CN100390537C - Method for predicting ion molecular formula utilizing fragmental ion is otopic peak in tandem mass-spectrum - Google Patents

Abstract

The present invention discloses a method for predicting an ion molecular formula by using isotopic peaks of fragment ions in a tandem mass spectrum. In the method, the single isotopic quality of a fragment ion and the relative abundance of each isotopic peak relative to a single isotopic element are respectively obtained from the tandem mass spectrum and a universal molecular formula with undetermined atom number of each element; then the obtained quality and relative abundance are respectively matched to obtain the nonnegative integer solution of the atom number of each undetermined element in the universal molecular formula; finally the molecular formula of the fragment ion can be obtained. The method of the present invention uses the isotopic peak information of the fragment ion in the tandem mass spectrum to calculate the corresponding molecular formula of the fragment ion in a mode of the isotopic peak of the fragment ion in the tandem mass spectrum. The method of the present invention can provide the accurate molecular formula information of the fragment ion, identify a candidate sequence provided by a database searching method used for identifying a polypeptide sequence and provide references for a de novo method used for solving the polypeptide sequence to generate a high-reliability candidate sequence.

Description

Predict the method for ionic molecule formula with the isotopic peak of fragmention in the tandem mass spectrum

Technical field

The present invention relates to a kind of proteomic analysis methods, specifically, relate to the method for the molecular formula of the cracked back fragmention that produces of a kind of predicted polypeptide sequence.

Background technology

Utilizing peptide fingerprint mass spectrum and tandem mass spectrum technology and database search at present and directly separating preface (de novo) method and identify that the pre-service of mass spectrometric data and the aftertreatment of qualification result are extremely important in the research of peptide sequence and protein.

Certified polypeptide in mass spectrometer by cracked be fragmention, the quality and the abundance of these fragmentions are measured by mass spectrometer, form tandem mass spectrum.Each fragmention with and isotope ion all in tandem mass spectrum, form corresponding spectrum peak.The isotope summit of considering fragmention causes for the qualification process of peptide or protein to obscure, be about 0.34 such as of poor quality between some amino acid residue, 1 and 1.5da, and the monovalence of same fragmention, divalence, the peak-to-peak mass-to-charge ratio of the isotope of trivalent (m/z) difference is respectively 1,0.5 and 0.333, the m/z difference of these amino acid residue quality differences and isotopic peak is overlapping, and causing needs in qualification process to judge that a spectrum peak in the tandem mass spectrum is certain fragment ion peak or the isotopic peak of another fragmention; In addition, the overlapping phenomenon of the isotopic peak of a plurality of amino acid masses summation backs and certain fragmention can be more.Therefore, one of traditional data preprocessing tasks is to identify the isotopic peak of a fragmention and rejected.

Yet in fact, it is closely-related that the distribution pattern of the isotopic peak of the fragmention that shows in the mass spectrum and the atom of this fragmention are formed (being molecular formula).Therefore can utilize the isotopic peak of fragmention to predict the molecular formula of this fragmention with regard to needing a kind of method, like this, the molecular formula of the fragmention that dopes can provide more information more accurately for database search and the de novo method that peptide is identified on the one hand, on the other hand, for carrying out aftertreatment, qualification result provides more foundation.

Summary of the invention

The object of the present invention is to provide a kind of isotopic peak that utilizes the fragmention in the tandem mass spectrum to predict the method for the molecular formula of this fragmention.

To achieve these goals, the invention provides a kind of method of using the isotopic peak prediction ionic molecule formula of fragmention in the tandem mass spectrum, comprising:

Step 1): from tandem mass spectrum, obtain single isotope and at least one isotopic spectrum peak thereof of a fragmention, calculate the peak-to-peak relative abundance of at least one isotopic spectrum of the list isotopic spectrum peak and the described fragmention of the isotopic quality of list of described fragmention, described fragmention;

Step 2) provide a general molecular formula of fragmention, each atoms of elements number is undetermined in the described general molecular formula;

Step 3): obtain the isotopic quality of theoretic list of fragmention, single isotope and its at least one abundance ratio of isotopes of fragmention with described general molecular formula; The single isotope of the isotopic quality of described theoretic list, fragmention and the relative abundance of its at least one isotope ion are the function of atom number undetermined in the described general molecular formula;

Step 4): will do coupling from tandem mass spectrum quality and relative abundance in the quality that obtain in the step 3) and relative abundance and the step 1), separate with the nonnegative integer that obtains each atoms of elements number undetermined in the described general molecular formula, thereby obtain the molecular formula of described fragmention.

In technique scheme, at least one isotope of the fragmention described in step 1) and the step 3) comprises first isotope and second isotope of fragmention.

In technique scheme, the peak-to-peak relative abundance of at least one isotopic spectrum of the list isotopic spectrum peak of the isotopic quality of list of the described fragmention that obtains in the step 1), described fragmention and described fragmention is constituted the isotopic distribution vector of an experiment; The isotopic quality of theoretic list, single isotope of fragmention and the isotopic distribution vector that its at least one abundance ratio of isotopes constitutes a theory with the fragmention that obtains in the step 3); Described coupling in the step 4) is as the coupling mark with the Euclidean distance between the isotopic distribution vector of the isotopic distribution vector of described experiment and described theory.

In technique scheme, comprise that also the chemical rule constraint condition of using the molecular formula that makes acquisition to meet chemical sense retrains described coupling.

In technique scheme, the nonnegative integer of each atoms of elements number undetermined is separated and is comprised in the described general molecular formula by described coupling acquisition: the real solution that obtains each atoms of elements number undetermined in the described general molecular formula by described coupling; Searching for the nonnegative integer that obtains each atoms of elements number undetermined in the described general molecular formula in the field of described real solution separates.

In technique scheme, also comprise the nonnegative integer of each atoms of elements number undetermined in the described general molecular formula that obtains in the step 4) is separated the step of filtering.Described filtration comprises average isotopic distribution mode method, and this method is filtered described nonnegative integer with single isotope of the isotopic quality of theoretic list of fragmention, fragmention and the statistical relationship between its at least one abundance ratio of isotopes and separated.Described filtration comprises that the chemical rule constraint condition that meets chemical sense with the molecular formula that makes acquisition filters described nonnegative integer and separate.Described filtration comprises separating with the nonnegative integer of two fragmentions to be carried out cross validation and separates with the nonnegative integer of filtering described two fragmentions.

The invention has the advantages that:

1) this method is that the isotope of fragmention in the tandem mass spectrum is composed making full use of of peak information;

2) this method can be composed the pattern at peak by the isotope of tandem mass spectrum fragmention, calculates the molecular formula (order of accuarcy is relevant with mass spectral precision, and precision is high more, and the molecular formula that calculates is reliable more) of this fragmention correspondence rapidly and accurately;

3) this method can provide the information of molecular formula accurately of fragmention, and the candidate sequence that can provide the database search method of identifying peptide sequence is differentiated;

4) the ionic molecule formula that calculates of this method can instruct the de novo method of finding the solution peptide sequence to produce highly reliable candidate's sequence.

Embodiment

Below in conjunction with the drawings and specific embodiments the present invention is described in further detail.

Single isotope of a fragmention is designated as P, and first isotope of this fragmention is designated as P ₁, the second isotope fragmention is designated as P ₂, the rest may be inferred, and the N isotope ion is designated as P _NHere, fragmention list isotope P is meant that the various components at this ion are single isotope (being that proton number is identical with neutron number).And the isotope of fragmention be meant with single isotope fragmention have identical molecular formula, but have than single isotope and more to many ion of extra neutrons, for example first isotope P of fragmention ₁Single isotope P than fragmention has an extra neutron more, the second isotope P ₂Have two extra neutrons than single isotope P, the rest may be inferred more.In the present invention, the isotope of fragmention is the ion that has extra neutron on the whole than single isotope of fragmention.

Peptide sequence enters mass spectrometer and is ionized, and in mass spectrometer, (Collision-InducedDissociation CID) is cracked into a plurality of fragmentions under the effect to have the separation that the peptide ion (these peptide ions also have identical amino acid sequence usually) of specific mass-to-charge ratio (m/z) colliding-inducing.Thereby tested the measuring of the m/z of these fragmentions forms tandem mass spectrum, and in a tandem mass spectrum, its horizontal ordinate is represented the mass-to-charge ratio (m/z) of fragmention, and its ordinate is the abundance of detected fragmention.

In tandem mass spectrum, the single isotope P that picks out a fragmention with and isotope P ₁～P _NIn the spectrum peak of at least one correspondence, target of the present invention then is to predict the molecular formula of single isotope P correspondence of fragmention by the distribution situation of these isotopic peaks.In one embodiment of the invention, the single isotope P that from tandem mass spectrum, only picks out this fragmention with and the first isotope P ₁With the second isotope P ₂Those skilled in the art will readily appreciate that in the description from behind, in other embodiments of the invention,, also can only pick out an isotopic spectrum peak of fragmention for the isotope fragmention---the first isotope fragmention P for example ₁, perhaps also can pick out more isotopic spectrum peak, choosing of different number isotope fragmentions can realize method of the present invention, complexity and the precision calculated in the time of still can having influence on the invention process.

From tandem mass spectrum, can also obtain the mass of ion M of single isotope fragmention P _e, this is that those skilled in the art is known.

In following calculating, at first define the isotopic distribution vector eIPV=(M of an experiment for convenience _e, I ₁, I ₂), wherein, M _eBe the mass of ion of single isotope P of the fragmention that from tandem mass spectrum, obtains, I ₁And I ₂The first isotope P of the corresponding fragmention of difference ₁With the second isotope P ₂The spectrum peak with respect to the relative abundance at the spectrum peak of single isotope P, these data all can obtain from tandem mass spectrum.

Then, define isotopic distribution vector tIPV=(M, the T of a theory again ₁, T ₂), this theory isotopic distribution vector tIPV can obtain from the general molecular formula of fragmention.If the general molecular formula of fragmention is C _N1H _N2N _N3O _N4S _N5, wherein n1～the n5 of each atom composition number of expression is a undetermined parameter in this molecular formula.Like this, in theoretical isotopic distribution vector tIPV, M is the quality from the fragmention of general molecular formula acquisition, T ₁And T ₂Be respectively the first isotope fragmention that obtains from the general molecular formula and the second isotope fragmention about single isotope fragmention relative abundance.Theoretical isotopic distribution vector tIPV can specifically can obtain by formula:

M＝V×X (1)

T ₁＝n ₁q _C+n ₂q _H+n ₃q _N+n ₄q _O1+n ₅q _S1 (2)

$T_2=n_4{q}_{O2}+n_5{q}_{S2}+\frac{1}{2}{T}_1^2-\frac{1}{2}(n_1{q}_C^2+n_2{q}_H^2+n_3{q}_N^2+n_4{q}_{O1}^2+n_5{q}_{S1}^2)---\left(3\right)$

V=[12 wherein, 1,14,16,32], the numeral among the V is each atoms of elements amount, X=[n1, n2, n3, n4, n5] ^Tq _C, q _HAnd q _NIt is respectively occurring in nature ¹³C with respect to ¹²C, D with respect to H, ¹⁴N with respect to ¹⁵The relative abundance of N, q ₀₁And q ₀₂It then is respectively occurring in nature ¹⁷O with respect to ¹⁶O, ¹⁸O with respect to ¹⁶The relative abundance of O, q _S1And q _S2It is occurring in nature ³³S with respect to ³²S, ³⁴S with respect to ³²The relative abundance of S, these relative abundances are known numeric value.

As seen, for the isotopic distribution of theory vectorial tIPV=(M, T ₁, T ₂), M wherein, T ₁And T ₂Be X=[n1, n2, n3, n4, n5] function.

In the present invention, with the isotopic distribution of theory vectorial tIPV=(M, T ₁, T ₂) and experiment isotopic distribution vector eIPV=(M _e, T ₁, T ₂) do coupling, so that obtain the molecular formula that the isotopic distribution vector with experiment mates most, also be the atom composition of vector X=[n1 in the general molecular formula, n2, n3, n4, n5] and a nonnegative integer separate.

In one embodiment of the invention, with the coupling mark of the Euclidean distance E between the isotopic distribution vector eIPV of theoretical isotopic distribution vector tIPV and experiment as tIPV and eIPV:

$E=\sqrt{{\mathrm{\δ}}_m^2+{\mathrm{\δ}}_1^2+{\mathrm{\δ}}_2^2}=\sqrt{{\left({M-M}_e\right)}^2+{(T_1-I_1)}^2+{(T_2-I_2)}^2}---\left(4\right)$

With formula (1)～(3) substitution (4), obtain

δ _m＝n ₁*12+n ₂*1+n ₃*14+n ₄*16+n ₅*32-M _e1， (5)

δ ₁＝n ₁*q _C+n ₂*q _H+n ₃*q _N+n ₄*q _O1+n ₅*q _S1-I ₁， (6)

${\mathrm{\δ}}_2=n_4*q_{O2}+n_5*q_{S2}-\frac{1}{2}(n_1*q_C^2+n_2*q_H^2+n_3*q_N^2+n_4*q_{O1}^2+n_5*q_{S1}^2)$

$+(n_1*q_C+n_2*q_H+n_3*q_N+n_4*q_O+n_5*q_S)*I_1-\frac{1}{2}{I_1}^2-I_2+\frac{1}{2}{{\mathrm{\δ}}_1}^2.---\left(7\right)$

Ignore in the formula (7)

, [δ is then arranged _mδ ₁δ ₂]=AX+B obtains

$Q\left(X\right)=E^2=\left[\begin{array}{ccc}{\mathrm{\δ}}_m& {\mathrm{\δ}}_1& {\mathrm{\δ}}_2\end{array}\right]\left[\begin{array}{c}{\mathrm{\δ}}_m\\ {\mathrm{\δ}}_1\\ {\mathrm{\δ}}_2\end{array}\right]=X^T{A}^T\mathrm{AX}+2B^T\mathrm{AX}+B^{T}B,---\left(8\right)$

Then have:

$E=\sqrt{Q\left(X\right)}=\sqrt{X^T{A}^T\mathrm{AX}+{2B}^T\mathrm{AX}+B^{T}B}---\left(9\right)$

Here in formula (9), X=[n1, n2, n3, n4, n5] ^TBe the atom composition of vector of fragmention undetermined, A and B are the constant matricess that is made of known quantity, and known quantity comprises the M that obtains from tandem mass spectrum here _e, I ₁And I ₂, and V=[12,1,14,16,32] and formula (2) and (3) in each abundance ratio of isotopes.

Described Euclidean distance E minimizes with formula (9), separates for one that can obtain X.Usually,, preferably also to some chemical rule constraint conditions be set to formula (9) for the molecular formula that makes acquisition meets chemical sense, for example:

● the fragmention quality of the molecular formula correspondence that obtains with X must be at scope [M _e-δ, M _e+ δ] in, δ is the maximum magnitude of m/z error, δ can be determined by mass spectrometric measuring accuracy.Just to satisfy | VX-M _e|≤δ.

● for certain element in the fragmention molecular formula, the m/z that uses ion is divided by the minimum isotopic mass number of this element quality, and the integral part of getting the gained result is exactly the upper limit of this element number.For example the atomic weight of element O is 16, if the mass-to-charge ratio of ion is m/z, then in the fragmention number of O element on be limited to

Promptly in X

Similarly, also can obtain similar constraint condition for other element in the fragmention.

● in fragmention, the number of C necessarily less than the number of H (promptly in X the number of n1＜n3), O and N necessarily less than the number of C (promptly at n4＜n1 and n3＜n1) or the like.These constraint conditions lie in the composition mode of the molecular composition mode of amino acid residue and leading ion type, and those skilled in the art is easy to sum up out according to their characteristics.

● in the ion with an electronics, the number sum of H and N is an odd number.Reason is if ion has an electric charge, so just has unsatisfied chemical bond to exist, and H and N have the odd number quantivalency and C, O, S have the even number quantivalency.

Should be appreciated that purpose that those skilled in the art also can meet chemical sense from the molecular formula that makes fragmention constructs other constraint condition.

In above-mentioned constraint condition or other constraint condition part or all can be expressed as a linear inequality DX≤G.Like this,, can solve this minimization problem of Euclidean distance E by the QUADRATIC PROGRAMMING METHOD FOR of standard in conjunction with formula (9), as shown in Equation (10):

The optimum solution of the X that obtains with QUADRATIC PROGRAMMING METHOD FOR from formula (10) is the X that separates in the real number field _R, in order to seek real molecular formula, can be with X _RBe used as starting point, then the nonnegative integer candidate solution of Local Search X in its neighborhood.Exactly, be exactly to each and X _RExist one to give a mark, use formula (9) to estimate the matching degree of these nonnegative integer candidate solutions in other words apart from the nonnegative integer candidate's candidate solution molecular formula in the scope of d.The value of d adapts with mass range of ions.Avoided enumerating all possible molecular formula like this, can in big mass range, predict the ionic molecule formula and guarantee higher reliability and operational efficiency.

Through Local Search, still can produce the candidate molecules formula of some, comprising some illegal and with experiment tandem mass spectrum unmatched molecular formula (can be called invalid with impossible molecular formula), in order to improve the degree of accuracy of prediction, preferably need eliminating as much as possible they.Can utilize one or more methods that comprise in average isotopic distribution pattern, chemical rule constraint and the cross validation to filter the candidate molecules formula in the present invention.These methods specifically describe as follows:

A. average isotopic distribution pattern

Said average isotopic distribution pattern is theoretical isotopic distribution vector tIPV=(M, T ₁, T ₂) in ingredient M, T ₁And T ₂Between statistical relationship.In order to seek the theoretical average isotopic distribution pattern of fragmention, the isotopic of theoretical fragmention of polypeptide that the inventor has calculated the trypsin hydrolysis correspondence of all proteins in the existing Protein Data Bank is evenly distributed and standard deviation, disclosed ingredient M, the T of tIPV ₁And T ₂Between relation.Specifically, the inventor at first carries out the protein among the SWISS-PROT theoretical enzyme and cuts and calculate polypeptide; Select quality in that (polypeptide in the 60u～3000u), this scope correspondence Q-TOF MS/MS and tested mass spectral critical field then.In addition, it should be noted that the isotope of S ^{+ 2}Very high (probability of appearance is 0.04210 to S, approximately is at the content of occurring in nature ¹⁸And in most cases can to comprise the polypeptide of the S more than five very rare 20 times of O).Therefore, we can be divided into six classes: S with above-mentioned molecular formula ⁰, S ¹, S ², S ³, S ⁴And S ⁵⁺, the number of corresponding contained S is 0,1,2,3 respectively, the peptide section more than 4 and 5 and 5.The inventor by these six classifications to adding up.Statistical result showed T ₁Linear with mass M, T ₂Then be the secondary relation with M, and T ²Along with T ₁Increase and increase and and T ₁Become quadratic function relation.

Like this, pass through T ₁, T ₂Can filter the candidate molecules formula with the above-mentioned distribution relation of M, to get rid of those invalid and/or impossible molecular formula.

B. chemical rule constraint

The chemical rule constraint here is similar with the constraint condition DX≤G in the formula (10), and its difference is: in formula (10), constraint condition DX≤G is used for constraint formulations

$E=\sqrt{Q\left(X\right)}=\sqrt{X^T{A}^T\mathrm{AX}+{2B}^T\mathrm{AX}+B^{T}B}$

So that obtain the X that separates in the real number field of X under this constraint condition _RAnd here, these constraint conditions are used to constrain in X _RThe field in the nonnegative integer that obtains of search separate the candidate molecules formula so that these candidate molecules formulas are filtered.

C. cross validation

Especially, the fragmention of the b series of a peptide section all is a homology, comprises b-, a-, and b*-, a*-, b °-, a ° of type ion, they share an identical original amino acid, can infer that thus their isotopic distribution pattern is very similar.Y series ion also is like this.If the M of certain two fragmention in the mass spectrum _eDiffer 28,17 or 18, and the I of these two fragmentions ₁And I ₂Very approaching, just can think the eIPV homology of two fragmention correspondences.Then, we just can use the eIPV of homology to carry out cross validation to predicting the outcome.For example, in the candidate molecules formula tabulation in a fragmention C is arranged for two fragmentions of homology _A1H _A2N _A3O _A4S _A5If, C _A1-1H _A2N _A3O _A4-1S _A5Do not appear in the candidate molecules formula tabulation of another fragmention, so just can think candidate molecules formula C _A1H _A2N _A3O _A4S _A5Be the result of mating at random and it is got rid of.

Claims (8)

1. method with the isotopic peak of fragmention in tandem mass spectrum prediction ionic molecule formula comprises:

Step 1): from tandem mass spectrum, obtain single isotope and at least one isotopic spectrum peak thereof of a fragmention, calculate the isotopic distribution vector of the peak-to-peak relative abundance of at least one isotopic spectrum of the list isotopic spectrum peak of the isotopic quality of list of described fragmention, described fragmention and described fragmention as experiment;

Step 2): a general molecular formula of fragmention is provided, and each atoms of elements number is undetermined in the described general molecular formula;

Step 3): obtain single isotope of the isotopic quality of theoretic list, fragmention of fragmention and its at least one abundance ratio of isotopes isotopic distribution vector with described general molecular formula as theory; Single isotope of the isotopic quality of described theoretic list, fragmention and its at least one abundance ratio of isotopes are the function of atom number undetermined in the described general molecular formula;

Step 4): the Euclidean distance of the isotopic distribution vector of the described experiment that obtains from tandem mass spectrum in the isotopic distribution vector of the described theory that obtains in the step 3) and the step 1) is minimized, obtain the molecular formula of the real number field of described fragmention;

Step 5): Local Search obtains the candidate molecules formula in nonnegative integer territory in the neighborhood of the molecular formula of described real number field, and obtains the predictive molecule formula of described fragmention from described candidate molecules formula;

Wherein, at least one isotope of the fragmention described in described step 1) and the step 3) comprises first isotope and second isotope of fragmention; And

The isotopic distribution vector eIPV=(M of described experiment _e, I ₁, I ₂), wherein, M _eBe the isotopic mass of ion of list of the fragmention that from tandem mass spectrum, obtains, I ₁And I ₂First isotope of corresponding fragmention and the second isotopic spectrum peak are with respect to the relative abundance at single isotopic spectrum peak respectively;

Isotopic distribution vector tIPV=(M, the T of described theory ₁, T ₂) obtain in the following way:

M＝V×X (1)

T ₁＝n ₁q _C+n ₂q _H+n ₃q _N+n ₄q _O1+n ₅q _S1 (2)

$T_2=n_4{q}_{O2}+n_5{q}_{S2}+\frac{1}{2}{T_1}^2-\frac{1}{2}(n_1{q}_C^2+n_2{q}_H^2+n_3{q}_N^2+n_4{q}_{O1}^2+n_5{q}_{S1}^2)---\left(3\right)$

Wherein, described general molecular formula is C _N1H _N2N _N3O _N4S _N5, n1～n5 is that each atom is formed number in the described general molecular formula of described fragmention, M be the quality from the fragmention of described general molecular formula acquisition, T ₁And T ₂Be respectively the first isotope fragmention that obtains from described general molecular formula and the second isotope fragmention relative abundance about single isotope fragmention, the row that V is made up of each atoms of elements amount in the described general molecular formula is vectorial, X=[n1, n2, n3, n4, n5] ^Tq _C, q _HAnd q _NIt is respectively occurring in nature ¹³C with respect to ¹²C, D with respect to H, ¹⁴N with respect to ¹⁵The relative abundance of N, q ₀₁And q ₀₂It then is respectively occurring in nature ¹⁷O with respect to ¹⁸O, ¹⁸O with respect to ¹⁶The relative abundance of O, q _S1And q _S2It is occurring in nature ³³S with respect to ³²S, ³⁴S with respect to ³²The relative abundance of S.

2. the method for predicting the ionic molecule formula with the isotopic peak of fragmention in the tandem mass spectrum according to claim 1, it is characterized in that, with the coupling mark of the Euclidean distance E between the isotopic distribution vector eIPV of theoretical isotopic distribution vector tIPV and experiment as tIPV and eIPV:

$E=\sqrt{{\mathrm{\δ}}_m^2+{\mathrm{\δ}}_1^2+{\mathrm{\δ}}_2^2}=\sqrt{{(M-M_e)}^2+{(T_1-I_1)}^2+{(T_2-I_2)}^2}---\left(4\right)$

3. the method for predicting the ionic molecule formula with the isotopic peak of fragmention in the tandem mass spectrum according to claim 1 and 2, it is characterized in that, in described step 4), also comprise with making the molecular formula of acquisition meet the chemical rule of chemical sense as the minimized constraint condition of described Euclidean distance.

4. the method for predicting the ionic molecule formula with the isotopic peak of fragmention in the tandem mass spectrum according to claim 1, it is characterized in that Local Search described in the step 5) is meant the candidate molecules formula of searching for the nonnegative integer territory in there is the scope of a distance in the molecular formula with described real number field.

5. the method for using the isotopic peak prediction ionic molecule formula of fragmention in the tandem mass spectrum according to claim 1 is characterized in that, also comprises the step that the candidate molecules formula in described nonnegative integer territory is filtered in step 5).

6. the method for predicting the ionic molecule formula with the isotopic peak of fragmention in the tandem mass spectrum according to claim 5, it is characterized in that, described filtration step comprises average isotopic distribution mode method, and this method is with the isotopic quality of theoretic list of fragmention, single isotope of fragmention and the candidate molecules formula that the statistical relationship between its at least one abundance ratio of isotopes filters described nonnegative integer territory.

7. the method for predicting the ionic molecule formula with the isotopic peak of fragmention in the tandem mass spectrum according to claim 5, it is characterized in that described filtration step comprises that the chemical rule constraint condition that meets chemical sense with the molecular formula that makes acquisition filters the candidate molecules formula in described nonnegative integer territory.

8. the method for predicting the ionic molecule formula with the isotopic peak of fragmention in the tandem mass spectrum according to claim 5, it is characterized in that described filtration step comprises with the candidate molecules formula in the nonnegative integer territory of two fragmentions and carries out the candidate molecules formula of cross validation with the nonnegative integer territory of filtering described two fragmentions.