CN100390537C - A Method for Predicting the Molecular Formula of Ions Using Isotope Peaks of Fragment Ions in Tandem Mass Spectrometry - Google Patents

Abstract

The invention discloses a method for predicting the molecular formula of an ion by using the isotope peak of the fragment ion in the tandem mass spectrum. The method obtains the monoisotope mass of the fragment ion and the mass of each The relative abundance of the isotopic peak relative to the monoisotope; the mass and relative abundance obtained respectively are matched to obtain the non-negative integer solution of the number of atoms of each element to be determined in the general molecular formula, and the molecular formula of the fragment ion is obtained. The method of the present invention utilizes the isotope spectrum peak information of the fragment ion in the tandem mass spectrum, and calculates the molecular formula corresponding to the fragment ion through the mode of the isotope spectrum peak of the fragment ion in the tandem mass spectrum. The method of the present invention can provide accurate molecular formula information of fragment ions, can identify candidate sequences provided by the database search method for identifying polypeptide sequences, and provide a basis for generating highly reliable candidate sequences by de novo method for solving polypeptide sequences.

Description

A Method for Predicting the Molecular Formula of Ions Using Isotope Peaks of Fragment Ions in Tandem Mass Spectrometry

technical field

The invention relates to a proteome analysis method, in particular to a method for predicting the molecular formula of fragment ions generated after peptide sequence fragmentation.

Background technique

In the current research of identifying peptide sequences and proteins using peptide fingerprint mass spectrometry and tandem mass spectrometry technology, database search and direct de novo (de novo) methods, the preprocessing of mass spectrometry data and the postprocessing of identification results are very important.

The identified peptides are fragmented into fragment ions in the mass spectrometer, and the mass and abundance of these fragment ions are measured by the mass spectrometer to form a tandem mass spectrum. Each fragment ion and its isotopic ion form a corresponding peak in the tandem mass spectrometer. Considering that the isotope peaks of fragment ions confuse the identification process of peptides or proteins, for example, the mass difference between some amino acid residues is about 0.34, 1 and 1.5da, while the monovalent, divalent, trivalent The mass-to-charge ratio (m/z) differences between the isotopic peaks of the valence are 1, 0.5, and 0.333, respectively, and these amino acid residue mass differences overlap with the m/z differences of the isotopic peaks, resulting in the need to judge the tandem mass spectrum in the identification process Is one of the peaks in a fragment ion peak or the isotope peak of another fragment ion; in addition, the overlap of the isotope peak of a fragment ion after summing the masses of multiple amino acids will be more. Therefore, one of the traditional data preprocessing tasks is to identify isotopic peaks of a fragment ion and remove them.

However, in fact, the distribution pattern of the isotopic peaks of the fragment ions shown in the mass spectrum is closely related to the atomic composition (ie molecular formula) of the fragment ions. Therefore, there is a need for a method that can use the isotope peaks of fragment ions to predict the molecular formula of the fragment ion. In this way, the predicted molecular formula of the fragment ion can provide more and more accurate information for the database search and de novo method of peptide identification on the one hand. , on the other hand, provide more basis for the post-processing of identification results.

Contents of the invention

The purpose of the present invention is to provide a method for predicting the molecular formula of the fragment ion by using the isotope peak of the fragment ion in the tandem mass spectrum.

In order to achieve the above object, the present invention provides a method for predicting ion molecular formula with the isotope peak of fragment ion in tandem mass spectrometry, comprising:

Step 1): Obtain a monoisotopic peak of a fragment ion and at least one isotopic peak of the fragment ion from the tandem mass spectrometer, calculate the mass of the monoisotope of the fragment ion, the monoisotopic spectral peak of the fragment ion, and the fragment ion The relative abundance between the spectral peaks of at least one isotope of

Step 2) providing a general molecular formula of fragment ions, the number of atoms of each element in the general molecular formula is to be determined;

Step 3): use the general molecular formula to obtain the theoretical monoisotopic mass of the fragment ion, the monoisotope of the fragment ion and the relative abundance of at least one isotope thereof; The relative abundance of an isotope and at least one isotopic ion thereof is a function of the number of atoms to be determined in said general formula;

Step 4): Match the mass and relative abundance obtained in step 3) with the mass and relative abundance of the tandem mass spectrum in step 1), so as to obtain the non-negative number of atoms of each element to be determined in the general molecular formula Integer solution to obtain the molecular formula of the fragment ion.

In the above technical solution, at least one isotope of the fragment ion described in step 1) and step 3) includes the first isotope and the second isotope of the fragment ion.

In the above technical scheme, the relative abundance between the mass of the monoisotope of the fragment ion obtained in step 1), the spectrum peak of the monoisotope of the fragment ion and the spectrum peak of at least one isotope of the fragment ion Constitute an experimental isotope distribution vector; The mass of the theoretical monoisotope of the fragment ion obtained in step 3), the monoisotope of the fragment ion and the relative abundance of at least one isotope thereof constitute a theoretical isotope distribution vector; Step 4 The matching in ) uses the Euclidean distance between the experimental isotope distribution vector and the theoretical isotope distribution vector as the matching score.

In the above technical solution, it is also included to constrain the matching with chemical rules and constraints that make the obtained molecular formula conform to the chemical meaning.

In the above technical solution, the non-negative integer solution of the number of atoms of each element to be determined in the general molecular formula obtained through the matching includes: obtaining the number of atoms of each element to be determined in the general molecular formula through the matching The real number solution; search in the field of the real number solution to obtain the non-negative integer solution of the number of atoms of each element to be determined in the general molecular formula.

In the above technical solution, it also includes the step of filtering the non-negative integer solutions of the atomic number of each element to be determined in the general molecular formula obtained in step 4). The filtering includes an average isotope distribution pattern method that filters the non-negative integer solution using a theoretical monoisotopic mass of the fragment ion, a statistical relationship between the monoisotope of the fragment ion and the relative abundance of at least one isotope thereof . The filtering includes filtering the non-negative integer solutions with chemical rule constraints that make the obtained molecular formula conform to chemical meaning. The filtering includes cross-validating with the non-negative integer solutions of the two fragment ions to filter the non-negative integer solutions of the two fragment ions.

The advantages of the present invention are:

1) This method is the full utilization of the isotopic spectrum peak information of fragment ions in the tandem mass spectrometry;

2) This method can quickly and accurately calculate the molecular formula corresponding to the fragment ion through the isotope spectrum peak mode of the tandem mass spectrometry fragment ion (the degree of accuracy is related to the accuracy of the mass spectrum, the higher the accuracy, the more reliable the calculated molecular formula);

3) The method can provide accurate molecular formula information of fragment ions, and can identify candidate sequences provided by the database search method for identifying polypeptide sequences;

4) The ion molecular formula calculated by this method can guide the de novo method for solving the polypeptide sequence to generate highly reliable candidate sequences.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

The monoisotope of a fragment ion is marked as P, the first isotope of this fragment ion is marked as P ₁ , the second isotope fragment ion is marked as P ₂ , and so on, and the Nth isotope ion is marked as _PN . Here, the monoisotopic P of the fragment ion means that the various constituent elements of the ion are monoisotopic (that is, the number of protons and neutrons is the same). The isotope of the fragment ion refers to the ion that has the same molecular formula as the monoisotopic fragment ion, but has more extra neutrons than the monoisotope. For example, the first isotope _P of the fragment ion has more than the monoisotope P of the fragment ion. One extra neutron, the second isotope _P2 has two more neutrons than the monoisotope P, and so on. In the present invention, an isotope of a fragment ion is an ion that, as a whole, has an extra neutron than a monoisotope of the fragment ion.

The peptide sequence enters the mass spectrometer and is ionized, and in the mass spectrometer, peptide ions with a specific mass-to-charge ratio (m/z) (these peptide ions usually also have the same amino acid sequence) undergo collision-induced separation (Collision-InducedDissociation, CID) under the action of fragmentation into multiple fragment ions. The m/z of these fragment ions are detected and measured to form a tandem mass spectrum. In a tandem mass spectrometer, the abscissa represents the mass-to-charge ratio (m/z) of the fragment ions, and the ordinate represents the abundance of the detected fragment ions .

In the tandem mass spectrometry, the monoisotope P of a fragment ion and at least one corresponding spectral peak among its isotopes P ₁ to _PN are selected. The object of the present invention is to predict the monoisotope of the fragment ion through the distribution of these isotope peaks. The molecular formula corresponding to P. In one embodiment of the present invention, only the monoisotope P of the fragment ion and its first isotope P ₁ and second isotope P ₂ are selected from the tandem mass spectrum. Those skilled in the art can easily understand from the following description that in other embodiments of the present invention, for isotopic fragment ions, it is also possible to select only the spectral peak of one isotope of the fragment ion—for example, the first isotopic fragment ion P ₁ , or more isotopic spectral peaks can be selected, and the selection of different numbers of isotopic fragment ions can realize the method of the present invention, but it will affect the complexity and accuracy of calculation when the present invention is implemented.

The ion mass _Me of the monoisotopic fragment ion P can also be obtained from the tandem mass spectrometry, which is well known to those skilled in the art.

In order to facilitate the following calculations, first define an experimental isotope distribution vector eIPV=(M _e , I ₁ , I ₂ ), where Me _is the ion mass of the monoisotope P of the fragment ion obtained from the tandem mass spectrometry, I ₁ and _I2 correspond to the relative abundance of the peaks of the first isotope _P1 and the second isotope _P2 of the fragment ions, respectively, relative to the peaks of the monoisotope P, both of which can be obtained from tandem mass spectrometry.

Then, define a theoretical isotope distribution vector tIPV=(M, T ₁ , T ₂ ), which can be obtained from the general molecular formula of fragment ions. The general molecular formula of fragment ions is assumed to be C _n1 H _n2 N _n3 O _n4 S _n5 , wherein n1 to n5 representing the number of each atom in the molecular formula are undetermined parameters. Thus, in the theoretical isotope distribution vector tIPV, M is the mass of the fragment ion obtained from the general formula, and _T1 and _T2 are respectively the first isotopic fragment ion and the second isotopic fragment ion obtained from the general molecular formula with respect to the monoisotopic fragment ion relative abundance. The theoretical isotope distribution vector tIPV can be specifically obtained by the formula:

M=V×X (1)

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

${\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; o</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 5</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; S</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; C</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; h</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; N</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; o</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 5</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; S</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 3</span>}<span\; class="notranslate">\; )</span>$

Wherein V=[12,1,14,16,32], the number in V is the atomic weight of each element, X=[n1, n2, n3, n4, n5] ^T ; q _C , q _H and q _N are respectively The relative abundance of ¹³ C relative to ¹² C, D relative to H, ¹⁴ N relative to ¹⁵ N in nature, q ₀₁ and q ₀₂ are the relative abundance of ¹⁷ O relative to ¹⁶ O, ¹⁸ O relative to ¹⁶ O in nature, respectively Abundance, q _s1 and q _s2 are the relative abundances of ³³ S relative to ³² S and ³⁴ S relative to ³² S in nature, and these relative abundances are all known values.

It can be seen that for the theoretical isotope distribution vector tIPV=(M, T ₁ , T ₂ ), M, T ₁ and T ₂ are all functions of X=[n1, n2, n3, n4, n5].

In the present invention, the theoretical isotope distribution vector tIPV=(M, T ₁ , T ₂ ) is matched with the experimental isotope distribution vector eIPV=(M _e , T ₁ , T ₂ ), so as to obtain the experimental isotope distribution vector The most matching molecular formula, that is, a non-negative integer solution of the atom composition vector X=[n1, n2, n3, n4, n5] in the general molecular formula.

In one embodiment of the present invention, the Euclidean distance E between the theoretical isotope distribution vector tIPV and the experimental isotope distribution vector eIPV is used as the matching score of tIPV and eIPV:

$\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; =</span>\sqrt{{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}<span\; class="notranslate">\; =</span>\sqrt{{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; m</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; e</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 4</span>}<span\; class="notranslate">\; )</span>$

Substituting formulas (1)~(3) into (4), we get

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{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; o</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 5</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; S</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; C</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; h</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; N</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; o</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 5</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; S</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; )</span>$

$<span\; class="notranslate">\; +</span><span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; C</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; h</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; N</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; o</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 5</span>}}<span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; S</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; *</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}{{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; .</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 7</span>}<span\; class="notranslate">\; )</span>$

项，则有[δ_m δ₁ δ₂]＝AX+B，得到Ignoring the formula (7)

item, then [δ _m δ ₁ δ ₂ ]=AX+B, we get

$\mathrm{<span\; class="notranslate">\; Q</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; E.</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>\left[\begin{array}{ccc}{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}& {\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}& {\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}\end{array}\right]\left[\begin{array}{c}{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}\\ {\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}\\ {\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}\end{array}\right]<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; x</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}{\mathrm{<span\; class="notranslate">\; A</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; AX</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; 2</span>}{\mathrm{<span\; class="notranslate">\; B</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; AX</span>}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; B</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; B</span>}<span\; class="notranslate">\; ,</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 8</span>}<span\; class="notranslate">\; )</span>$

Then there are:

$\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; =</span>\sqrt{\mathrm{<span\; class="notranslate">\; Q</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; )</span>}<span\; class="notranslate">\; =</span>\sqrt{{\mathrm{<span\; class="notranslate">\; x</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}{\mathrm{<span\; class="notranslate">\; A</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; AX</span>}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; 2</span>}\mathrm{<span\; class="notranslate">\; B</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; AX</span>}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; B</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; B</span>}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 9</span>}<span\; class="notranslate">\; )</span>$

Here in formula (9), X=[n1, n2, n3, n4, n5] ^T is the atomic composition vector of undetermined fragment ion, and A and B are the constant matrix that known quantity is made of, and here known quantity comprises _Me , I ₁ and I ₂ obtained from tandem mass spectrometry, and V=[12, 1, 14, 16, 32] and the relative abundance of each isotope in formulas (2) and (3).

A solution of X can be obtained by minimizing the Euclidean distance E described by formula (9). Usually, in order to make the obtained molecular formula conform to the chemical meaning, it is preferable to set some chemical rule constraints on the formula (9), for example:

●The mass of the fragment ion corresponding to the molecular formula obtained by X must be within the range [M _e -δ, M _e +δ], δ is the maximum range of m/z error, and δ can be determined by the measurement accuracy of the mass spectrometer. That is to satisfy |VX-M _e |≤δ.

即在X中

类似地，对于碎片离子中其它元素也可获得相似的约束条件。●For a certain element in the fragment ion molecular formula, divide the m/z of the ion by the mass number of the isotope with the lowest mass of this element, and take the integer part of the result as the upper limit of the number of this element. For example, the atomic weight of element O is 16, if the mass-to-charge ratio of ions is m/z, the upper limit of the number of O elements in fragment ions is

i.e. in X

Similarly, similar constraints can be obtained for other elements in the fragment ions.

●In fragment ions, the number of C must be less than the number of H (that is, n1<n3 in X), and the number of O and N must be less than the number of C (that is, in n4<n1 and n3<n1 )etc. These constraints are implicit in the molecular composition of amino acid residues and the composition of main ion types, and those skilled in the art can easily summarize them based on their characteristics.

●In an ion with one electron, the sum of the numbers of H and N is an odd number. The reason is that if the ion has a charge, then there is an unsaturated chemical bond, and both H and N have odd valences and C, O, and S have even valences.

It should be understood that those skilled in the art can also construct other constraint conditions for the purpose of making the molecular formula of the fragment ion conform to the chemical meaning.

Part or all of the above constraints or other constraints can be expressed as a linear inequality DX≤G. In this way, combined with formula (9), the standard quadratic programming method can be used to solve the minimization problem of Euclidean distance E, as shown in formula (10):

The optimal solution of X obtained from formula (10) by quadratic programming method is a solution X _R in the real number field. In order to find the real molecular formula, X _R can be used as the starting point, and then search locally in its neighborhood Non-negative integer candidate solutions for X. To be precise, it is to score each non-negative integer candidate solution formula within a distance d from X _R , or use formula (9) to evaluate the matching degree of these non-negative integer candidate solutions. The value of d is adapted to the ion mass range. This avoids enumerating all possible molecular formulas, enables prediction of ion molecular formulas in a large mass range and ensures high reliability and operating efficiency.

After a local search, a certain number of candidate molecular formulas will still be produced, including some illegal and mismatched molecular formulas with the experimental tandem mass spectrum (respectively called invalid and impossible molecular formulas), in order to improve the accuracy of prediction, preferably They need to be excluded as much as possible. In the present invention, one or more methods including average isotope distribution pattern, chemical rule constraint and cross-validation can be used to filter candidate molecular formulas. These methods are described in detail as follows:

A. Average isotope distribution pattern

The mean isotope distribution pattern is the statistical relationship between the components M, T1 and _T2 in the theoretical isotope distribution vector tIPV=(M, _{T1 , T2} ). In order to find the theoretical average isotope distribution pattern of fragment ions, the inventors calculated the average distribution and standard deviation of the isotopes of the theoretical fragment ions of polypeptides corresponding to trypsin hydrolysis of all proteins in the existing protein database, revealing the components of tIPV M, Relationship between _T1 and _T2 . Specifically, the inventors first theoretically digested the proteins in SWISS-PROT to obtain polypeptides; then selected polypeptides with a mass within (60u~3000u), which corresponds to the standard range of Q-TOF MS/MS experimental mass spectrometry . In addition, it is worth noting that the isotope of S ^{+ 2} S is very high in nature (the probability of occurrence is 0.04210, which is about 20 times that of ¹⁸ O), and in most cases, peptides that can contain more than five S are very rare . Therefore, we can divide the above molecular formula into six categories: S ⁰ , S ¹ , S ² , S ³ , S ⁴ and S ⁵⁺ , corresponding to the number of S contained in 0, 1, 2, 3, 4 and 5 and more than 5 peptides. The inventors made statistics according to these six categories. Statistical results show that T ₁ has a linear relationship with mass M, T ₂ has a quadratic relationship with M, and T ² increases with T ₁ and has a quadratic function with T ₁ .

In this way, the candidate molecular formulas can be filtered through the above distribution relationship of T ₁ , T ₂ and M to exclude those invalid and/or impossible molecular formulas.

B. Chemical rule constraints

The chemical rule constraint here is similar to the constraint condition DX≤G in formula (10), the difference is that in formula (10), the constraint condition DX≤G is used to constrain the formula

$\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; =</span>\sqrt{\mathrm{<span\; class="notranslate">\; Q</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; )</span>}<span\; class="notranslate">\; =</span>\sqrt{{\mathrm{<span\; class="notranslate">\; x</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}{\mathrm{<span\; class="notranslate">\; A</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; AX</span>}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; 2</span>}\mathrm{<span\; class="notranslate">\; B</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; AX</span>}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; B</span>}}^{\mathrm{<span\; class="notranslate">\; T</span>}}\mathrm{<span\; class="notranslate">\; B</span>}}$

In order to obtain the solution X _R in a real field of X under this constraint. Here, these constraint conditions are used to constrain the candidate molecular formulas of non-negative integer solutions searched in the domain of _XR , so as to filter these candidate molecular formulas.

C. Cross Validation

In particular, the b-series fragment ions of a peptide are all homologous, including b-, a-, b*-, a*-, b°-, a° type ions, which share the same original amino acid sequence , so it can be speculated that their isotope distribution patterns are very similar. The same is true for the y-series ions. If the _Me of two fragment ions in the mass spectrum differ by 28, 17 or 18, and the I ₁ and I ₂ of the two fragment ions are very close, it can be considered that the eIPVs corresponding to the two fragment ions are homologous. Then, we can use the homologous eIPV to cross-validate the prediction results. For example, for two homologous fragment ions, there is C _a1 H _a2 N _a3 O _a4 S _a5 in the list of candidate molecular formulas in one fragment ion, if C _a1-1 H _a2 N _a3 O _a4-1 S _a5 does not appear in In the candidate molecular formula list of another fragment ion, then the candidate molecular formula C _a1 H _a2 N _a3 O _a4 S _a5 can be considered as the result of random matching and excluded.

Claims (8)

1. A method for predicting ion molecular formula with the isotope peak of fragment ion in tandem mass spectrometry, comprising: Step 1): Obtain a monoisotopic peak of a fragment ion and at least one isotopic peak of the fragment ion from the tandem mass spectrometer, calculate the mass of the monoisotope of the fragment ion, the monoisotopic spectral peak of the fragment ion, and the fragment ion The relative abundance between the spectral peaks of at least one isotope of is used as the isotope distribution vector of the experiment; Step 2): providing a general molecular formula of fragment ions, the number of atoms of each element in the general molecular formula is to be determined; Step 3): use the general molecular formula to obtain the theoretical monoisotopic mass of the fragment ion, the monoisotope of the fragment ion and the relative abundance of at least one isotope thereof as a theoretical isotope distribution vector; the theoretical monoisotopic the mass, monoisotope of the fragment ion and the relative abundance of at least one isotope thereof as a function of the number of atoms to be determined in said general formula; Step 4): The Euclidean distance between the theoretical isotope distribution vector obtained in step 3) and the experimental isotope distribution vector obtained from tandem mass spectrometry in step 1) is minimized to obtain the real number domain of the fragment ions Molecular formula; Step 5): local search in the neighborhood of the molecular formula of the real number field to obtain the candidate molecular formula of the non-negative integer field, and obtain the predicted molecular formula of the fragment ion from the candidate molecular formula; Wherein, at least one isotope of the fragment ion described in the step 1) and step 3) includes the first isotope and the second isotope of the fragment ion; and The experimental isotope distribution vector eIPV=(M _e , I ₁ , I ₂ ), wherein, Me _is the ion mass of the monoisotope of the fragment ion obtained from the tandem mass spectrometry, and I ₁ and I ₂ correspond to the ion mass of the fragment ion The relative abundance of the peaks of the first isotope and the second isotope relative to the peaks of the monoisotope; The theoretical isotope distribution vector tIPV=(M, T ₁ , T ₂ ) is obtained as follows: M＝V×X (1) T ₁ =n ₁ q _C +n ₂ q _H +n ₃ q _N +n ₄ q _O1 +n ₅ q _S1 (2) ${\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; o</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 5</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; S</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}{{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; C</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; h</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; N</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; o</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; 5</span>}}{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; S</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 3</span>}<span\; class="notranslate">\; )</span>$ Wherein, the general molecular formula is C _n1 H _n2 N _n3 O _n4 S _n5 , n1～n5 is the number of atoms in the general molecular formula of the fragment ions, and M is the number of fragment ions obtained from the general molecular formula. Mass, T _{and T} are the relative abundance of the first isotopic fragment ion and the second isotopic fragment ion obtained from the general molecular formula, respectively, with respect to the monoisotopic fragment ion, and V is composed of the atomic weight of each element in the general molecular formula The row vector of X=[n1, n2, n3, n4, n5] ^T ; q _C , q _H and q _N are the ratios of ¹³ C relative to ¹² C, D relative to H, and ¹⁴ N relative to ¹⁵ N in nature q ₀₁ and q ₀₂ are the relative abundances of ¹⁷ O relative to ¹⁸ O and ¹⁸ O relative to ¹⁶ O in nature, respectively; q _s1 and q _s2 are the relative abundances of ³³ S relative to ³² S and ³⁴ S in nature The relative abundance of ³² S. 2. the method for predicting ion molecular formula with the isotope peak of fragment ion in the tandem mass spectrum according to claim 1, is characterized in that, uses the Euclidean distance E between the isotope distribution vector tIPV of theory and the isotope distribution vector eIPV of experiment as Matching scores of tIPV and eIPV: $\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; =</span>\sqrt{{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; \&delta;</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}<span\; class="notranslate">\; =</span>\sqrt{{<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; m</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; e</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; +</span>{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 4</span>}<span\; class="notranslate">\; )</span>$ 3. according to claim 1 or 2 described method with the isotope peak prediction ion molecular formula of fragment ion in tandem mass spectrometry, it is characterized in that, in described step 4) also comprise using the chemical rule that the molecular formula that obtains meets chemical meaning As a constraint condition for the minimization of the Euclidean distance. 4. the method for predicting ion molecular formula with the isotope peak of fragment ion in tandem mass spectrometry according to claim 1, is characterized in that, step 5) described in local search refers to that there is a distance with the molecular formula of described real number field Search for candidate formulas in the domain of non-negative integers in the range. 5. The method for predicting ion molecular formulas with the isotope peaks of fragment ions in tandem mass spectrometry according to claim 1, characterized in that, in step 5) also includes the step of filtering the candidate molecular formulas in the non-negative integer field. 6. the method for predicting ion molecular formula with the isotope peak of fragment ion in tandem mass spectrometry according to claim 5, is characterized in that, described filtering step comprises average isotope distribution mode method, and this method uses the theoretical monoisotope of fragment ion The statistical relationship between the mass of , the monoisotope of the fragment ion and the relative abundance of at least one isotope thereof filters the candidate molecular formulas of the non-negative integer domain. 7. the method for predicting ion molecular formula with the isotope peak of fragment ion in tandem mass spectrometry according to claim 5, is characterized in that, described filtering step comprises using the chemical rule constraints that make the molecular formula obtained meet chemical meaning to filter described non- Candidate formulas for the domain of negative integers. 8. the method for predicting ion molecular formula with the isotope peak of fragment ion in tandem mass spectrometry according to claim 5, is characterized in that, described filtering step comprises the candidate molecular formula of the non-negative integer domain with two fragment ions to carry out cross validation to Candidate formulas for the non-negative integer domain of the two fragment ions are filtered.