JP2008128849A - Peptide identification method using mass spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately and efficiently determine a predicted value of a plurality of kinds of fragment ion strengths occurring following cleavage of peptide. <P>SOLUTION: The effective atomic number obtained by multiplying the atomic number by a scaling factor is considered about each of amino acid residues constituting an fragment ion, the sum of effective atomic numbers of respective amino acid residues is used as an effective atomic number of the fragment ion (S1), and proton affinity is determined by substituting the effective atomic number into a predetermined logarithm equation (S2). Proton affinity can vary by varying the scaling factor in response to the specificity or the like of amino acid sequence, even when the constitution of the amino acid residues are the same. The fragment ion total amount calculated from activation energy is distributed in response to the proton affinity of a plurality of fragment ions produced by the same fragmenting reaction, and a further accurate fragment ion strength is determined. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for mass-analyzing a sample containing a peptide mixture and identifying the amino acid sequence of each peptide using mass spectral data obtained thereby.

In recent years, protein structures and functions have been rapidly analyzed as post-genomic research. As one of such protein structure / function analysis methods (proteome analysis), protein expression analysis and primary structure analysis using mass spectrometers are widely performed, and a quadrupole ion trap So-called MS ⁿ analysis (n is an integer of 2 or more), which captures and cleaves a specific peak by, for example, collision induced decomposition (CID), is effective. In general, in MS ² (= MS / MS) analysis, an ion having a specific mass-to-charge ratio (m / z) is first selected from an analysis object as a precursor ion, and the precursor ion is cleaved by CID. Then, information on the mass and chemical structure of the target ion can be obtained by mass analysis of ions (product ions) generated by cleavage.

When the amino acid sequence of a protein is determined by MS ⁿ analysis as described above, the protein is first digested with an appropriate enzyme to form a mixture of peptide fragments, and then the peptide mixture is subjected to mass spectrometry. At this time, since stable isotopes having different masses exist in the elements constituting each peptide, a plurality of peaks having different mass-to-charge ratios depending on the isotopic composition of peptides having the same amino acid sequence. Arise. The plurality of peaks are composed of peaks of ions (main ions) composed only of isotopes having the maximum natural abundance ratio, and peaks of ions containing other isotopes (isotope ions), which are from 1 Da. A peak group composed of a plurality of peaks arranged at intervals of several Da is formed.

Subsequently, from a mass spectrum data of the peptide mixture as described above, a set of isotope peaks derived from a single peptide is selected as a precursor ion, and ions obtained by cleaving the precursor ion ( Product ion) mass analysis (MS ² analysis).

  The amino acid sequence of the test peptide can be determined by subjecting the spectral pattern of the product ion obtained as described above and the spectral pattern of the precursor ion to a database search. Standard software for identifying peptides based on mass spectral data obtained by mass spectrometry is conventionally used. For example, as a typical database search engine for proteome analysis, MASCOT provided by Matrix Science is well known.

  In the analysis using MASCOT, first, the peptide with the matching mass is searched from the peptides registered in the database using the mass of the fragment ion appearing in the obtained mass spectrum as a key. A plurality of candidate peptide names are ranked and listed in order of matching degree. Analysts use this list to estimate peptides. However, in reality, the highest rank in the list of candidate peptides is often not the correct substance, and the search using the software is not necessarily highly reliable. The reason for this is considered to be that although matching of fragment ion mass is considered in the above software, matching with the peak intensity of fragment ions is not considered at all or hardly.

  Apart from MASCOT, a database search engine called SEQUEST provided by Thermo Electron is also used. This software takes into account the matching of both the fragment ion mass and its peak intensity. However, basically, just like MASCOT, the database has only mass-related data, and the strength is very simple (specifically, the b / y ion intensity is 50, Dehydration / desalting ionic strength is only evaluated by matching with mass spectral data obtained by measurement by giving fragment ionic strength of candidate peptides under 10). Therefore, the reliability cannot be expected with respect to the intensity matching.

  In view of the conventional problems as described above, the present inventor predicts a mass spectrum pattern for an amino acid sequence given as a candidate peptide and calculates a correlation with the actually measured fragment ion intensity, thereby A method of evaluating the reliability and identifying the peptide has been proposed (see Non-Patent Document 1, etc.). In such a technique, it is important to predict the peptide fragment intensity with high accuracy in order to predict the mass spectrum pattern. A basic method for predicting the fragment strength of a conventional peptide will be described.

The process of cleaving peptide molecular ions is known as a chemical reaction called a unimolecular reaction, and the reaction rate is given using the Arrhenius equation, which is a predictive equation for a chemical reaction at a certain temperature.
Fragment ion intensity ∝ reaction constant = A · exp (−E _a / R · T) (1)
Here, E _a is an apparent activation energy, R is a gas constant, T is an absolute temperature, and A is a value called a frequency factor. Alternatively, Gibbs free energy ΔG _a is used instead of activation energy E _a ,
Fragment ion intensity ∝A ₀ · exp (−ΔG _a / R · T) (2)
It can also be expressed as Here, _A0 is a frequency factor. Equation (2) is a modification of Equation (1), and the frequency factor differs in form depending on this variation.

  Such a fragmentation reaction is not one kind, and when a peptide molecular ion is cleaved into several fragment ions, there is a fragmentation reaction corresponding to each cleavage. Among them, there are fragment ions generated by fragmentation reaction by cleavage of the same bond, such as b fragment ion and y fragment ion.

  In many fragmentation reactions, the lengths of amino acid sequences are different, and y-type and b-type fragment ions are often generated simultaneously as described above. A molecular kinetic method called Kinetic Method is used to distribute the intensity of individual fragment ions from the total amount of fragment ions, that is, the intensity of y fragment ions and b fragment ions in the above example. Is known (see Non-Patent Document 2).

  On the other hand, recently, assuming a mathematical calculation model consisting of several tens of parameters, and providing a means to predict the spectrum pattern of fragment ion intensity under this assumption, the mass and intensity of fragment ions were combined. A technique for performing matching is proposed in Non-Patent Document 3. The calculation model described in this document employs the above-mentioned Arrhenius equation and a model equation that is formally matched to the kinetic method, and basically obtains the mass spectral intensity of the peptide by the following method. be able to.

The basic formula is the following formula (3).
[Fragment ion intensity] = [total amount of fragment ions generated by the relevant fragmentation reaction] × [distribution to fragment ions according to gas phase basicity] (3)
Here, gas-phase basicity (hereinafter abbreviated as GB) is an index representing the strength of the base in the gas phase. The two terms on the right side of the above equation (3) are obtained by the following procedure.

[First step: calculation of the total amount of fragment ions generated in the fragmentation reaction at the relevant binding position]
The total amount of fragment ions generated in the relevant fragmentation reaction is multiplied by the rate of excitation from the reaction position to the activated state by the probability of the molecular configuration (hereinafter referred to as reaction configuration) as the premise of the fragmentation reaction of interest. By seeking. The probability of becoming a reaction position is determined from the gas phase basicity for the peptide bond cleaved by the fragmentation reaction. On the other hand, the proportion excited from the reaction position to the activated state is obtained by applying the activation energy to the Arrhenius equation. Then, the total amount of fragment ions cleaved at the binding position of interest is obtained by multiplying the probability of the reaction position by the obtained ratio value.

[Second step: Calculation of distribution according to gas phase basicity to fragment ions]
In order to individually determine the amount of a plurality of fragment ions generated in the fragmentation reaction of interest, a method based on the kinetic method is used to determine the total amount of fragment ions determined in the first step, that is, the gas phase base of fragment ions. Exp {GB / (R · T _eff )} (where T _eff is called the effective temperature, depending on the magnitude of the nature, the type and pressure of the collision gas, the size of the peptide molecule, the ion before decomposition, A weight of a value that depends on the number of charges, etc.) is assigned and distributed to each fragment ion. At this time, the gas phase basicity of the fragment ion is obtained by using the gas phase basicity of the amino acid residue and assuming the following equation (4).
exp {GB / (R · T _eff )} = Σexp {GB _i / (R · T _eff )} (4)
Here, GB is the gas phase basicity of the fragment ion to be obtained, and i is a serial number assigned to the amino acid residue constituting the peptide molecule currently assumed. That is, GB _i means the gas phase basicity of the i-th amino acid residue. Also in the following description, for convenience, it is assumed that sequential numbers are given in order from the amino acid residue on the N-terminal side.

  As described above, in the method described in Non-Patent Document 3, the fragment ion intensity is calculated using physical quantities such as gas phase basicity, activation energy, and effective temperature. The physical quantity can be calculated by a simple calculation formula or evaluation formula, and the mass spectrum intensity of the peptide molecule can be obtained by substituting these physical quantities into the assumed calculation formula. At this time, parameters used in simple calculation formulas and evaluation formulas for obtaining these physical quantities are determined in advance by a technique such as parameter fitting so as not to contradict several fragment ion intensity observation cases. Further, one physical quantity is divided into two or more parameters so that as many cases as possible can be dealt with under the assumed calculation formula, and the parameter values are determined by parameter fitting.

However, in the conventional method for calculating the fragment ion intensity described above, some assumptions and simplifications in formulating the calculation formula and the evaluation formula are unreasonable, and therefore, the accuracy may be impaired. Specific examples include the following.
[1] The evaluation value of gas phase basicity of a single amino acid is inconsistent with standard literature data. For example, based on Non-Patent Document 3, the evaluation value of gas phase basicity of glycine, alanine and isoleucine is only about 1 kJ / mol, but the standard data published by NIST (Non-Patent Document) 4)), the difference is significantly different between glycine and alanine, about 15 kJ / mol, and between glycine and isoleucine, about 30 kJ / mol.
[2] Regarding the dependence of the fragment gas phase basicity on the type of fragment ion, for example, in the case of the same amino acid sequence, the gas phase basicity of the b type fragment ion and the y fragment ion Is only slightly considered for the contribution of terminal NH ₂ and COOH, and does not fully reflect the contribution due to the characteristic C-terminal structure of b-type fragment ions.

  [3] Equation (4) considers only the dependency of the type of amino acid residue constituting the fragment ion, and does not consider much the dependency of the amino acid residue sequence. That is, the influence of the amino acid residue adjacent on the C-terminal side is only considered slightly. However, this is clearly lacking in generality. For example, proline (pyrrolidine-2-carboxylic acid), which contains amino acid residues that are characteristically different from other amino acids and have a characteristic molecular bond that has a molecular structure with an amino group nitrogen in the ring. The ionization energy state should be different depending on where the amino acid residue (eg, proline) is located in the amino acid sequence. Therefore, it is reasonable to consider that the gas phase basicity varies depending on the amino acid residue sequence when at least the above-mentioned specific amino acid residues are included.

この表により、同じアミノ酸の組み合わせであってもプロリンの位置によってプロトン親和力が相違することは明らかであり、同様のことは気相塩基性度についても予想される。なお、この表において、Ｐ−ＸとＸ−ＰとのＰＡの値の開きは大きな場合で１０〜１５kcal/mol即ち４０〜６０kJ/molほどあり、このような大きな相違は非特許文献３による評価方法では説明できない。また非特許文献３に基づいて、Ｇ−Ｐ、Ａ−Ｐ、Ｉ−Ｐ、Ｌ−ＰやＶ−Ｐなどの気相塩基性度を評価しても、互いの評価値には殆ど差がなく１kJ/mol程度であるが、表１に示すＰＡの計算結果ではその差が最大１３kcal/mol即ち５０kJ/molほどにも及ぶ。 Table 1 shows proton affinities of various dipeptides containing proline (P) when proline is present on the N-terminal side (PX amino acid sequence) and when it is present on the C-terminal side (XP amino acid sequence). (Force of attracting protons) The results obtained by calculating PA by molecular dynamics method are shown.

From this table, it is clear that even in the same amino acid combination, the proton affinity differs depending on the position of proline, and the same is expected for the gas phase basicity. In this table, the difference in PA value between PX and XP is about 10 to 15 kcal / mol, that is, about 40 to 60 kJ / mol, and such a large difference is evaluated by Non-Patent Document 3. It cannot be explained by the method. Moreover, even if gas-phase basicity, such as GP, AP, IP, LP, and VP, is evaluated based on Non-Patent Document 3, there is almost no difference between the evaluation values. Although it is about 1 kJ / mol, the difference in the calculation results of PA shown in Table 1 reaches a maximum of 13 kcal / mol, that is, about 50 kJ / mol.

Sugawara, Ishida, Takeuchi, “Peptide fragment prediction method using first-principles calculation and its application to qualitative analysis”, 54th General Meeting of Mass Spectrometry, Osaka, 2006 Julia Laskin and one other, "The Theoretical Basis of The Kinematic Method From The Point Of View Of The Finite Heat Bath Theory the Kinematic Method from the Point of the View of Finite heat bath Theory), Journal of Phys. Chem. A, 2000, pp. 8829-8837. Z. Zhang, “Predication of Low-Energy Collision-Induced Dissociation Spectra of Peptides” Analytical Chemistry, Vol.76, No.14, July 15, 2004, pp.3908-3922 "National Institute of Standards and Technology", NIST, [searched November 15, 2006], Internet <URL: http://webbook.nist.gov/>

  The present invention has been made to solve the above-mentioned problems, and the object of the present invention is to predict the fragment ionic strength of a given peptide more accurately than before, and thereby to analyze proteins and proteins based on mass spectral data. It is an object of the present invention to provide a peptide identification method that can further improve the accuracy of determining the amino acid sequence of a peptide.

The present invention made to solve the above problems is a peptide identification method for determining the amino acid sequence of a peptide mixture in a test sample based on mass spectral data obtained by MS ⁿ analysis, comprising: In a peptide identification method for determining the validity of a candidate peptide by calculating the mass spectrum pattern by predicting the fragment ion intensity and comparing this with the actually measured mass spectrum, each of the plurality of fragment ions generated in the fragmentation reaction of the peptide To predict fragment ionic strength,
a) an intensity calculation step for determining the intensity of fragment ions generated in the fragmentation reaction;
b) a distribution step of distributing the fragment ionic strength according to the proton affinity or gas phase basicity given to each fragment ion type;
When calculating the proton affinity or gas phase basicity for each type of fragment ion, a correction factor that reflects the specificity of the amino acid sequence in the actual number of atoms for each amino acid residue constituting the fragment ion The number of effective atoms multiplied is calculated, and the number of effective atoms of the fragment ion obtained from the sum of the number of effective atoms for each amino acid residue is applied to a predetermined approximate calculation formula to calculate proton affinity or gas phase basicity. It is characterized by that.

  As one aspect of the peptide identification method according to the present invention, the predetermined approximate calculation formula may be a formula that approximates the nonlinear relationship between the number of effective atoms and proton affinity or gas phase basicity logarithmically.

  When calculating the fragment ion intensity for each type of fragment ion (for example, y-type and b-type fragment ions) generated in a fragmentation reaction for a peptide molecule having a certain amino acid sequence, the proton affinity (or gas phase base) of each fragment ion is calculated. However, conventionally, values such as proton affinity and gas phase basicity have been given only roughly. On the other hand, the peptide identification method according to the present invention provides a method for obtaining proton affinity and gas phase basicity with higher accuracy than before and with a practical calculation amount and calculation time.

  In other words, considering the number of effective atoms obtained by multiplying the number of atoms by an appropriate correction factor for each amino acid residue, even if the types of amino acid residues contained in the amino acid sequence are the same, the proton affinity or When the gas phase basicity is affected, the number of effective atoms is changed by correcting the correction coefficient according to each, so that the proton affinity and the gas phase basicity are also changed. Specifically, for example, when a basic amino acid is present in an amino acid sequence, a different correction coefficient may be given depending on whether the basic amino acid is present at the N-terminus. In addition, when proline is present in the amino acid sequence, different correction factors may be given depending on whether the proline is present at the N-terminus in combination with other amino acid residues adjacent to the proline.

  In addition, the accuracy of proton affinity and gas phase basicity can be further increased by changing the number of effective atoms depending on the type of fragment ions generated from the same fragmentation reaction. For example, in the by fragmentation reaction, a y fragment ion and a b fragment ion are generated, but even if they have the same amino acid sequence, the latter has three fewer atoms than the former, and therefore reflects the number of effective atoms.

  Conventionally, as shown in the equation (4), the gas phase basicity (same as proton affinity) has not been considered in consideration of the type of fragment ion or the sequence of amino acid residues. According to the peptide identification method, proton affinity and gas phase basicity can be determined with higher accuracy in consideration of the influence of the type of fragment ion and the sequence of amino acid residues. Thereby, the fragment ion intensity can be calculated with high accuracy for each type of fragment ion generated in the fragmentation reaction of the peptide, and therefore the reliability of peptide identification based on this can be improved.

  In addition, it is sufficient to prepare different values for the correction coefficients as described above only for those that have a large influence on proton affinity and gas phase basicity, and are not calculated one by one during the identification process, but are calculated in advance. It is possible to obtain an appropriate value by searching the database by making the database into a database and performing the identification process. Therefore, it is possible to avoid an enormous amount of computation and computation time on the computer while improving accuracy as described above, and it is possible to improve throughput by executing efficient identification processing.

  First, the overall procedure of the peptide identification method according to an embodiment of the present invention will be described. FIG. 1 is a schematic flowchart of analysis processing in the peptide identification method of the present embodiment. Actually, this analysis processing is executed by operating new proteome analysis software on a computer.

First, a substance (peptide) to be analyzed is subjected to mass analysis (MS ⁿ analysis) using, for example, an ion trap time-of-flight mass spectrometer, and mass spectrum data is collected (step S1). Next, an analysis process is performed on the mass spectrum data using existing database search software for proteome analysis (for example, MASCOT). That is, using the mass of fragment ions appearing in the mass spectrum (strictly speaking, the mass-to-charge ratio, m / z value) as a key, a peptide whose mass matches is searched from peptides registered in the database (step S2). Then, the plurality of peptide molecules obtained by the search are ranked and listed in order of match degree (step S3). Conventionally, it has been necessary to identify from the listed peptide molecules by the analyst's own judgment (for example, selecting the one with the highest score). According to the analysis processing according to the present invention, Further, candidates can be further narrowed down (in other words, more highly accurate determination material can be provided) by the processing after step S4.

  That is, among candidate peptides listed, a plurality of candidate peptides with clearly higher scores than others are selected, and the mass spectrum pattern is predicted by calculating the fragment ion intensity for each amino acid sequence of each candidate. . For this purpose, first, the total amount of fragment ions generated in the fragmentation reaction is calculated (step S4), and further, a plurality of fragment ions generated in the same fragmentation reaction according to the proton affinity (or gas phase basicity) determined by the calculation are calculated. The intensity distribution is performed to determine the fragment ion intensity (step S5). The mass spectrum pattern of each candidate peptide is predicted from the fragment ion intensity thus determined, and intensity matching evaluation is performed by correlation with the mass spectrum obtained by actual measurement (step S6). Then, by comparing the correlation evaluation results, the most likely candidate peptide is selected and presented, for example, on a display screen of a monitor (step S7). The new function by these steps S4 to S7 is combined with the function by the conventional standard database search software, so that an identification result with higher reliability than the conventional can be expected.

  The peptide identification method according to the present embodiment is particularly characterized by the processes in steps S4 and S5. These will be described in detail below.

(1) Method for calculating the total amount of fragment ions generated in the fragmentation reaction FIG. 3 is a principle explanatory view showing the method for calculating the fragment ion intensity in terms of energy levels, (a) based on proton affinity, (b) FIG. 2 is a diagram illustrating the principle based on gas phase basicity. Although the expressions are different, each uses a fragment ion intensity calculation formula based on the Arrhenius reaction formula shown in Eq. (1).

The total amount of fragment ions generated in the fragmentation reaction such as CID is determined using the Arrhenius reaction equation shown in equation (1). At that time, it is necessary to provide the activation energy E _a of the associated fragment as one of the parameters, the activation energy is the energy from the initial state of the peptide, the activation state of fragment ions of interest, i.e. to the transition state It is also possible to obtain the difference directly by calculation, but here we consider the sum of the two state energy differences. That is, for example, as shown in FIG. 3A, the proton (H ⁺ ) moves from the precursor ion (XY-H ⁺ ), which is the initial state, to the peptide bond position (called the reactive molecule position) that is cleaved. energy variation for migration and moving energy _{E H,} the transition state from the reaction molecules position ^{(X-H + -Y) (} X-H + -Y *) (hereinafter, referred to as the activation energy _{E a} ') Perform the calculation separately.

After dividing the energy in this way, as an approximate calculation, the activation energy E _a ′ is obtained by simplifying the molecular model of two or three amino acid sequences existing on both sides of the peptide bond to be cleaved. The reason for this simplification is that the fragmentation reaction can be regarded as a local reaction near the peptide bond. As for the movement energy E _H proton, obtained by simplifying the molecular model dipeptides formed by combining the first and last the presence of protons to the amino acid of the mobile. In this way, the molecular model can be simplified because protons are strongly bonded to nearby atoms, and the bond strength with distant atoms and molecules can be neglected. This is because the portion not to be omitted may be omitted.

And simplified to two or three amino acid sequences molecular models as described above activation energy E _{a 'in} order to determine the movement energy E _H of and proton activation energy E _a for each combination of pre-amino acid sequence', The proton transfer energy E _H is calculated and stored in a database, and when a bond is given, the corresponding energy E _a ′ and E _H are derived from the database. Although there are at most 20 types of common amino acids, for example, even if only a combination of 5 amino acid sequences is considered, if two types of b fragment ions and y fragment ions are considered, the energy calculation target is 20 ⁵ × 2 = It becomes a huge number of 64 × 10 ^5, and it takes a lot of time to calculate all of this. On the other hand, by simplifying to 2 or 3 amino acid sequence molecular models as described above, the number of objects to be calculated can be greatly reduced, and calculation can be performed in a relatively short time using a computer. Therefore, the database for calling the activation energy E _a ′ and the proton transfer energy E _H can be constructed relatively easily.

In the past research, it is known that the fragmentation reaction is roughly divided into two reaction mechanisms called the mobile proton model or the remote proton model. In the remote proton model, protons are used in the fragmentation reaction. In some cases, the reaction mechanism does not move (see, for example, Japanese Patent Application No. 2006-4553). In this case directly without performing the calculation of the moving energies E _H proton, but calculates only activation energy E _a, calculation method even in this case it may be Junjire the calculation of E _{a '.}

Further, when the fragmentation reaction process is considered not by proton affinity but by gas phase basicity, as shown in FIG. 3 (b) in contrast to FIG. 3 (a), the proton transfer energy E _H is used instead. Proton transfer free energy (ΔG _H ), activation free energy when decomposing via the transition state (XH ⁺ -Y ^* ) from the reactive molecule position (XH ⁺ -Y) in place of E _a ′ ( The activation free energy (ΔG _a ) may be expressed by the sum of ΔG _a ′).

If the activation energy E _a (or activation free energy ΔG _a ) is obtained as described above, the other parameters, gas constant R, temperature T, and frequency factor A (or A ₀ ) are known, so Arrhenius The fragment ion intensity, that is, the total amount of fragment ions of X ions and Y ions generated simultaneously in the fragmentation reaction can be calculated from the above reaction formula.

(2) Fragment ion intensity distribution calculation method according to proton affinity (or gas phase basicity) to multiple types of fragment ions To obtain a mass spectrum pattern, multiple types generated by the fragmentation reaction of interest It is necessary to individually determine the amount of generated fragment ions for each type (for example, b fragment ions and y fragment ions). Therefore, the total amount of fragment ions obtained in step S4 is calculated based on the kinetic method, that is, exp {PA / (R · T) according to the proton affinity PA (or gas phase basicity GB) of the fragment ions. _eff )} (or exp {GB / (R · T _eff )}).

For example, in a by fragmentation reaction, which is a typical fragmentation reaction process, b fragment ions and y fragment ions are generated, but the proton affinity (or gas phase basicity) of b fragment ions and y fragment ions is increased. When PAb and PAy (or GBb and GBy) are used, the distribution ratio is
b Fragment ion distribution ratio = expPAb / (expPAb + expPAy) (5)
y fragment ion distribution ratio = expPAy / (expPAb + expPAy) (6)
It becomes. When the gas phase basicity GB is used, GBb and GBy may be used instead of PAb and PAy in the above formulas (5) and (6). This allocation method itself is the kinetic method itself, but the proton affinity (or gas phase basicity) of the fragment ions used for this calculation is obtained by the following characteristic method.

  Past work (John L. Hohmes, Christiane Aubry, and Paul M. Mayer), “Proton Affinities of Primary Alkanols: An Appraisal of the Kinematics・ Proton Affinities of Primary Alkanols: An Appraisal of the Kinematic Method ”, Journal Physics Chemistry (J. Phys. Chem.), A, 103, pp.705-709 (1999)) For the same series of primary alkanols (Primary Alkanols), assuming that there is a curve relationship between the number of atoms and proton affinity, this curve is used to predict the proton affinity of unknown alkanols belonging to the same series. is doing.

  The peptide identification method according to the present embodiment also basically uses this principle to determine the proton affinity from the number of atoms of the fragment ion, but it is not the mere number of atoms but the effective number of atoms considering the type and sequence of amino acid residues. Introduce a new concept. That is, for each amino acid residue constituting the fragment ion, the effective number of atoms obtained by multiplying the number of atoms by a correction coefficient (hereinafter referred to as a scaling factor) is given, and the sum of the effective number of atoms of each amino acid residue is given as the fragment ion. The number of effective atoms. In addition, a nonlinear relationship between the proton affinity (or gas phase basicity) of the fragment ion and the number of effective atoms is represented by a logarithmic expression, and the proton affinity (or gas phase base is substituted by substituting the effective number of atoms into this logarithmic expression. (Sensitivity) is easily obtained.

Specifically, considering the case of the y fragment ion, for example, since the y fragment ion generally has the structure of a peptide molecule ion consisting of n amino acid residues, the number of effective atoms can be calculated by the following equation (7). N is represented.
N = P ₁ · L ₁ + P ₂ · L ₂ +... + P _n · L _n +3 (7)
Here, L _i and P _i are respectively the number of atoms and the scaling factor of the i-th amino acid residue counted from the N-terminal side of the amino acid sequence. If all the scaling factors P _i are 1, the number of effective atoms N is the same as the number of atoms of the y fragment ion. Here, “3” at the right end of the right side of the formula (7) is the number of atoms of the hydroxyl group (—OH) bonded to the C-terminal and the hydrogen group (—H) bonded to the N-terminal. The proton affinity of the y fragment ion is expressed by the following equation (8) using the effective number of atoms N defined by the equation (7).
PA = a · ln {b · (N−3)} = a · ln {b · (P ₁ · L ₁ + P ₂ · L ₂ +... + P _n · L _n )} (8)
Here, a and b are constants and are determined in advance by parameter fitting so as to match the actual proton affinity value as much as possible.

  In the logarithmic expression shown in the equation (8), (N-3) is used as a variable without using the effective number of atoms N as it is, but this may be N. In this case, however, the constants a and b may be determined by new parameter fitting.

On the other hand, in the case of b fragment ions, the proton affinity PAb is calculated by the following equation (9).
PAb = a · ln {b · (N−6)} = a · ln {b · (P ₁ · L ₁ + P ₂ · L ₂ +... + P _n · L _n −3)} (9)
In the y fragment ion and the b fragment ion composed of the same amino acid residue, the b fragment ion has three fewer atoms, so the equation (9) takes this into account.

  In the above formulas (8) and (9), proton affinity is defined as a form that depends on the type of amino acid residues constituting the fragment ion but does not depend on the order of the amino acid residues. However, it is conventionally known that the value of proton affinity is somewhat affected by the order of amino acid residues. In particular, proton affinity is considered to be easily affected by the molecular structure being interrupted at the N-terminal and C-terminal at both ends of the amino acid sequence. Among them, the N-terminal has a proton at the beginning of the fragmentation reaction in the mobile proton model. Since it is a molecular position, if a specific amino acid such as proline or basic amino acid is present at the N-terminus, it is expected that the proton affinity changes to a level that cannot be ignored.

  Therefore, considering the magnitude of the influence at the N-terminal where the molecular structure is interrupted, a scaling factor different from that when there is no such amino acid at the N-terminal is assigned when a basic amino acid is present at the N-terminal. In addition, regarding amino acids having a characteristic molecular structure different from other amino acids such as proline, the proton affinity is considered to be influenced by the combination with adjacent amino acids. Therefore, when a unique amino acid such as proline is present, a different scaling factor is given to each combination of the proline and the adjacent amino acid. If the scaling factor changes in this way, the effective number of atoms changes even if the original number of atoms is the same, and the proton affinity also changes accordingly.

The gas phase basicity GB can be expressed similarly to the proton affinity PA. That is, for the y fragment ion and the b fragment ion, the gas phase basicities GBy and GBb are respectively expressed by the following equations.
GBy = a ′ · ln {b ′ · (P ₁ ′ · L ₁ + P ₂ ′ · L ₂ +... + P _n ′ · L _n )} (10)
GBb = a ′ · ln {b ′ · (P ₁ ′ · L ₁ + P ₂ ′ · L ₂ +... + P _n ′ · L _n −3)} (11)
Here, a ′ and b ′ are constants, and are determined in advance by curve fitting so as to match the actual gas phase basicity as much as possible. P _i ′ and L _i are the number of atoms of the i-th amino acid residue and the scaling factor, respectively. Similarly to the proton affinity, the gas phase basicity is changed by assigning different scaling factors depending on whether a specific amino acid is at the N-terminus or not. The same applies to proline.

Specifically, the scaling factor P _i (or P _i ′) used in the equations (8) to (11) should be determined so as to match the experimental data together with the constants a and b (or a ′ and b ′). Since they are constants, these values can be obtained in advance by the least square method in comparison with many observation data. The obtained values are compiled into a database in a table format, for example, and appropriate values are read according to the given amino acid sequence, and the proton affinity (or gas phase) is calculated for each fragment ion using the above formula. Basicity) can be calculated.

Also, the scaling factor P _i can be determined by another method. That is, when n = 1, if attention is paid to the fact that the y fragment ion is an amino acid ion, the proton affinity of the amino acid is well known, and conversely, the scaling factor can be obtained from the proton affinity of the amino acid. In this case, assuming a y fragment ion (= amino acid ion) consisting of only one i-th amino acid residue in the equation (8) and setting the proton affinity of the amino acid ion as PA _i , the equation (8) is PA _i = a · ln (b · P _i · L _i ) (12)
That means
P _i = {1 / (b · L _i )} exp (PA _i / a) (13)
And can be transformed. Therefore, the scaling factor P _i can be obtained by substituting the value of proton affinity PA _i of amino acid ions obtained from experiments and theoretical considerations into the equation (13).

Also, if equation (8) is transformed,
exp (PA / a) = b · (P ₁ · L ₁ + P ₂ · L ₂ +... + P _n · L _n ) (14)
become that way. Substituting the above equation (13) into this equation,
exp (PA / a) = exp (PA 1 / a) + exp (PA 2 / a) + ... + exp (PA n / a) ... (15)
It becomes. This formula is similar in form to equation (4), but the meaning is completely different. That is, the denominator a of the exponential factor in the equation (15) is a constant, while the effective temperature _Teff is the denominator R · _Teff of the exponential factor in the equation (4), which is the mass-to-charge ratio of the peptide molecule, m / z value, etc. Is a constant that is not a constant (see Non-Patent Document 3 above).

Incidentally, not necessarily (13) the not necessary to determine the scaling factor P _i with may be determined by a parameter fitting or the like so as to match as possible to the actual proton affinity of fragment ions (or gas phase basicity).

A specific example of calculating the proton affinity for each fragment ion by the above method will be described. Table 2 shows the proton affinity of peptides composed of amino acid sequences of small molecular weight, such as polyglycine (G, GG, GGG, GGGG, GGGGG) and polyalanine (A, AA, AAA, AAAA), B3LYP / 6- The result calculated | required by the molecular dynamics calculation using the density functional method in the level of 311 + G (2d, p) is shown.

Using this result, the parameters a and b in the equation (8) can be determined from curve fitting of a curve with the horizontal axis representing the number of atoms N-3 and the vertical axis representing the proton affinity. That is, glycine Doing curve fitting assuming (G) and are both 1 for scaling factor P _i of amino acids such as alanine (A), the result is
PA [kcal / mol] = 11.395 · ln (N−3) +190.86 (16)
And obtained. If this is transformed into the form of equation (8), the constants a and b are
a = 11.395 [kcal / mol], b = 1.88016 × 10 ⁷
It is obtained.

このように定数ａ、ｂ及びスケーリング因子Ｐを決めておけば、(8)式を用いて任意のｙフラグメントイオンのプロトン親和力を、(9)式を用いて任意のｂフラグメントイオンのプロトン親和力をそれぞれ予測値として計算することができる。 Table 3 is an example in which the scaling factors were obtained from the proton affinity values of amino acids using the values of the constants a and b for various amino acids other than glycine and alanine. Scaling factors can be obtained in the same manner for amino acids not shown here.

If the constants a and b and the scaling factor P are determined in this way, the proton affinity of an arbitrary y fragment ion can be calculated using the equation (8), and the proton affinity of an arbitrary b fragment ion can be calculated using the equation (9). Each can be calculated as a predicted value.

Table 4 is an example in which a predicted value of proton affinity PA for y fragment ions composed of two amino acids, in other words, dipeptide ions, was calculated using the above results. In this example, the proton affinity of a dipeptide containing alanine (A) is predicted, but the result obtained from molecular dynamics calculation using the density functional method at the level of B3LYP / 6-311 + G (2d, p). It can be confirmed that the proton affinity prediction method in the present embodiment as described above is practically effective.

Table 5 shows the results of predicting the proton affinity of the b fragment ion composed of two glycines (G) using the formula (9) and comparing it with the calculated value by molecular dynamics. Also in this case, it can be seen that both agree relatively well.

プロリン以外のアミノ酸残基のスケーリング因子は既に決定されているものとし、表１に示したジペプチドのプロトン親和力を(8)式に代入することにより、プロリンのスケーリング因子を決定することができる。 As described above, Table 1 shows molecular dynamics calculation values of proton affinity for dipeptides in combination of proline (P) and other amino acids. The proline scaling factor obtained from the results of Table 1 is shown in Table 1. Is shown in Table 6.

It is assumed that scaling factors for amino acid residues other than proline have already been determined, and the proline scaling factor can be determined by substituting the proton affinity of the dipeptide shown in Table 1 into equation (8).

  The proline scaling factor when proline is present at the N-terminus can be obtained by substituting the value of proton affinity of the dipeptide of the PX mode in Table 1 into the formula (8). The scaling factor shown on the left side of Table 6 is given according to the type of amino acid residue that binds to proline. Further, the scaling factor when proline is not at the N-terminus can be obtained by substituting the value of proton affinity of the XP peptide in Table 1 into the formula (8), and is shown on the right side in Table 6. A scaling factor is given depending on the type of amino acid residue that binds to proline.

  As exemplified above, an appropriate scaling factor can be determined according to the amino acid sequence. FIG. 2 is a flowchart showing a processing procedure when the distribution calculation of fragment ion intensity is actually performed by a computer. That is, when a peptide molecule to be evaluated is given, a scaling factor is determined according to a predetermined algorithm in accordance with the amino acid sequence (step S11). Specifically, for example, when an amino acid sequence is input, a condition that affects the scaling factor is extracted as described above, for example, and a scaling factor for each amino acid residue is obtained by a database search corresponding to the condition. Next, the proton affinity of each fragment ion is calculated based on the equations (8) and (9) from the scaling factor of each amino acid residue and the number of atoms (step S12). Alternatively, the gas phase basicity of each fragment ion may be calculated based on the equations (10) and (11).

  If the proton affinity or gas phase basicity of each fragment ion is found, for example, if it is a by fragment, the fragment ion intensity is allocated based on the equations (5) and (6) (step S13). . Of course, it is natural that even if it is not a by-y fragment, it can be similarly distributed.

  As described above, according to the peptide identification method of the present invention, the fragment ionic strength is a simple model equation in accordance with the well-known standard theory of chemical reaction, that is, in accordance with the Arrhenius reaction rate equation and the kinetic method. Can be obtained by approximate calculation. If an attempt is made to directly obtain the fragment ion intensity by a computer without using such a model formula, since the size of the amino acid sequence of the peptide is generally large, the calculation amount becomes enormous and processing in a practical time becomes difficult. On the other hand, according to the method employed in the above embodiment, the activation energy and proton affinity (or gas phase basicity) necessary for the calculation are efficiently applied to the peptide molecule, that is, practical. It is possible to obtain within a wide range of time. In particular, proton affinity (or gas phase basicity) tends to increase as the size of the amino acid sequence increases, and does not tend to converge to a certain value. Therefore, it can be said that it is very effective that the proton affinity and the gas phase basicity can be obtained from a simple evaluation formula with a small amount of calculation.

  In addition, when calculating the proton affinity of each fragment ion, the value is corrected in consideration of the type of the fragment ion and the specificity of the amino acid sequence (for example, the presence or location of proline), so with higher accuracy Thus, the fragment ion intensity can be determined, and as a result, improvement in peptide identification accuracy can be expected.

  It should be noted that the above embodiment is merely an example of the present invention, and it is a matter of course that modifications, corrections, additions, and the like are appropriately included within the scope of the present invention within the scope of the present invention.

The schematic flowchart of the analysis process in the peptide identification method by one Embodiment of this invention. The flowchart which shows the process sequence at the time of performing distribution calculation of fragment ion intensity. Explanatory drawing which showed the calculation method of fragment ion intensity with the energy profile along the reaction path.

Claims (4)

A peptide identification method for determining the amino acid sequence of a peptide mixture in a test sample on the basis of mass spectral data obtained by MS ⁿ analysis, wherein the fragment ion intensity of a candidate peptide is predicted to obtain a mass spectral pattern. In the peptide identification method that judges the validity of a candidate peptide by comparing the measured mass spectrum with the actually measured mass spectrum, in order to predict the fragment ion intensity for each of multiple types of fragment ions generated in the peptide fragmentation reaction,
a) an intensity calculation step for determining the intensity of fragment ions generated in the fragmentation reaction;
b) a distribution step of distributing the fragment ionic strength according to the proton affinity or gas phase basicity given to each fragment ion type;
When calculating the proton affinity or gas phase basicity for each type of fragment ion, multiply the actual number of atoms for each amino acid residue constituting the fragment ion by a correction factor that reflects the specificity of the amino acid sequence. The effective number of atoms of the fragment ion obtained from the sum of the effective number of atoms for each amino acid residue is applied to a predetermined approximate calculation formula to calculate proton affinity or gas phase basicity. A peptide identification method using mass spectrometry.   2. The peptide using mass spectrometry according to claim 1, wherein the predetermined approximate calculation formula is a formula that approximates the nonlinear relationship between the number of effective atoms and proton affinity or gas phase basicity logarithmically. Identification method.   The mass spectrometry according to claim 1 or 2, wherein when a basic amino acid is present in the amino acid sequence, a different correction coefficient is given depending on whether or not the basic amino acid is present at the N-terminus. Peptide identification method.   When a proline is present in an amino acid sequence, a correction factor that differs depending on whether the proline is present at the N-terminus or not in combination with another amino acid residue adjacent to the proline is provided. A peptide identification method using the mass spectrometry according to 1 or 2.

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