JP2005283430A - Method for analyzing structure of biological sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To facilitate the interpretation of mass spectral data at the determination of the amino acid sequences of proteins and peptides through the use of mass spectrometry. <P>SOLUTION: Hydrogen atoms capable of replacing proteins and peptides are substituted into deuterium atoms in heavy water of 50% to analyze the proteins and the peptides in a mass spectroscope. A series of peak groups are formed in an acquired mass spectrum according to a ratio of deuterium substitution. By selecting a peak of 50% deuterium substitution at the center of the peak groups and performing MS/MS analysis, product ions form an isotope pattern due to the polymerization degree of deuterium. The spectral data is interpreted by considering the isotope pattern to identify the amino acid sequences. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for analyzing structural information of biological samples such as proteins, peptides, sugar chains, etc. using mass spectrometry.

  A protein has a certain amino acid sequence defined by genetic information. However, since the protein that is actually expressed has undergone various modifications after translation, the amino acid sequence of the expressed protein is determined experimentally in order to have a one-to-one correspondence between specific genetic information and the protein. Must.

In recent years, as a method for determining the amino acid sequence of such a protein, analysis using a mass spectrometer has been widely performed, such as PSD (Post-source decay) and QIT (quadrupole ion trap). : MS / MS analysis (or MS ⁿ analysis) that captures a specific peak and secondary cleavage by a quadrupole ion trap) is effective.
As a method for obtaining an amino acid sequence from a precursor ion or product ion spectrum pattern obtained by such a method, a method for identifying a test peptide by subjecting the obtained product ion spectrum pattern to a sequence database search, There is a method for estimating a structure by mathematically analyzing a spectral pattern using computer software.

  However, the method using database search has a problem that identification cannot be performed for proteins that are not registered in the database. In addition, sequence analysis by computer software has the following problems.

  There are various patterns of precursor ion cleavage patterns, and it is very difficult to predict the ion species of product ions obtained by cleavage. For example, fragmentation that occurs around peptide bonds is defined as shown in FIG. 1 for convenience. Here, r represents the total number of amino acid residues of this peptide, and n represents the residue number counted from the N-terminus. In general, the y-ion and b-ion series are best observed, but in practice, the product obtained by fragmentation or dehydration of one of the side chains is added to this, so that the resulting mass is obtained. The spectrum becomes even more complicated. Therefore, in the analysis by the computer software as described above, it is difficult to assign peaks, and accurate sequencing cannot be performed. In addition, a method of chemically treating a sample to increase or eliminate a specific series of peak intensities has been performed. However, when such chemical modification is performed, a modification reaction, desalting, etc. A complicated operation was required.

  Furthermore, as a method for facilitating interpretation of MS / MS spectral data, as described in Non-Patent Document 1 and Non-Patent Document 2, 100% replacement of exchangeable hydrogen atoms of proteins and peptides with deuterium. A method has been proposed. This utilizes the fact that the number of exchangeable hydrogen atoms is determined by the amino acid, and that the molecular weight of the sample shifts depending on the amino acid composition by deuterium substitution.

All hydrogen atoms covalently bonded to nitrogen, oxygen and sulfur atoms constituting the protein easily exchange with water hydrogen atoms in an aqueous solution. It is said that such a hydrogen atom can be exchanged. Specifically, R-NH ₂ (amino group), R-OH (hydroxyl group), R-C00H (carboxyl group), R-SH (thiol group), R—CO—NH ₂ and R—NH—CO—R ′ (primary and secondary amide groups), R—NH—R ′ (imino group), R—NH—C (═NH) NH ₂ (guanidino group) ) And the like.

There is always one exchangeable hydrogen atom in the main chain of the peptide chain, excluding proline, per amino acid residue. Therefore, all exchanges are possible by dissolving a peptide with molecular weight M, number of amino acid residues r, and the number of exchangeable hydrogen atoms of each amino acid (j) δ _j in heavy water (100% D ₂ 0). When a hydrogen atom is replaced with deuterium, the position of the molecular ion peak is
[M + D + r + Σδ _j ] ⁺ − [M + H] ⁺ = r + Σδ _j +1 (1)
Only, ie, the number of exchangeable hydrogen atoms moves to the higher mass side. Here, [M + H] ⁺ is the m / z ratio of the molecular ion peak of the natural abundance ratio, and the number of exchangeable hydrogen atoms (δ _j ) of each amino acid side chain is
δ _j = -1; Pro
_{δ j = 0; Gly, Ala} , Val, Leu, Ile, Met, Cys / 2, Phe
δ _j = 1; Ser, Thr, Asp, Glu, Cys, Tyr, Trp, His
δ _j = 2; Asn, Gln, Lys
δ _j = 4; Arg
It is. In Arg, protonated Arg ⁺ (δ _j = 5) is chemically stable, but in mass spectrometry, 1 to several protons are assigned to the entire ion according to the electric charge, so Arg, Lys, Side chains of basic amino acids such as His are considered electrically neutral.

Which protein corresponds to the molecular ion from the amino acid sequence on the database using the result of the formula (1) obtained from the sample labeled with deuterium and the sample not labeled with the value of molecular weight M, which is also determined by the amino acid composition A method of searching for is considered.
However, in the above method, all exchangeable hydrogen atoms of proteins and peptides are substituted with deuterium, so although peak shift occurs, the obtained spectral pattern is essentially the same as that without deuterium substitution. Therefore, it cannot be expected that the attribution of the peak will be greatly facilitated.
Spengler, B., Luetzenkirchen, F., and Kaufmann, R. On target deuteration for peptide sequencing by laser mass spectrometry. Org. Mass Spectrom. 28, 1482-1490 (1993). Sepetov, NF, Issakova, OL, Lebl, M., Swiderek, K., Stahl, DC, and Lee, TD The use of hydrogen-deuterium exchange to facilitate peptide sequencing by electrospray tandem mass spectrometry.Rapid Commun. Mass Spectrom. 7 , 58-62 (1993).

  The problem to be solved by the present invention is to provide a method for analyzing the structure of a biological sample, in which assignment of a peak of a mass spectrum obtained by mass spectrometry is easy and accurate structure determination can be performed.

The structure analysis method of a biological sample according to the present invention, which has been made to solve the above problems, replaces a replaceable hydrogen atom of a biological sample with a deuterium atom in water containing D ₂ O at a predetermined ratio. The structural information is obtained by analyzing the biological sample with a mass spectrometer.

As described above, the exchangeable hydrogen atoms of proteins and peptides are replaced with deuterium atoms in water containing D ₂ O at a predetermined ratio, and MS / MS analysis is performed. Shows a peculiar isotope pattern. By taking into account the isotope pattern of this product ion, it is possible to easily interpret MS / MS spectral data, and it is possible to accurately determine the amino acid sequence of proteins and peptides that are not registered in the database. Become.

  First, the principle of the structure analysis method for a biological sample according to the present invention will be described.

When a replaceable hydrogen atom of a protein is replaced with a predetermined ratio, for example, 50%, the molecular ion peak containing s = r + Σδ _j +1 in the above formula (1) is r + Σδ _j + 2 = s + 1 Split into two. At this time the peak intensity ratio of is represented by the binomial coefficient of Pascal's triangle, or (p + q) ^s, it is half of the exchangeable hydrogen atoms contained in the ion corresponding to one peak in natural abundance of strength 1 When replaced with deuterium, the intensity of the t-th peak of s + 1 produced by splitting is
_s C _t (p) ^st (q) ^t = _s C _t (1/2) ^s (t = 0,1,2, ..., s-1, s) (2)
It becomes.

Here, since the ratio of deuterium substitution is 50%, p = q = 1/2, and a series of peaks is t = s / 2 (s is an even number), or t = (s + 1) / 2 (s is an odd number) Symmetrical and bell-shaped contours are shown. As a result, even if s increases, (1/2) ^s ≈ 0 near both ends corresponding to the bottom of the bell-shaped curve, so that the observed peak range does not unnecessarily widen. According to the central limit theorem, when these peaks show a Gaussian distribution with a bell-shaped curve, the line width (w _1/2 ) of this curve is
w _1/2 = s ^1/2 / 2 (3)
It is estimated that

For example, when s = 200, the main peak is at most ± 7 mass units, and the center can be specified from the symmetry of the peak group. Moreover, the intensity I (t = s / 2) of the peak at the center, that is, the binomial coefficient of the equation (2) is
_{I (t) = s C t} (1/2) s ≒ 1 / (πs) -1/2 (4)
Therefore, when s = 10, it is about 25% of the single peak, and even when s = 200, it is reduced only to 4%. Since the original 1% is s> 3000, that is, a considerably large protein exceeding about 2000 amino acid residues, such a decrease in sensitivity is not a problem.

When 50% of the exchangeable hydrogen atoms of a peptide are replaced with deuterium, the mass spectrum is expressed by the following formula (2), from those containing almost no deuterium to those almost completely substituted with deuterium. Reflects the abundance ratio of molecular species with various isotope contents represented. The maximum peak at the center of the peak group corresponds to a molecular species in which exchangeable hydrogen atoms are replaced with deuterium at exactly 50%. However, this peak of maximum intensity includes various molecular species with different distributions of hydrogen and deuterium even though the masses are exactly the same, and the distribution shows that the number of exchangeable hydrogen atoms is s. If there are pcs, _s C _{s / 2} .

For example, if s = 6 simply,
HHHDDD HHDHDD HHDDHD HHDDDH
HDHHDD HDHDHD HDHDDH HDDHHD
HDDHDH HDDDHH DHHHDD DHHDHD
DHHDDH DHDHHD DHDHDH DHDDHH
DDHHHD DDHHDH DDHDHH DDDHHH
₆ C ₃ = 20 kinds of ion species are included in the peak of the same mass of ions in single.

If the above peak is taken out by an ion trap and cleaved into an N-terminal part containing two exchangeable hydrogen atoms and the remaining C-terminal part by MS / MS, the N-terminal side is
HH (4); HD, DH (12); DD (4)
From the existence probability, the strength ratio is 1: 3: 1 triplet (triplet: triplet)
3H + 1D (4); 2H + 2D (12); 1H + 3D (4)
Is observed as a triplet with an intensity ratio of 1: 3: 1. At this time, the number of MS / MS peaks after cleavage and the intensity ratio can be easily predicted by the number of exchangeable hydrogen atoms of the smaller fragment due to the symmetry of the equation (2). In the above example, since there are two exchangeable hydrogen atoms at the N-terminal, the number of peaks generated is 2 + 1 = 3, and the intensity ratio of this triplet is ₂ C ₀ · ₄ C ₃ : ₂ C ₁ · _{4 C 2: 2 C 2 · 4} C 1 = 4: 12: 4 = 1: 3: 1 and can be stochastically predicted.

These numbers and equations are expressed as follows: s is the number of exchangeable hydrogen atoms in the first ion trapped peak, s is the number of exchangeable hydrogen atoms in the smaller fragment, and v is the rest (vs = u). When generalized, the number of peaks generated by cleavage is u + 1, and each peak intensity I _j is
I _j = _u C _j · _v C _{(s / 2-j)} (j = 0,1,…, u) (5)
It becomes. Although s is considered as an even number, when s is an odd number, theoretically, the peaks to be ion trapped are both two peaks of equal intensity appearing at the center of the peak group. However, if s is sufficiently larger than 1, s >> u, and the two items in the product of Eq. (5) are almost unaffected by changes in j, so the peak intensity I _j is determined only by the binomial coefficient. For example, if u = 2, a triplet of I ₀ : I ₁ : I ₂ = 1: 2: 1 is observed.

  If one fragment is a b-ion and the other fragment is a y-ion, the pair of b-ions and y-ions generated simultaneously by the same bond cleavage are exactly the same on the mass spectrum as shown in FIG. Indicates a pattern. This makes it very easy to find such b-ion and y-ion (or a-ion and z-ion) pairs from a complex spectrum in which multiple bonds are cleaved and many peaks appear.

In general, if this method is applied to a peptide with the number of amino acid residues r and molecular weight M, and a series of b-ions and y-ions is ideally observed by MS / MS, it shows the same cleavage pattern and the mass of each pair. B ₁ -ion (m / z = M (b ₁ )) and y _r-1 -ion (m / z = M (y _r-1 )), b ₂ , whose number is the same as the mass of the precursor ion -Ion (m / z = M (b ₂ )) and y _r-2 -Ion (m / z = M (y _r-2 )) (where M (b ₁ ) + M (y _r-1 ) = M By tracing (b ₂ ) + M (y _r-2 ) = M) on the spectrum, you can easily read the amino acid sequence and, of course, computer data processing for peak assignment that automates this The algorithm accuracy is also expected to improve. Needless to say, the above-mentioned mass number M means that one half of exchangeable hydrogen atoms are replaced by deuterium.

  Moreover, even from only one ion series, such as only y-ions, the amino acid sequence can be estimated relatively easily by following the peaks that increase the degree of division as the mass gradually decreases.

Furthermore, since the multiplicity of the peak is δ _j +2, which is obtained by adding 1 to the number of exchangeable hydrogen atoms unique to each amino acid residue given by the equation (1), the peak assignment result also from this point Can be verified. For example, glutamic acid (Glu) and glutamine (Gln) have molecular weights of 129 and 128, respectively, but only 1 mass unit, but by performing 50% deuterium substitution, the peak from which Glu is eliminated is a triplet, Since the peak from which Gln is eliminated becomes a quartet, the difference between the two becomes clear.

As described above, the feature of the amino acid sequencing method for proteins and peptides based on the above 50% deuterium substitution is that a peak in which 50% of exchangeable hydrogen atoms are substituted with deuterium is specifically detected by PSD or QIT. with capture caused by MS / MS or MS ^n, a peak pairs from each other, with the mark in the cleavage pattern or multiplicity, lies in that to facilitate and ensure the determination of the amino acid sequence.

If the multiplicity of a fragment far from the end in one MS / MS analysis is too large, one central peak of a specific ion group is trapped again among appropriate secondary ions. MS ³ analysis may be performed.

Deuterium substitution at a predetermined ratio of the biological sample as described above can be performed by dissolving the biological sample in water (H ₂ O / D ₂ O) containing D ₂ O at a predetermined ratio. As a sample for mass spectrometry, a lyophilized biological sample subjected to the deuterium substitution is used. The mass spectrometer used for the analysis is not particularly limited, but it is desirable to use an ion trap mass spectrometer.

  The structure analysis method of a biological sample based on deuterium substitution at a predetermined ratio of the present invention can be used for structure analysis of sugar chains in addition to proteins and peptides. In particular, since there are a large number of exchangeable hydrogen atoms in sugars, this method is effective in identifying peptides with sugar chains and determining molecular weight.

The ratio of substitution with deuterium is not limited to the above 50%. For example, in the case of a pure polysaccharide, the mixing ratio of H ₂ O and D ₂ O is changed to 4: 1 so as not to branch. If one deuterium atom corresponds to one pyranose ring, the approximate number of pyranose rings can be estimated.

  Hereinafter, 50% of exchangeable hydrogen atoms are substituted with deuterium for insulin B chain (amino acid residue number 30, molecular weight 3496, exchangeable hydrogen atom number 49) having the amino acid sequence as shown in FIG. Examples of mass spectrometry are shown below.

First, 2 mg of insulin B chain was dissolved in 0.5 ml of 50% heavy water and then freeze-dried to obtain an insulin B chain sample in which 50% of exchangeable hydrogen atoms were deuterated.
MS / MS analysis of the insulin B chain sample with 50% deuterium substitution and the insulin B chain sample with no deuterium substitution was performed. The center of the peak group (m / z = 3496.71) divided into eight according to the body abundance ratio is 50% deuterium substituted (lower part of Fig. 4). /z=3521.78). This is 50% of the increase in mass expected from equation (1) (r + Σδ _j +1 = 49 + 1)
3521.78−3496.71 ＝ 25.07 ≒ (49 + 1) / 2
Was found to match exactly.

The figure which shows the pattern of the cleavage which arises around a peptide bond. Schematic which shows the isotope pattern of the product ion in the structural analysis method of this invention. Amino acid sequence of insulin B chain. Mass spectrum by 50% deuterium substitution of insulin B chain.

Claims (5)

Substituting deuterium atoms for exchangeable hydrogen atoms in a biological sample in water containing D ₂ O at a predetermined ratio, and analyzing the biological sample with a mass spectrometer to obtain structural information thereof Structural analysis method for biological samples. Determining the amino acid sequence of a protein or peptide by substituting deuterium atoms for exchangeable hydrogen atoms in water containing D ₂ O at a predetermined ratio and analyzing the protein or peptide with a mass spectrometer A method for determining an amino acid sequence. In a water containing D ₂ O at a predetermined ratio, a replaceable hydrogen atom of a protein or peptide is replaced with a deuterium atom, and the protein or peptide is subjected to MS / MS (PSD) analysis with a mass spectrometer, An amino acid sequence determination method characterized by determining an amino acid sequence from the abundance ratio of product ion isotopes. In a water containing D ₂ O at a predetermined ratio, the exchangeable hydrogen atom of a sugar chain is replaced with a deuterium atom, and the structure is determined by analyzing the sugar chain with a mass spectrometer. Characteristic method for analyzing sugar chain structure.   5. The method for analyzing a structure of a biological sample according to claim 1, wherein the predetermined ratio is 50%.

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