment of the invention, from step d the method is implemented for all of the primary ions to be analysed, without primary mass selection.

It is also possible to carry out steps g and h before steps d, e and f. Furthermore, in general, it is possible to carry out all acquisition steps - b, (d, e), and (g, h) - in any possible order and computation steps - c, f, i and j - once the corresponding acquisition steps have been done.

According to the graphical representation conventionally used by the person skilled in the art, each mass dissociation peak is generally represented with two perpendicular axes, with the measured characteristic function values in abscissa axis, and the corresponding occurrences in ordinate axis.

According to one preferred embodiment of the invention, if the ion source 1 can produced both mono-charged and doubly charged primary ions, the dissociation device 2 can be used as the ionization device 3. The first dissociation mass spectrum without using ionized neutral fragments is produced with mono charged primary ions, and the second dissociation mass spectrum using ionized neutral fragments is produced with doubly charged primary ions of the same molecules forming the previous mono charged ions. The ionized neutral fragments of the second dissociation mass spectrum are produced by the dissociation of the doubly charged fragments in charged fragment pairs using the dissociation device 2.

As previously described, the method according to the invention allows a concurrent generation, in a single acquisition and without mass selection, of all the mass dissociation spectra corresponding to all the primary mass peaks of the primary mass spectrum. It is possible that the mass peaks of interest, for which a dissociation mass spectrum is wanted, be only part of the set of primary mass peaks obtained in step (b).

It is possible to use a selection device 5 positioned between the ion source 1 and the dissociation device 2, as illustrated in figure 3, to simultaneously select the primary peaks of interest before the injection of the primary ions in the dissociation device 2, and eliminate the others. It is then possible to simultaneously generate the set of dissociation spectra of interest according to the method of the invention. The aforementioned selection device 5 can be a quadrupolar mass spectrometer, which selects a large mass band comprising simultaneously several primary mass peaks of interest. This kind of device 5 allows the reduction of the number of primary peaks selected, but can only select a part of all primary mass peaks of interest at each acquisition, what can require the generation of several dissociation spectra to study all the primary peaks of interest on all the primary mass range.

By contrast, the use of an ion trap, or a temporal gate, as selection device 5 enables a selection of a set of primary mass peaks of interest by successive individual or more primary mass peaks covering all the primary mass range in a single dissociation mass spectrum.

In this embodiment, the ions generated by the ion source 1 are stored in the ion trap 5 until their ejection out of the trap towards the dissociation device 2 in relation to their mass-to-charge ratio, as known by the person skilled in the art. A device made of, for example, a pair of deflection plates subject to a variable tension and positioned at the exit of the ion trap 5, can deviate the ejected primary ions that are of no interest, and only enables the primary ions corresponding to the primary mass peaks of interest to pass through.

In this embodiment, the ion trap 5 used as tandem mass spectrometer in the MSⁿ mode, as known by the person skilled in the art, enables using the method of the invention not only by selecting the ions of a group of primary mass to be injected into the dissociation device 2 to produce the two dissociation mass spectra (MS² mode) necessary to the method of the invention, but also by using the method of the invention by first selecting the charged fragments of a group of dissociation mass peaks produced by the dissociation of primary ions from a single primary peak selected in the ion trap 5 used as tandem mass spectrometer. These dissociated charged fragments are injected into the dissociation device 2, to be dissociated and to produce two dissociation mass spectra used in the method according to the invention in the MS³ mode.

Instead of selected primary ions to be dissociated in the ion trap, and producing MS³ dissociation mass spectra with the method of the invention, charged fragments from a single dissociation mass peak can be selected and dissociated by the ion trap 5, before being injected and dissociated in the dissociation device 2 to produce the two dissociation mass spectra necessary to the method of the invention in the MS⁴ mode, and so on in other MSⁿ mode.

In another embodiment, a temporal gate can be used with time-of-flight mass spectrometers to only select the ions of interest to be injected into the dissociation device 2.

It is known by the person skilled in the art using tandem mass spectrometers that in some ions sources, the dissociation of the primary ions can be produced inside the ion source by ISD (In Source Dissociation), instead of a dissociation device 2.

El (Electron Impact) ion source and MALDI (Matrix Assisted Laser Desorption) ion source are non limiting examples of this type of ions sources.

According to one preferred embodiment of the invention, the ion source 1 can be used in ISD (In Source Dissociation) mode both as dissociation device 2 and as ionization device 3 for dissociated neutral fragments.

According to one preferred embodiment of the invention, only one dissociation mass spectrum (MS-MS) using the ionization device 3 is produced to realize the method of the invention.

This another embodiment of the method of the invention is similar to the method described in the patent PCT/EP2007/056655 for multi charged primary ions dissociated directly in charged fragment pairs. In this particular case, the dissociation mass peaks produced by the charged fragments of the dissociated neutral/charged fragment pairs, cannot be distinguish from the dissociation mass peaks produced by the ionized neutral fragments. This increases the number of potential pairs by producing additional potential pairs forming by dissociation mass peak pairs each composed of one peak produced by charged fragments of neutral/charged fragment pairs from parent ions of one primary mass peak, and the second peak produced by charged fragments of neutral/charged fragment pairs from parent ions of another primary mass peak.

These additional potential pairs produce false real pair identifications, which do not exist in the method according to the invention with two successive dissociation spectra, the first one being produced without using the ionized neutral fragments, and the second one being produced using the ionized neutral fragments.

Real pairs obtained with the two successive dissociation mass spectra are always composed of one dissociation mass peak of the first dissociation mass spectrum and of a second dissociation mass peak of the second dissociation mass spectrum which does not exist in the first one. It will be understood that the concrete embodiment of the invention as described above is achieved typically by a digital computer such as a DSP (for "Digital Signal Processor") executing the appropriate programs.

In particular, the correlation laws are typically numerical data (such as the equation (1 )) with which numerical characteristic function data generated by the spectrometers and supplied to the computer is compared.

More practically, the present invention can be embodied in the form of a software module that is added to an existing mass spectrometry device, and interfaced with another software of this equipment so as to perform, for the most part, the establishment of the correlation laws data and collection of the characteristic function data in order to compare them with these correlation laws data. In any event, the professional in this field will understand that production of the primary mass spectrum and of the dissociation spectra obtained from charged primary ions dissociating into pairs of neutral/charged fragments, with ionization of the neutral fragments, provides the possibility of identifying the molecules studied.

Components and operation of spectrometers according to the invention Now, are described in greater detail, and by way of non-limiting examples, some preferred spectrometer components and spectrometer operations in a tandem mass spectrometer implementing the spectrometry method according to the invention.

The ion source 1 can be continuous or pulsed, such as an ESI or nano-ESI (Electro-Spray lonisation) ion source, a MALDI (Matrix Assisted Laser Desorption lonisation) pulsed laser ion source, an APCI (Atmospheric Pressure Chemical lonisation) ion source, an APPI (Atmospheric Pressure Photo lonisation) ion source, a LDI (Laser Desorption lonisation) ion source, an ICP (Inductively Coupled Plasma) ion source, en El (Electron Impact) ion source, a Cl (Chemical lonisation) ion source, a Fl (Field lonisation) ion source, a FAB (Fast Atom Bombardment) ion source, a LSIMS (Liquid Secondary Ion Mass Spectrometry) ion source, an API (Atmospheric Pressure lonisation) ion source, a FD (Field Desorption) ion source, a DIOS (Desorption lonisation On Silicium) ion source, a MAB (Metastable Atom Bombardment) ion source, or any other type of ion source producing charged ions.

The selection device 5 used to select and inject the primary ions corresponding the primary mass peaks of interest into the dissociation device 2 can be: a quadrupolar mass spectrometer, a 3D ion trap with a hyperbolic geometry, a linear 2D ion trap with a cylindrical geometry, or any other type of ion trap. The device 5 can be a temporal gate to select the primary peaks of interest before the dissociation into the device 2. The dissociation device 2 can be a multipolar waveguide, an ion trap, a

Fourier Transform mass spectrometer, or any other device allowing the generation of dissociated charged fragments. The dissociation of the primary ions in the dissociation device 2 can be implemented with a collision chamber containing gas that allows dissociation by CID/CAD (Collision Induced Dissociation/Collision Activated Dissociation), a time-of-flight space allowing spontaneous dissociation (PSD or Post Source Decay) after increasing the internal energy of the primary molecule ionised in the ion source or over the time-of-flight path by photo ionisation, or with the SID (Surface Induced Dissociation) technique, the ECD (Electron Capture Dissociation) technique, the ETD (Electron Transfer Dissociation) technique, the IRMPD (Infra Red Multi Photon Dissociation) technique, the PD (Photo Dissociation) technique, the BIRD (Back Body Infra Red Dissociation) technique, or again any technique of fragmentation of the primary ions.

The ionization device 3 can be any type of ion source able to ionize the neutral fragments produced by the dissociation of the primary ions in the dissociation device 2. The ionization device 3 can be a MALDI (Matrix Assisted Laser Desorption

Ionisation) pulsed laser ion source, an ESI or nano-ESI (Electro-Spray Ionisation) ion source, an APCI (Atmospheric Pressure Chemical Ionisation) ion source, an Pl (Photo Ionisation) ion source, an APPI (Atmospheric Pressure Photo Ionisation) ion source, an LDI (Laser Desorption Ionisation) ion source, an ICP (Inductively Coupled Plasma) ion source, en El (Electron Impact) ion source, a Cl (Chemical Ionisation) ion source, a Fl (Field Ionisation) ion source, an API (Atmospheric Pressure Ionisation) ion source, a MAB (Metastable Atom Bombardment) ion source, or any other type of ion source producing charged ions with the dissociated neutral fragments. The mass spectrometer 4 used to generate the primary mass spectrum and the dissociation mass spectra without primary mass selection can be one of the following group: a time-of-flight mass spectrometer, a magnetic sector mass spectrometer, a quadrupolar mass spectrometer, an ion trap, a FTICR mass spectrometer, or any other type of mass spectrometer. For time-of-flight mass spectrometer 4, a time-of-flight space between an ion packet pulsation and a ion detector measuring the time-of-flight of ions can be rectilinear, or equipped with a reflectron. In this case, the reflectron can be of the single-stage or two-stage type, of the Curved Field Reflectron (CFR) type, or a quadratic or any other type of reflectron.

The pulse of each ion packets to measure the time-of-flight of the ions can be implemented in the ion source 1 , between the ion source 1 and the dissociation device 2, or between the dissociation device 2 and the time-of- flight space.

The pulse of the ion packet, which is necessary for a time-of-flight mass spectrometer when the ion source is continuous, can be implemented by one of the following techniques: scan of the continuous beam of ions through a notch, application of a variable electric field between two deflection plates, orthogonal injection by application of a variable electric field between two electrodes perpendicularly to the continuous ions beam.

If the mass spectrometer 4 is an ion trap, it can be chosen amongst: a 3D ion trap with a hyperbolic geometry, a linear 2D ion trap with a cylindrical geometry, or any other type of ion trap.

The Fourier Transform mass spectrometer 4 can be a FTICR mass spectrometer that uses a static magnetic field or a radial logarithmic electrical field to store the ions. Now will be described two non-limiting embodiments of the ionization device 3 using ion source to ionize dissociated neutral fragments in gaseous phase.

First embodiment of ionization device 3 with ionization of the neutral fragments inside the dissociation device 2

A first embodiment of ionization device 3 with ionization of the neutral fragments inside the dissociation device 2 according to the invention is illustrated in figure 7. The dissociation device 2 of figure 7 is a multipolar waveguide q containing gas producing dissociation of the primary ions by CID at low kinetic energy. As known by the person skilled in the art who uses tandem mass spectrometers, the primary ions and the dissociated charged fragments crossing the dissociation device 2 are focused toward a centre 6 of the multipolar waveguide q by electric fields. The ionization device 3 of figure 7 is a Pl (Photo lonisation) ion source using a laser 7 as light source of ionisation. The laser light for ionizing the neutral fragments is transported by optical fibers 8 up to an ionisation area 9 situated inside a volume of the multipolar ion guide q2 surrounding the centre 6 of the dissociation device 2 where the ions are focused. The primary ions M⁺ crossing a central part of the multipolar waveguide q2 are dissociated into neutral/charged fragment pairs (m⁺,m') by CID (Collision induced Dissociation) with the gas.

The dissociated neutral fragments m' not sensitive to the focusing electric fields, can be transported from the dissociation area at centre 6 of the multipolar waveguide q 2 to the ionisation area 9, where they are ionized by the laser light.

The ionized neutral fragments m'⁺ are focused toward the centre of the multipolar waveguide q2 by the electric fields and collisional cooling with the gas, before being injected with the dissociated charged fragments in the mass spectrometer 4 to produce the dissociation mass spectrum.

In one embodiment of the first embodiment of the ionization device 3, the multipolar waveguide q can be replaced by an ion trap.

In this embodiment of the invention the charged fragments and the ionized neutral fragments are trapped in the ion trap 2 before being injected into the mass spectrometer 4.

Second embodiment of the ionization device 3 with ionization of neutral fragments outside the dissociation device 2

A second embodiment of ionization device 3 with ionization of the neutral fragments outside the dissociation device 2 according to the invention is illustrated in figure 8. The dissociation device 2 of figure 8 is a multipolar waveguide q containing gas producing dissociation of the primary ions by CID at low kinetic energy.

The ionization device 3 of figure 8 is an El (Electron Impact) ion source, as known by the person skilled in the art who uses tandem mass spectrometers, positioned in a chamber outside the dissociation device 2 with a connection 10 between the dissociation device 2 and the ionization device 3.

The primary ions M⁺ crossing a central part of the multipolar waveguide q2 are dissociated into neutral/charged fragment pairs (m⁺,m') by CID (Collision induced Dissociation) with the gas. The dissociated neutral fragments m' not sensitive to the focusing electric fields, can be transported by the gas via the connection 10 from a dissociation area at the centre 6 of the multipolar waveguide q2, to the ionisation area 9 of the El (electron Impact) ion source 3, where they are ionized by the electrons e^" of the ion source 3. The ionized neutral fragments produced are then injected from the El

(electron Impact) ion source 3 into the mass spectrometer 4.

In one variant of the second embodiment of ionization device 3, the ionized neutral fragments are injected from the El (electron Impact) ion source 3 in the dissociation device 2, before to be injected into the mass spectrometer 4. In another variant of the second embodiment of ionization device 3, the multipolar waveguide q can be replaced by an ion trap.

In this variant of the invention the charged fragments and the ionized neutral fragments are trapped in the ion trap 2 before being injected into the mass spectrometer 4.

Now will be described four non-limiting embodiments of mass spectrometers based on the use of the components illustrated in figures 2 and 3.

First embodiment of tandem mass spectrometer using time-of-f light mass spectrometer with low kinetic energy dissociation A first embodiment of a tandem mass spectrometer according to the invention is illustrated in figure 2.

It includes, in succession, in a general direction of movement of the primary ions, an ion source 1 (it can be anyone of the ion sources listed before), a dissociation device 2 and an ionization device 3, both identical to the ones described previously in the first embodiment of ionization device 3 with the ionization of the neutral fragments inside the dissociation device 2, illustrated in figure 7, and a time-of-flight mass spectrometer 4 including a device for pulsing an ion beam by orthogonal injection and a time-of-flight space with a reflectron and an ion detector.

This embodiment for a tandem mass spectrometer is known by the person skilled in the art who uses tandem mass spectrometers with primary mass selection as Q-q-TOF, further equipped with a quadrupolar mass spectrometer, in order to implement the selection of primary mass in mode MS-MS.

The first embodiment is consequently similar to said devices, except that it does not comprise the quadrupolar mass spectrometer Q, and it comprises a secondary ion source 3 to ionize dissociated neutral fragment in the dissociation device 2. First, the primary mass spectrum is generated with the time-of-flight mass spectrometer 4, without mass dissociation into the multipolar waveguide q 2.

The first dissociation mass spectrum is generated without primary mass selection, still in the time-of-flight mass spectrometer 4, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy.

Then, the second dissociation mass spectrum containing both peaks from dissociated charged and ionized neutral fragments is generated without primary mass selection, still in the time-of-flight mass spectrometer 4, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy, and the laser photo ionisation of the dissociated neutral fragments inside the dissociation device 2 by the ionization device 3. The real pairs of dissociation mass peaks corresponding to each primary mass peak are identified, according to the method of the invention, and finally each dissociation spectra, comprising the real pairs of dissociation mass peaks of a corresponding primary mass peak, is generated. In one variant of the first embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 3, the device is equipped with either a quadrupolar mass spectrometer or an ion trap 5, positioned between the ion source 1 and the dissociation device 2, in order to select simultaneously, with a large mass window, several primary mass peaks of interest, and to reduce the number of primary peaks of interest if necessary.

In the particular case of using an ion trap as device 5 to select the primary ions of interest, several primary peaks can be individually selected on all the mass range in multiplex mode, instead of to be selected in one large mass selection window, as explained previously in the description. In addition, if the ion trap is a tandem mass spectrometer equipped to dissociate ions by CID (Collision Induced Dissociation), it can be used according to the invention in MSⁿ mode, as also described previously.

In another variant of the first embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 8, the device 3 previously described in the first embodiment of the ionization device 3, is replaced by the ionization device 3 previously described in the second embodiment of the ionization device 3.

Second embodiment of tandem mass spectrometer using ion trap mass spectrometer

A second embodiment of a tandem mass spectrometer according to the invention is illustrated in figure 2.

It includes, in succession, in a general direction of movement of the primary ions, an ion source 1 (it can be anyone of the ion sources listed before), a dissociation device 2 and a ionization device 3 identical to the one described previously in the first embodiment of ionization device 3 with the ionization of the neutral fragments inside the dissociation device 2, illustrated in figure 7, and an ion trap mass spectrometer 4.

The ion trap can be 2D or 3D ion trap, and is known by the person skilled in the art who uses identical mass spectrometer to generate mass spectra. First, the primary mass spectrum is generated with the ion trap mass spectrometer, without mass dissociation into the multipolar waveguide q 2.

The first dissociation mass spectrum is generated without primary mass selection, still in the ion trap mass spectrometer, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy.

Then, the second dissociation mass spectrum containing peaks from dissociated charged and ionized neutral fragments is generated without primary mass selection, still in the ion trap mass spectrometer, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy, and the laser photo ionisation of the dissociated neutral fragments inside the dissociation device 2 by the ionization device 3.

The real pairs of dissociation mass peaks corresponding to each primary mass peak are identified, according to the method of the invention, and finally each dissociation spectra, comprising the real pairs of dissociation mass peaks of a corresponding primary mass peak, is generated.

In one variant of the second embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 3, the device is equipped with either a quadrupolar mass spectrometer or an ion trap 5, positioned between the ion source 1 and the dissociation device 2, in order to select simultaneously, with a large mass window, several primary mass peaks of interest, and to reduce the number of primary peaks of interest if necessary.

In the particular case of using an ion trap as selection device 5 to select the primary ions of interest, several primary peaks can be individually selected on all the mass range in multiplex mode, instead of being selected in one large mass selection window, as explained previously in the description. In addition, if the ion trap is a tandem mass spectrometer equipped to dissociate ions by CID (Collision Induced Dissociation), it can be used according to the invention in MSⁿ mode, as also described previously.

In another variant of the second embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 8, the ionization device 3 previously described in the first embodiment of the ionization device 3, can be replaced by the ionization device 3 previously described in the second embodiment of the ionization device 3.

Third embodiment of tandem mass spectrometer using Fourier Transform mass spectrometer

A third embodiment of a tandem mass spectrometer according to the invention is illustrated in figure 2. It includes, in succession, in a general direction of movement of the primary ions, an ion source 1 (it can be anyone of the ion sources listed before), a dissociation device 2 and a ionization device 3 identical to the one described previously in the first embodiment of ionization device 3 with the ionization of the neutral fragments inside the dissociation device 2, illustrated in figure 7, and an Fourier Transform mass spectrometer (FTICR) 4.

The Fourier Transform mass spectrometer can be a FTICR mass spectrometer that uses a static magnetic field or a radial logarithmic electrical field to store the ions, and is known by the person skilled in the art who uses identical mass spectrometer to generate mass spectra. First, the primary mass spectrum is generated with Fourier Transform trap mass spectrometer, without mass dissociation into the multipolar waveguide q 2.

The first mass dissociation spectrum is generated without primary mass selection, still in the Fourier Transform mass spectrometer, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy. Then, the second mass dissociation spectrum containing mass peaks from dissociated charged and ionized neutral fragments is generated without primary mass selection, still in the Fourier Transform mass spectrometer, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy, and the laser photo ionisation of the dissociated neutral fragments inside the dissociation device 2 by the ionization device 3.

In one variant of the third embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 3, the device is equipped with either a quadrupolar mass spectrometer or an ion trap 5, positioned between the ion source 1 and the dissociation device 2, in order to select simultaneously, with a large mass window, several primary mass peaks of interest, and to reduce the number of primary peaks of interest if necessary.

In the particular case of using an ion trap as selection device 5 to select the primary ions of interest, several primary peaks can be individually selected on all the mass range in multiplex mode, instead of to be selected in one large mass selection window, as explained previously in the description.

In addition, if the ion trap is a tandem mass spectrometer equipped to dissociate ions by CID (Collision Induced Dissociation), it can be used according to the invention in MSⁿ mode, as also described previously. In another variant of the third embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 8, the ionization device 3 previously described in the first embodiment of the ionization device 3, can be replaced by the ionization device 3 previously described in the second embodiment of the ionization device 3. Fourth embodiment of tandem mass spectrometer using time-of-flight mass spectrometer with high kinetic energy dissociation

A fourth embodiment of a tandem mass spectrometer according to the invention is illustrated in figure 3. It includes, in succession, in a general direction of movement of the primary ions, a MALDI ion source 1 , a linear time-of-flight space, a dissociation device 2 comprising a collision cell containing gas producing dissociation of the primary ions by CID (Collision Induced Dissociation) at high kinetic energy, also used as ionization device 3 to produce the ionized neutral fragments, a time-of-flight space with a reflectron device and an ion detector 4.

This embodiment for a tandem mass spectrometer is known by the person skilled in the art who uses tandem mass spectrometers with primary mass selection as MALDI-TOF-TOF, further equipped with a temporal gate device 5 positioned before the dissociation device 2, in order to implement the selection of primary mass in dissociation mode.

A first variant is consequently identical to said devices, except that it uses the temporal gate device 5 for multiplexing primary mass selection to produce the dissociation mass spectra necessary to the method of the invention. First, the primary mass spectrum is generated with the time-of-flight mass spectrometer 4 without mass dissociation into the collision cell.

The first dissociation mass spectrum without using ionized neutral fragments is generated with multiplexing primary mass selection of mono charged ions of several primary mass peaks of interest, still in the time-of- flight mass spectrometer 4, after the primary ions dissociation into neutral/charged fragment pairs inside the collision cell 2 by CID (Collision Induced dissociation) at high kinetic energy.

Then the second dissociation mass spectrum using ionized neutral fragments is generated with the multiplexing mass selection of the doubly charged ions of the corresponding mono charged ions used for the first dissociation mass spectrum, still in the time-of-flight mass spectrometer 4, after the primary doubly charged ion dissociations into charged fragment pairs inside the collision cell (2 and 3) by CID at high kinetic energy.

Fifth embodiment of tandem mass spectrometer using αuadripolar mass spectrometer

A fifth embodiment of a tandem mass spectrometer according to the invention is illustrated in figure 2.

It includes, in succession, in a general direction of movement of the primary ions, an ion source 1 (it can be anyone of the ion sources listed before), a dissociation device 2 and an ionization device 3, both identical to the ones described previously in the first embodiment of ionization device 3 with the ionization of the neutral fragments inside the dissociation device 2, illustrated in figure 7, and a quadrupolar mass spectrometer 4.

This embodiment for a tandem mass spectrometer is known by the person skilled in the art who uses tandem mass spectrometers with primary mass selection as Q-q-Q, further equipped with a quadrupolar mass spectrometer, in order to implement the selection of primary mass in mode MS-MS.

A first variant is consequently similar to said devices, except that it does not comprise the quadrupolar mass spectrometer Q, and it comprises a secondary ion source 3 to ionize dissociated neutral fragment in the dissociation device 2.

First, the primary mass spectrum is generated with the quadrupolar mass spectrometer 4, without mass dissociation into the multipolar waveguide q 2.

The first dissociation mass spectrum is generated without primary mass selection, still in the quadrupolar mass spectrometer 4, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy. Then, the second dissociation mass spectrum containing both peaks from dissociated charged and ionized neutral fragments is generated without primary mass selection, still in the quadrupolar mass spectrometer 4, after the primary ions dissociation into charged fragments inside the multipolar waveguide q 2 by CID at low kinetic energy, and the laser photo ionisation of the dissociated neutral fragments inside the dissociation device 2 by the ionization device 3.

In one variant of the fifth embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 3, the device is equipped with either a quadrupolar mass spectrometer or an ion trap 5, positioned between the ion source 1 and the dissociation device 2, in order to select simultaneously, with a large mass window, several primary mass peaks of interest, and to reduce the number of primary peaks of interest if necessary.

In addition, if the ion trap is a tandem mass spectrometer equipped to dissociate ions by CID (Collision Induced Dissociation), it can be used according to the invention in MSⁿ mode, as also described previously. In another variant of the fifth embodiment of the tandem mass spectrometer according to the invention, the device is equipped with an ion trap to replace the quadrupolar mass spectrometer 4 to produce the MS and the MS-MS spectra.

In another variant of the fifth embodiment of the tandem mass spectrometer according to the invention, as illustrated in figure 8, the ionization device 3 previously described in the first embodiment of the ionization device 3, can be replaced by the ionization device 3 as previously described in the second embodiment of the ionization device 3.

Sixth embodiment of tandem mass spectrometer using ion trap mass spectrometer

A sixth embodiment of a tandem mass spectrometer according to the invention is illustrated in figure 3.

It includes, in succession, in a general direction of movement of the primary ions, an ion source 1 (it can be anyone of the ion sources listed before), an ion trap mass spectrometer 4, including a dissociation device 2 and an ionization device 3, both identical to the ones described previously in the first embodiment of ionization device 3 with the ionization of the neutral fragments inside the dissociation device 2, illustrated in figure 7.

The sixth embodiment is consequently similar to ion trap device, known by the person skilled in the art, except it comprises a secondary ion source 3 to ionize dissociated neutral fragment.

First, the primary mass spectrum is generated with the ion trap mass spectrometer 4, without mass dissociation.

The first dissociation mass spectrum is generated with a large primary mass selection, still in the ion trap mass spectrometer 4, after the primary ions dissociation into charged fragments by CID at low kinetic energy inside the ion trap used as dissociation device 2.

Then, the second dissociation mass spectrum containing both peaks from dissociated charged and ionized neutral fragments is generated with large primary mass selection, still in the ion trap mass spectrometer 4, after the primary ions dissociation into charged fragments by CID at low kinetic energy inside the ion trap mass spectrometer used as dissociation device 2, and the laser photo ionisation of the dissociated neutral fragments inside the ion trap by the ionization device 3 positioned inside the ion trap. The real pairs of dissociation mass peaks corresponding to each primary mass peak are identified, according to the method of the invention, and finally each dissociation spectra, comprising the real pairs of dissociation mass peaks of a corresponding primary mass peak, is generated.

Finally, a computer program can be designed to perform the necessary steps of the method according to the invention. The necessary steps comprise:

- controlling the system so that it generates, from a source of charged primary ions to be analysed, a primary mass spectrum of said primary ions, without dissociation, where this spectrum contains peaks of occurrences of primary ions, - performing a data acquisition of this spectrum, including characteristic function values at maxima of at least some of the primary mass peaks, the corresponding mass-to-charge ratio values, and primary electric charges, associated to said peaks,

- from said data, determining correlation laws that all possible pair of said values, corresponding to pairs of charged fragment peaks resulting from the dissociation of parent primary ions of interest corresponding to said primary mass peaks have to meet,

- controlling the system so that it generates concurrent dissociation of primary ions of interest associated to primary mass peaks so as to obtain peaks of charged fragments from said parent primary ions, and to generate characteristic function values at maxima of said dissociated charged fragment peaks and corresponding mass-to-charge ratio values,

- forming every potential pair of said values,

- controlling the system so that it generates the concurrent dissociation of primary ions of interest associated to primary mass peaks in order to produce neutral/charged fragment pairs and ionizing the dissociated neutral fragment to produce dissociated charged fragment pairs, and obtain charged fragment peak pairs from said parent primary ions, and to generate characteristic function values at maxima of said dissociated charged fragments mass peaks and ionized neutral fragment mass peaks and their corresponding mass-to- charge ratio values, - identifying, from amongst said potential pairs, using a proximity criterion, each real pair of charged fragment peaks corresponding to the parent primary ions by comparing which one of the potential pairs of values corresponds to a real dissociation mass peak pair in the dissociation mass spectrum produced with ionized neutral fragments, and

- generating dissociation mass spectra corresponding respectively to the primary mass peak of interest, comprising the real pairs of dissociation mass peaks identified.

BIBLIOGRAPHICAL REFERENCES

[1] J. D. Pinston et al, Rev. Sci.lnstrum., 57 (4), (1983), p.583.

C. G. Enke et al, US Patent 4,472,631 (1984). [2] S. Della-Negra and Y. Leybec, Anal. Chem., 57 (11 ), (1985), p. 2035.

K.G. Standing et al, Anal. Instrumen., 16, (1987), p; 173.

R.J. Conzemius US patent 4,894,536 (1990). [3] Alderdice et al, US patent 5,206,508 (1993).

[4] R.H. Bateman, J. M. Brown, D. J. Kenny, US patent 2005/0098721 A1 5).

[5] C. G. Enke, patent PCT/US 2004/008424.

[6] D.Scigocki, International patent application WO-A-2007/077245.

[7] R.W. Vachet and J. Wilson US patent 7,141 ,784 (2006).

Claims

1. Method of tandem mass spectrometry for use in a mass spectrometer having a known characteristic function of mass-to-charge ratios of ions to be analysed, characterized in that it comprises the following steps:

(a) providing a primary charged ions source to be analysed,

(b) generating a primary mass spectrum of the primary ions, without dissociation, wherein said spectrum contains primary ion peaks of occurrence,

(c) from said characteristic function values at maxima of at least some of said primary mass peaks, the corresponding mass-to-charge ratio values, and primary electric charges, determining correlation laws that all possible pairs of mass-to-charge values, corresponding to pairs of charged fragments resulting from the dissociation of parent primary ions of interest corresponding to said primary mass peaks, have to meet, (d) dissociating primary ions of interest associated to said primary mass peaks, in order to produce neutral/charged fragment pairs and obtain dissociated charged fragment mass peaks from said parent primary ions,

(e) generating said characteristic function values at maxima of said dissociated charged fragment mass peaks and said corresponding mass-to- charge ratio values,

(f) forming every potential pair of said characteristic function values and said corresponding mass-to-charge ratio values, with said possible pairs of each said correlation law corresponding to each primary mass peak of interest, (g) dissociating primary ions of interest associated to primary mass peaks, in order to produce neutral/charged fragment pairs, and ionizing said dissociated neutral fragment to produce dissociated charged fragment pairs, and obtain corresponding charged fragment peak pairs from said parent primary ions, (h) generating said characteristic function values at maxima of the dissociated charged fragments mass peaks and ionized neutral fragment mass peaks, and said corresponding mass-to-charge ratio values, and (i) identifying, from amongst said potential pairs, using a proximity criterion, each real pair of charged fragment peaks corresponding to said parent primary ions by comparing which one of said potential pairs of values corresponds to a real dissociation mass peak pair in said dissociation mass spectrum produced with ionized neutral fragments.

(j) generating dissociation mass spectra corresponding respectively to said parent primary ions of interest, comprising said real pairs of dissociation mass peaks identified.

2. Method according to claim 1 , wherein said step of determining said correlation laws is performed before said step of generating said characteristic function values for said dissociated fragments.

3. Method according to any one of claims 1 to 2, wherein said step of determining said correlation laws is performed subsequently to said step of generating said characteristic function values for said dissociated fragments.

4. Method according to any one of claims 1 to 3, wherein said step of forming said every potential pair is performed after said step of generating characteristic function values at maxima of said dissociated charged fragments mass peaks and ionized neutral fragment mass peaks and said corresponding mass-to-charge ratio values,

5. Method according to any one of claims 1 to 4, wherein said proximity criterion is adjustable.

6. Method according to any one of claims 1 to 5, wherein said proximity criterion is an accuracy of said values at maxima of said primary mass peaks of interest and/or an accuracy of said values at maxima of said dissociated charged fragment mass peaks.

7. Method according to any one of claims 1 to 6 further comprising a step of selecting a group of different primary ions of interest by primary mass selection.

8. Method according to claim 7, wherein said step of selection of said primary ions of interest is implemented before said step of generating said characteristic function values for said dissociated fragments.

9. Tandem mass spectrometer, comprising: (a) a source (1 ) of charged primary ions to be analysed,

(b) a device (4) for generating a primary mass spectrum of the primary ions, without dissociation, where said spectrum contains primary ion peaks of occurrence,

(c) a set of correlation laws determined from the characteristic function values at maxima of at least some of said primary mass peaks, their corresponding mass-to-charge ratio values, and their primary electric charge, and that all possible pairs of mass-to-charge ratio values, corresponding to pairs of charged fragment peaks resulting from the dissociation of parent primary ions of interest corresponding to said primary mass peaks, have to meet,

(d) a dissociation device (2) adapted to dissociate primary ions of interest associated to primary mass peaks, in order to produce neutral/charged fragment pairs, and obtain dissociated charged fragments from each of said parent primary ions, (e) a device for generating and storing characteristic function values at maxima of said dissociated charged fragment mass peaks and their corresponding mass-to-charge ratio values,

(f) an ionization device (3) to ionize neutral fragments produced by said dissociation in neutral/charged fragment pairs of primary ions of interest associated to primary mass peaks in the dissociation device (2), (g) a device for generating and storing characteristic function values at maxima of said dissociated charged fragment mass peaks and ionized neutral fragment mass peaks and their corresponding mass-to-charge values,

(h) a processing device for forming every potential pair of said mass-to- charge ratio values, for identifying, from amongst said potential pairs, using a proximity criterion, each real pair of charged fragment peaks corresponding to the parent primary ions by comparing which one of the potential pairs of values corresponds to a real dissociation mass peak pair in the dissociation mass spectrum produced with ionized neutral fragments, and for generating dissociation mass spectra corresponding respectively to the parent primary ions of interest, comprising the real pairs of dissociation mass peaks identified.

10. Spectrometer according to claim 9, further comprising a primary mass selection device for selecting a group of different primary ions of interest.

11. Spectrometer according to claim 10, wherein said primary mass selection device comprises an ion trap.

12. Spectrometer according to claim 11 , wherein said ion trap uses MSⁿ mode to select dissociated charged fragments to be injected into the dissociation device for dissociation into neutral/charged fragment pairs.

13. Spectrometer according to claim 10, wherein said primary mass selection device comprises a quadrupolar.

14. Spectrometer according to claim 10, wherein said primary mass selection device comprises a temporal gate.

15. Spectrometer according to any one of claims 9 to 14, wherein said dissociation device is a multipolar wave guide.

16. Spectrometer according to any one of claims 9 to 14, wherein said dissociation device is an ion trap.

17. Spectrometer according to any one of claims 9 to 16, wherein said ionization device is an ion source ionizing dissociated neutral fragments in gaseous phase.

18. Spectrometer according to any one of claims 9 to 17, wherein said dissociated neutral fragments are ionized inside the dissociation device.

19. Spectrometer according to any one of claims 9 to 17, wherein said dissociated neutral fragments are ionized outside said dissociation device.

20. Spectrometer according to any one of claims 9 to 19, wherein said dissociation device dissociating said primary ions is said ionization device ionizing said dissociated neutral fragments.

21. Spectrometer according to any one of claims 9 to 20, wherein said first dissociation mass spectrum is produced with mono charged primary ions, and said second dissociation mass spectrum is produced with doubly charged primary ions.

22. Computer program designed to be implemented in a mass spectrometry system comprising a mass spectrometer having a known characteristic function of mass-to-charge ratios of ions, including a set of instructions adapted to perform the following steps:

(a) controlling the system so that it generates, from a source of charged primary ions to be analysed, a primary mass spectrum of said primary ions, without dissociation, where this spectrum contains peaks of occurrences of primary ions,

(b) performing a data acquisition of this spectrum, including characteristic function values at maxima of at least some of the primary mass peaks, the corresponding mass-to-charge ratio values, and primary electric charges, associated to said peaks,

(c) from said data, determining correlation laws that all possible pair of said values, corresponding to pairs of charged fragment peaks resulting from the dissociation of parent primary ions of interest corresponding to said primary mass peaks have to meet,

(d) controlling the system so that it generates concurrent dissociation of primary ions of interest associated to primary mass peaks so as to obtain peaks of charged fragments from said parent primary ions, and to generate characteristic function values at maxima of said dissociated charged fragment peaks and corresponding mass-to-charge ratio values,

(e) forming every potential pair of said values,

(f) controlling the system so that it generates the concurrent dissociation of primary ions of interest associated to primary mass peaks in order to produce neutral/charged fragment pairs and ionizing the dissociated neutral fragment to produce dissociated charged fragment pairs, and obtain charged fragment peak pairs from said parent primary ions, and to generate characteristic function values at maxima of said dissociated charged fragments mass peaks and ionized neutral fragment mass peaks and their corresponding mass-to-charge ratio values,

(g) identifying, from amongst said potential pairs, using a proximity criterion, each real pair of charged fragment peaks corresponding to the parent primary ions by comparing which one of the potential pairs of values corresponds to a real dissociation mass peak pair in the dissociation mass spectrum produced with ionized neutral fragments.

(h) generating dissociation mass spectra corresponding respectively to the primary mass peak of interest, comprising the real pairs of dissociation mass peaks identified.